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Sample records for one-step rt-pcr assay

  1. Establishment and validation of two duplex one-step real-time RT-PCR assays for diagnosis of foot-and-mouth disease.

    Science.gov (United States)

    Gorna, K; Relmy, A; Romey, A; Zientara, S; Blaise-Boisseau, S; Bakkali-Kassimi, L

    2016-09-01

    Two duplex one-step TaqMan-based RT-PCR protocols for detection of foot-and-mouth disease virus (FMDV) were established and validated. Each RT-PCR test consists of a ready-to-use master mix for simultaneous detection of the well established 3D or IRES FMDV targets and incorporates the host β-actin mRNA as an internal control target, in a single-tube assay. The two real-time RT-PCR 3D/β-actin and IRES/β-actin tests are highly sensitive and able to detect up to 7TCID50/ml of FMDV and 10 copies/1μl of viral RNA. In field epithelium samples, the diagnostic sensitivity was 100% (95% CI; 91-100%) for the 3D/β-actin test and 97% (95% CI; 87-100%) for the IRES/β-actin test. The diagnostic specificity was 100% (95% CI; 95-100%) for both RT-PCRs. In addition, the two protocols proved to be robust, showing inter-assay coefficients of variation ranging from 1.94% to 6.73% for the IRES target and from 2.33% to 5.42% for the 3D target for different RNA extractions and different RT-PCR conditions. The internally controlled one-step real-time RT-PCR protocols described in this study provide a rapid, effective and reliable method for the detection of FMDV and thus may improve the routine diagnosis for foot-and-mouth disease.

  2. Development and evaluation of novel one-step TaqMan realtime RT-PCR assays for the detection and direct genotyping of genogroup I and II noroviruses

    DEFF Research Database (Denmark)

    Schultz, Anna Charlotte; Vega, Everado; Dalsgaard, Anders;

    2011-01-01

    , polioviruses, and rotaviruses. L-RT-qPCR products were typed by sequencing. ResultsThe novel GI and GII L-RT-qPCR assays detected and typed all but one of the NoV positive panel samples. As few as 5–500 RNA copies could be accurately typed by sequencing of amplicons. ConclusionsWe developed novel one-step Taq......BackgroundCurrent detection and genotyping methods of genogroup (G) I and II noroviruses (NoVs) consist of a 2-step approach including detection of viral RNA by TaqMan realtime RT-PCR (RT-qPCR) followed by conventional RT-PCR and sequencing of partial regions of ORF1 or ORF2. ObjectiveTo develop...... novel long-template one-step TaqMan assays (L-RT-qPCR) for the rapid detection and direct genotyping of GI and GII NoVs and to evaluate the sensitivity and specificity of the assays. Study designGI and GII-specific broadly reactive L-RT-qPCR assays were developed by combining existing NoV primers...

  3. Comprehensive multiplex one-step real-time TaqMan qRT-PCR assays for detection and quantification of hemorrhagic fever viruses.

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    Zheng Pang

    Full Text Available BACKGROUND: Viral hemorrhagic fevers (VHFs are a group of animal and human illnesses that are mostly caused by several distinct families of viruses including bunyaviruses, flaviviruses, filoviruses and arenaviruses. Although specific signs and symptoms vary by the type of VHF, initial signs and symptoms are very similar. Therefore rapid immunologic and molecular tools for differential diagnosis of hemorrhagic fever viruses (HFVs are important for effective case management and control of the spread of VHFs. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR assay is one of the reliable and desirable methods for specific detection and quantification of virus load. Multiplex PCR assay has the potential to produce considerable savings in time and resources in the laboratory detection. RESULTS: Primers/probe sets were designed based on appropriate specific genes for each of 28 HFVs which nearly covered all the HFVs, and identified with good specificity and sensitivity using monoplex assays. Seven groups of multiplex one-step real-time qRT-PCR assays in a universal experimental system were then developed by combining all primers/probe sets into 4-plex reactions and evaluated with serial dilutions of synthesized viral RNAs. For all the multiplex assays, no cross-reactivity with other HFVs was observed, and the limits of detection were mainly between 45 and 150 copies/PCR. The reproducibility was satisfactory, since the coefficient of variation of Ct values were all less than 5% in each dilution of synthesized viral RNAs for both intra-assays and inter-assays. Evaluation of the method with available clinical serum samples collected from HFRS patients, SFTS patients and Dengue fever patients showed high sensitivity and specificity of the related multiplex assays on the clinical specimens. CONCLUSIONS: Overall, the comprehensive multiplex one-step real-time qRT-PCR assays were established in this study, and proved to be

  4. Development of a One-Step Immunocapture Real-Time RT-PCR Assay for Detection of Tobacco Mosaic Virus in Soil

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    Jin-Guang Yang

    2012-12-01

    Full Text Available Tobacco mosaic virus (TMV causes significant losses in many economically important crops. Contaminated soils may play roles as reservoirs and sources of transmission for TMV. In this study we report the development of an immunocapture real-time RT-PCR (IC-real-time RT-PCR assay for direct detection of TMV in soils without RNA isolation. A series of TMV infected leaf sap dilutions of 1:101, 1:102, 1:103, 1:104, 1:105 and 1:106 (w/v, g/mL were added to one gram of soil. The reactivity of DAS-ELISA and conventional RT-PCR was in the range of 1:102 and 1:103 dilution in TMV-infested soils, respectively. Meanwhile, the detection limit of IC-real-time RT-PCR sensitivity was up to 1:106 dilution. However, in plant sap infected by TMV, both IC-real-time RT-PCR and real-time RT-PCR were up to 1:106 dilution, DAS-ELISA could detect at least 1:103 dilution. IC-real-time RT-PCR method can use either plant sample extracts or cultivated soils, and show higher sensitivity than RT-PCR and DAS-ELISA for detection of TMV in soils. Therefore, the proposed IC-real-time RT-PCR assay provides an alternative for quick and very sensitive detection of TMV in soils, with the advantage of not requiring a concentration or RNA purification steps while still allowing detection of TMV for disease control.

  5. Development of novel AllGlo-probe-based one-step multiplex qRT-PCR assay for rapid identification of avian influenza virus H7N9.

    Science.gov (United States)

    Zhang, Yanjun; Mao, Haiyan; Yan, Juying; Wang, Xinying; Zhang, Lei; Guus, Koch; Li, Hui; Li, Zhen; Chen, Yin; Gong, Liming; Chen, Zhiping; Xia, Shichang

    2014-07-01

    Recently, human deaths have resulted from infection with low-pathogenicity avian influenza virus H7N9 strains that have emerged recently in China. To strengthen H7N9 surveillance and outbreak control, rapid and reliable diagnostic methods are needed. To develop a sensitive quantitative real-time RT-PCR assay for rapid detection of H7N9 viral RNA, primers and AllGlo probes were designed to target the HA and NA genes of H7N9. Conserved sequences in the HA and NA genes were identified by phylogenic analysis and used as targets for H7N9 virus detection. The similarities of the targeted HA and NA gene sequences from different H7 and N9 influenza virus strains were 93.2-99.9 % and 96.0-99.6 %, respectively The specificity and sensitivity of the new multiplex real-time qRT-PCR was established. The test was used for the detection of viral RNA in human pharyngeal swabs and environmental samples. The detection limit of the multiplex qRT-PCR was estimated to be about 10(-1) TCID50/reaction. Finally, the diagnostic sensitivities of the multiplex qRT-PCR, virus isolation and TaqMan qRT-PCR were compared using pharyngeal swabs and environmental samples. These analyses yielded positive results in 46.7 %, 43.3 % and 20.0 % of the samples, respectively. The novel multiplex AllGlo qRT-PCR is a rapid and sensitive method to identify H7N9 virus in clinical and environmental samples and can be used to facilitate studies on the epidemiology of H7N9 virus.

  6. Specific detection of H5N1 avian influenza A virus in field specimens by a one-step RT-PCR assay

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    Gupta Sanjay

    2006-03-01

    Full Text Available Abstract Background Continuous outbreaks of the highly pathogenic H5N1 avian influenza A in Asia has resulted in an urgent effort to improve current diagnostics to aid containment of the virus and lower the threat of a influenza pandemic. We report here the development of a PCR-based assay that is highly specific for the H5N1 avian influenza A virus. Methods A one-step reverse-transcription PCR assay was developed to detect the H5N1 avian influenza A virus. The specificity of the assay was shown by testing sub-types of influenza A virus and other viral and bacterial pathogens; and on field samples. Results Validation on 145 field specimens from Vietnam and Malaysia showed that the assay was specific without cross reactivity to a number of other infuenza strains as well as human respiratory related pathogens. Detection was 100% from allantoic fluid in H5N1 positive samples, suggesting it to be a reliable sampling source for accurate detection. Conclusion The assay developed from this study indicates that the primers are specific for the H5N1 influenza virus. As shown by the field tested results, this assay would be highly useful as a diagnostic tool to help identify and control influenza epidemics.

  7. Establishment and Application of One-step RT-PCR Assay for BVDV Detection%牛病毒性腹泻病毒一步法.RT-PCR检测方法的建立及应用

    Institute of Scientific and Technical Information of China (English)

    于新友; 李天芝; 沈志强

    2015-01-01

    An one-step RT-PCR assay for detection of BVDV was established using a pair of primers based on the 5'-untranslated region gene of bovine viral diarrhea virus (BVDV), and the speciifcity, sensitivity were studied.This method speciifcally amplify a fragment from BVDV, but not from control virus, with a detection limit of 1pg RNA of BVDV.Therefore the established one-step RT-PCR technique provided a sensitive, speciifc, fast and reliablemethod for diagnosis and epizootic study of the BVDV.%根据牛病毒性腹泻病毒(BVDV)5′端非编码区基因保守序列设计引物,建立了检测BVDV一步法反转录-聚合酶链(RT-PCR)方法,并对其特异性、敏感性进行了研究。结果表明,该方法对BVDV检测的灵敏度达到1pg RNA,特异性强、敏感性高,可用于牛病毒性腹泻病的早期确诊和病毒鉴定。

  8. Detection of Zika virus by SYBR green one-step real-time RT-PCR.

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    Xu, Ming-Yue; Liu, Si-Qing; Deng, Cheng-Lin; Zhang, Qiu-Yan; Zhang, Bo

    2016-10-01

    The ongoing Zika virus (ZIKV) outbreak has rapidly spread to new areas of Americas, which were the first transmissions outside its traditional endemic areas in Africa and Asia. Due to the link with newborn defects and neurological disorder, numerous infected cases throughout the world and various mosquito vectors, the virus has been considered to be an international public health emergency. In the present study, we developed a SYBR Green based one-step real-time RT-PCR assay for rapid detection of ZIKV. Our results revealed that the real-time assay is highly specific and sensitive in detection of ZIKV in cell samples. Importantly, the replication of ZIKV at different time points in infected cells could be rapidly monitored by the real-time RT-PCR assay. Specifically, the real-time RT-PCR showed acceptable performance in measurement of infectious ZIKV RNA. This assay could detect ZIKV at a titer as low as 1PFU/mL. The real-time RT-PCR assay could be a useful tool for further virology surveillance and diagnosis of ZIKV.

  9. One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq® in stool samples

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    Rashi Gautam

    2016-01-01

    Full Text Available Background. Group A rotavirus (RVA infection is the major cause of acute gastroenteritis (AGE in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR offer several advantages including increased sensitivity, higher throughput, and faster turnaround time. Methods. In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq® rotavirus strains along with an internal processing control (Xeno or MS2 RNA. Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12 and VP4 (P[4], P[6] and P[8] genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostable rTth polymerase enzyme, dsRNA denaturation, reverse transcription (RT and amplification (PCR steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Results. The VP7 qRT-PCRs exhibited 98.8–100% sensitivity, 99.7–100% specificity, 85–95% efficiency and a limit of detection of 4–60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81–92% efficiency and limit of detection of 150–600 copies in multiplex reactions. The VP4 q

  10. One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples.

    Science.gov (United States)

    Gautam, Rashi; Mijatovic-Rustempasic, Slavica; Esona, Mathew D; Tam, Ka Ian; Quaye, Osbourne; Bowen, Michael D

    2016-01-01

    Background. Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time. Methods. In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostable rTth polymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Results. The VP7 qRT-PCRs exhibited 98.8-100% sensitivity, 99.7-100% specificity, 85-95% efficiency and a limit of detection of 4-60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81-92% efficiency and limit of detection of 150-600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98

  11. A one-step, triplex, real-time RT-PCR assay for the simultaneous detection of enterovirus 71, coxsackie A16 and pan-enterovirus in a single tube.

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    Shiyin Zhang

    Full Text Available The recent, ongoing epidemic of hand, foot, and mouth disease (HFMD, which is caused by enterovirus infection, has affected millions of children and resulted in thousands of deaths in China. Enterovirus 71 (EV71 and coxsackie A16 (CA16 are the two major distinct pathogens for HFMD. However, EV71 is more commonly associated with neurologic complications and even fatalities. Therefore, simultaneously detecting and differentiating EV71 and CA16 specifically from other enteroviruses for diagnosing HFMD is important. Here, we developed a one-step, triplex, real-time RT-PCR assay for the simultaneous detection of EV71, CA16, and pan-enterovirus (EVs in a single tube with an internal amplification control. The detection results for the serially diluted viruses indicate that the lower limit of detection for this assay is 0.001-0.04 TCID50/ml, 0.02 TCID50/ml, and 0.001 TCID50/ml for EVs, EV71, and CA16, respectively. After evaluating known HFMD virus stocks of 17 strains of 16 different serotypes, this assay showed a favorable detection spectrum and no obvious cross-reactivity. The results for 141 clinical throat swabs from HFMD-suspected patients demonstrated sensitivities of 98.4%, 98.7%, and 100% for EVs, EV71, and CA16, respectively, and 100% specificity for each virus. The application of this one-step, triplex, real-time RT-PCR assay in clinical units will contribute to HFMD surveillance and help to identify causative pathogen in patients with suspected HFMD.

  12. One-Step RT-PCR protocols improve the rate of dengue diagnosis compared to Two-Step RT-PCR approaches.

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    De Paula, Sérgio Oliveira; de Melo Lima, Cristiane; Torres, Maria Paula; Pereira, Márcia Rodrigues Garbin; Lopes da Fonseca, Benedito Antônio

    2004-08-01

    Dengue is the most important arboviral disease transmitted to humans. In our laboratory, we have been working on the standardization of the polymerase chain reaction (PCR) diagnosis of this disease. In this work, we compared five commercial kits regularly used on reverse-transcription polymerase chain reaction (RT-PCR) protocols: two Two-Step kits (SuperScript II RT/Super Mix kit and reverse transcription system/Taq DNA polymerase) and three One-Step kits (ready-to-go RT-PCR Beads kit, QIAGEN One-Step RT-PCR kit, and AcessQuick RT-PCR system). Thirty-one serum samples of patients with clinical diagnosis of dengue fever (DF) were analyzed by RT-PCR and serology. RNA extraction was done with the QIAamp Viral RNA kit, and cDNA synthesis and PCR done according to the manufacturer's protocol for the five kits. Out of the 31 serum samples collected from patients suspected of having dengue, 27 were IgM-positive, confirming the dengue diagnosis. Out of those, 24 were positive by the ready-to-go RT-PCR Beads kit, 25 were positive by AcessQuick RT-PCR system and 27 were positive by QIAGEN One-Step RT-PCR kit. On the other hand, only six samples were positive by the SuperScript II RT/Super Mix kits and 10 were positive by reverse transcription system/Taq DNA polymerase kit. The best performance observed with the One-Step kits was confirmed in spiked samples with known quantities of dengue-1 virus since they detected up 1 x 10(2) PFU/ml, while the most sensitive Two-Step kit detected up 1 x 10(4) PFU/ml. These data show that One-Step RT-PCR kits yielded a higher rate of dengue virus detection than the Two-Step kits and correlated well with the serological diagnosis.

  13. Molecular characterization of two Rocio flavivirus strains isolated during the encephalitis epidemic in São Paulo State, Brazil and the development of a one-step RT-PCR assay for diagnosis.

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    Coimbra, Terezinha Lisieux Moraes; Santos, Raimundo N; Petrella, Selma; Nagasse-Sugahara, Teresa Keico; Castrignano, Silvana Beres; Santos, Cecília L Simões

    2008-01-01

    Rocio virus (ROCV) was responsible for an explosive encephalitis epidemic in the 1970s affecting about 1,000 residents of 20 coastland counties in São Paulo State, Brazil. ROCV was first isolated in 1975 from the cerebellum of a fatal human case of encephalitis. Clinical manifestations of the illness are similar to those described for St. Louis encephalitis. ROCV shows intense antigenic cross-reactivity with Japanese encephalitis complex (JEC) viruses, particularly with Ilheus (ILHV), St. Louis encephalitis, Murray Valley and West Nile viruses. In this study, we report a specific RT-PCR assay for ROCV diagnosis and the molecular characterization of the SPAn37630 and SPH37623 strains. Partial nucleotide sequences of NS5 and E genes determined from both strains were used in phylogenetic analysis. The results indicated that these strains are closely related to JEC viruses, but forming a distinct subclade together with ILHV, in accordance with results recently reported by Medeiros et al. (2007).

  14. Development of one-step SYBR Green real-time RT-PCR for quantifying bovine viral diarrhea virus type-1 and its comparison with conventional RT-PCR

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    Lou Sai

    2011-07-01

    Full Text Available Abstract Background Bovine viral diarrhea virus (BVDV is a worldwide pathogen in cattle and acts as a surrogate model for hepatitis C virus (HCV. One-step real-time fluorogenic quantitative reverse transcription polymerase chain reaction (RT-PCR assay based on SYBR Green I dye has not been established for BVDV detection. This study aims to develop a quantitative one-step RT-PCR assay to detect BVDV type-1 in cell culture. Results One-step quantitative SYBR Green I RT-PCR was developed by amplifying cDNA template from viral RNA and using in vitro transcribed BVDV RNA to establish a standard curve. The assay had a detection limit as low as 100 copies/ml of BVDV RNA, a reaction efficiency of 103.2%, a correlation coefficient (R2 of 0.995, and a maximum intra-assay CV of 2.63%. It was 10-fold more sensitive than conventional RT-PCR and can quantitatively detect BVDV RNA levels from 10-fold serial dilutions of titrated viruses containing a titer from 10-1 to 10-5 TCID50, without non-specific amplification. Melting curve analysis showed no primer-dimers and non-specific products. Conclusions The one-step SYBR Green I RT-PCR is specific, sensitive and reproducible for the quantification of BVDV in cell culture. This one-step SYBR Green I RT-PCR strategy may be further optimized as a reliable assay for diagnosing and monitoring BVDV infection in animals. It may also be applied to evaluate candidate agents against HCV using BVDV cell culture model.

  15. Simultaneous Detection of Three Arboviruses Using a Triplex RT-PCR Enzyme Hybridization Assay

    Institute of Scientific and Technical Information of China (English)

    Dan Dong; Shi-hong Fu; Li-hua Wang; Zhi Lv; Tai-yuan Li; Guo-dong Liang

    2012-01-01

    Arboviruses represent a serious problem to public health and agriculture worldwide.Fast,accurate identification of the viral agents of arbovirus-associated disease is essential for epidemiological surveillance and laboratory investigation.We developed a cost-effective,rapid,and highly sensitive one-step "triplex RT-PCR enzyme hybridization"assay for simultaneous detections of Japanese Encephallitis virus (JEV,Flaviviridae)Getah virus (GETV,Togaviridae),and Tahyna virus (TAHV,Bunyaviridae) using three pairs of primers to amplify three target sequences in one RT-PCR reaction.The analytical sensitivity of this assay was 1 PFU/mL for JEV,10PFU/mL for GETV,and 10 PFU/mL for TAHV.This assay is significantly more rapid and less expensive than the traditional serological detection and single RT-PCR reaction methods.When “triplex RT-PCR enzyme hybridization” was applied to 29 cerebrospinal fluid(CSF)samples that were JEV-positive by normal RT-PCR assay,all samples were strongly positive for JEV,but negative for GETV and TAHV,demonstrating a good sensitivity,specificity,and performance at CSF specimen detection.

  16. Development of a highly sensitive real-time one step RT-PCR combined complementary locked primer technology and conjugated minor groove binder probe

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    Lee Sunhwa

    2011-06-01

    Full Text Available Abstract Background Enterovirus (EV infections are commonly associated with encephalitis and meningitis. Detection of enteroviral RNA in clinical specimens has been demonstrated to improve the management of patients, by ruling out other causes of disease. Method To develop a sensitive and reliable assay for routine laboratory diagnosis, we developed a real-time one step reverse transcription polymerase chain reaction (RT-PCR assay with minor groove binder probes and primers modified with complementary locked primer technology (TMC-PCR. We checked the sensitivity of the developed assay by comparing it to a previously published TaqMan probe real-time one-step RT-PCR (TTN-PCR procedure using enteroviral isolates, Enterovirus Proficiency panels from Quality Control on Molecular Diagnostics (QCMD-2007, and clinical specimens from patients with suspected EV infections. Results One hundred clinical specimens from 158 suspected viral meningitis cases were determined to be positive by the TMC-PCR assay (63.29%, whereas only 60 were found to be positive by the TTN-PCR assay (37.97%. The positive and negative agreements between the TMC-PCR and TTN-PCR assays were 100% and 59.2%, respectively. Conclusion This data suggest that the TMC-PCR assay may be suitable for routine diagnostic screening from patient suspected EV infection.

  17. Rapid diagnosis of acute hemorrhagic conjunctivitis due to coxsackievirus A24 variant by real-time one-step RT-PCR.

    Science.gov (United States)

    Lévêque, Nicolas; Lahlou Amine, Idriss; Tcheng, Remy; Falcon, Delphine; Rivat, Nathalie; Dussart, Philippe; Muyembe, Jean-Jacques; Chomel, Jean-Jacques; Norder, Helene; Eugene, Maxime; Lina, Bruno

    2007-06-01

    Coxsackievirus A24 variant is, together with enterovirus 70 and adenoviruses, the major etiological agent involved in acute hemorrhagic conjunctivitis outbreaks worldwide. However, the standard virus isolation method followed by serotyping or VP1 region sequencing is time-consuming. A rapid method for the detection of coxsackievirus A24 variant from conjunctival swab specimens would be useful in the context of explosive and extensive outbreaks. A one-step real-time RT-PCR assay based on TaqMan technology was thus developed and assessed on 36 conjunctival swabs from outbreaks of conjunctivitis in Morocco in 2004 due to a coxsackievirus A24 variant and in Corsica in 2006 due to adenovirus type 3, and 83 virus strains including 41 coxsackievirus A24 variant collected in French Guiana and Guadeloupe in 2003, in the Democratic Republic of the Congo in 2003, in Morocco in 2004 and 42 other virus species genetically close or known to be responsible for conjunctivitis. All the conjunctival swabs from coxsackievirus A24 variant related outbreak and the 41 coxsackievirus A24 variant strains were tested positive by the RT-PCR assay within 4h. This novel single-tube real-time RT-PCR assay is sensitive and specific, and consists in a reliable and faster alternative to the viral culture for recent and future acute hemorrhagic conjunctivitis outbreaks caused by coxsackievirus A24 variant.

  18. Real time TaqMan RT-PCR assay for the detection of Cucumber green mottle mosaic virus.

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    Hongyun, Chen; Wenjun, Zhao; Qinsheng, Gu; Qing, Chen; Shiming, Lin; Shuifang, Zhu

    2008-05-01

    A real time reverse-transcription polymerase chain reaction (RT-PCR) was developed for efficient detection of Cucumber green mottle mosaic virus (CGMMV). The method was designed to use a duo-primer system with a TaqMan probe targeting the conserved sequence in 3' noncoding region (NCR) of CGMMV to detect isolates of this virus collected in China. The sensitivity of the real time RT-PCR assay was 0.13 pg of total RNA or 50 molecules of RNA transcripts. This level of sensitivity indicated that the one step real time RT-PCR developed in the present study could be used for routine testing assays. The real time RT-PCR method could assist in the implementation of quarantine measures for prevention and control of the disease caused by CGMMV.

  19. A Rapid and Sensitive One Step-SYBR Green Based Semi Quantitative Real Time RT-PCR for the Detection of peste des petits ruminants Virus in the Clinical Samples

    Institute of Scientific and Technical Information of China (English)

    Vinayagamurthy Balamurugan; Arnab Sen; Gnanavel Venkatesan; Vinita Yadav; Vandna Bhanot; Veerakyathappa Bhanuprakash; Raj Kumar Singh

    2012-01-01

    A sensitive and rapid single step real time (rt) RT-PCR was standardized using one-step Brilliant SYBR Green kit(R) for detection and semi-quantitation of peste des petitis ruminants virus (PPRV) using the virus RNA and matrix (M) protein gene-specific primers and compared with established conventional RT-PCR and TaqMan RT-PCR.The assay amplifies a 124 bp fragment of the PPRV M gene with Tm of 78.28 to 78.50.The assay was linear within a range of 50 ng to 0.5 fg total virus RNA with a detection limit (sensitivity) of 0.5 fg.Based on the serial dilution of the live-attenuated PPR vaccine virus,the detection limit was ~0.0001 cell culture infectious dose 50% units (TCID50).Additionally,swab materials spiked with known titre of vaccine virus were equally well detected in the assay.The standardized rt RT-PCR was easily employed for the detection of PPRV nucleic acid directly in the field and experimental clinical samples.The assay detected the PPRV nucleic acid as early as 3 day post infection (dpi) and up to 20 dpi in swab materials from the experimental samples.The assay was rapid and more sensitive than TaqMan and conventional RT-PCR in the detection of PPRV nucleic acid from the PPR suspected clinical samples of sheep and goats.Therefore,the established,simplified SYBR green rt RT-PCR is an alternative test to the already existing various diagnostic assays and could be useful for rapid clinical diagnosis with advantage in reducing risk of contamination.

  20. Comparison of the conventional multiplex RT-PCR, real time RT-PCR and Luminex xTAG® RVP fast assay for the detection of respiratory viruses.

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    Choudhary, Manohar L; Anand, Siddharth P; Tikhe, Shamal A; Walimbe, Atul M; Potdar, Varsha A; Chadha, Mandeep S; Mishra, Akhilesh C

    2016-01-01

    Detection of respiratory viruses using polymerase chain reaction (PCR) is sensitive, specific and cost effective, having huge potential for patient management. In this study, the performance of an in-house developed conventional multiplex RT-PCR (mRT-PCR), real time RT-PCR (rtRT-PCR) and Luminex xTAG(®) RVP fast assay (Luminex Diagnostics, Toronto, Canada) for the detection of respiratory viruses was compared. A total 310 respiratory clinical specimens predominantly from pediatric patients, referred for diagnosis of influenza A/H1N1pdm09 from August 2009 to March 2011 were tested to determine performance characteristic of the three methods. A total 193 (62.2%) samples were detected positive for one or more viruses by mRT-PCR, 175 (56.4%) samples by real time monoplex RT-PCR, and 138 (44.5%) samples by xTAG(®) RVP fast assay. The overall sensitivity of mRT-PCR was 96.9% (95% CI: 93.5, 98.8), rtRT-PCR 87.9% (95% CI: 82.5, 92.1) and xTAG(®) RVP fast was 68.3% (95% CI: 61.4, 74.6). Rhinovirus was detected most commonly followed by respiratory syncytial virus group B and influenza A/H1N1pdm09. The monoplex real time RT-PCR and in-house developed mRT-PCR are more sensitive, specific and cost effective than the xTAG(®) RVP fast assay.

  1. Gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of Japanese encephalitis virus

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    Huang, Su-Hua; Yang, Tsuey-Ching; Tsai, Ming-Hong; Tsai, I.-Shou; Lu, Huang-Chih; Chuang, Pei-Hsin; Wan, Lei; Lin, Ying-Ju; Lai, Chih-Ho; Lin, Cheng-Wen

    2008-10-01

    Virus isolation and antibody detection are routinely used for diagnosis of Japanese encephalitis virus (JEV) infection, but the low level of transient viremia in some JE patients makes JEV isolation from clinical and surveillance samples very difficult. We describe the use of gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of JEV from its RNA genome. We tested the effect of gold nanoparticles on four different PCR systems, including conventional PCR, reverse-transcription PCR (RT-PCR), and SYBR green real-time PCR and RT-PCR assays for diagnosis in the acute phase of JEV infection. Gold nanoparticles increased the amplification yield of the PCR product and shortened the PCR time compared to the conventional reaction. In addition, nanogold-based real-time RT-PCR showed a linear relationship between Ct and template amount using ten-fold dilutions of JEV. The nanogold-based RT-PCR and real-time quantitative RT-PCR assays were able to detect low levels (1-10 000 copies) of the JEV RNA genomes extracted from culture medium or whole blood, providing early diagnostic tools for the detection of low-level viremia in the acute-phase infection. The assays described here were simple, sensitive, and rapid approaches for detection and quantitation of JEV in tissue cultured samples as well as clinical samples.

  2. Gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of Japanese encephalitis virus

    Energy Technology Data Exchange (ETDEWEB)

    Huang, S-H; Tsai, M-H; Lin, C-W [Department of Biotechnology, College of Health Science, Asia University, Wufeng, Taichung, Taiwan (China); Yang, T-C; Chuang, P-H [Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan (China); Tsai, I-S; Lu, H-C [Nanotechnology Research Center, Feng Chia University, Taichung, Taiwan (China); Wan Lei; Lin, Y-J [Department of Medical Genetics and Medical Research, China Medical University Hospital, Taichung, Taiwan (China); Lai, C-H [Department of Microbiology and Immunology, China Medical University, Taichung, Taiwan (China)], E-mail: cwlin@mail.cmu.edu.tw

    2008-10-08

    Virus isolation and antibody detection are routinely used for diagnosis of Japanese encephalitis virus (JEV) infection, but the low level of transient viremia in some JE patients makes JEV isolation from clinical and surveillance samples very difficult. We describe the use of gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of JEV from its RNA genome. We tested the effect of gold nanoparticles on four different PCR systems, including conventional PCR, reverse-transcription PCR (RT-PCR), and SYBR green real-time PCR and RT-PCR assays for diagnosis in the acute phase of JEV infection. Gold nanoparticles increased the amplification yield of the PCR product and shortened the PCR time compared to the conventional reaction. In addition, nanogold-based real-time RT-PCR showed a linear relationship between Ct and template amount using ten-fold dilutions of JEV. The nanogold-based RT-PCR and real-time quantitative RT-PCR assays were able to detect low levels (1-10 000 copies) of the JEV RNA genomes extracted from culture medium or whole blood, providing early diagnostic tools for the detection of low-level viremia in the acute-phase infection. The assays described here were simple, sensitive, and rapid approaches for detection and quantitation of JEV in tissue cultured samples as well as clinical samples.

  3. One-step multiplex quantitative RT-PCR for the simultaneous detection of viroids and phytoplasmas of pome fruit trees.

    Science.gov (United States)

    Malandraki, Ioanna; Varveri, Christina; Olmos, Antonio; Vassilakos, Nikon

    2015-03-01

    A one-step multiplex real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan chemistry was developed for the simultaneous detection of Pear blister canker viroid and Apple scar skin viroid along with universal detection of phytoplasmas, in pome trees. Total nucleic acids (TNAs) extraction was performed according to a modified CTAB protocol. Primers and TaqMan MGB probes for specific detection of the two viroids were designed in this study, whereas for phytoplasma detection published universal primers and probe were used, with the difference that the later was modified to carry a MGB quencher. The pathogens were detected simultaneously in 10-fold serial dilutions of TNAs from infected plant material into TNAs of healthy plant up to dilutions 10(-5) for viroids and 10(-4) for phytoplasmas. The multiplex real-time assay was at least 10 times more sensitive than conventional protocols for viroid and phytoplasma detection. Simultaneous detection of the three targets was achieved in composite samples at least up to a ratio of 1:100 triple-infected to healthy tissue, demonstrating that the developed assay has the potential to be used for rapid and massive screening of viroids and phytoplasmas of pome fruit trees in the frame of certification schemes and surveys.

  4. Rapid detection of equine influenza virus H3N8 subtype by insulated isothermal RT-PCR (iiRT-PCR) assay using the POCKIT™ Nucleic Acid Analyzer.

    Science.gov (United States)

    Balasuriya, Udeni B R; Lee, Pei-Yu Alison; Tiwari, Ashish; Skillman, Ashley; Nam, Bora; Chambers, Thomas M; Tsai, Yun-Long; Ma, Li-Juan; Yang, Pai-Chun; Chang, Hsiao-Fen Grace; Wang, Hwa-Tang Thomas

    2014-10-01

    Equine influenza (EI) is an acute, highly contagious viral respiratory disease of equids. Currently, equine influenza virus (EIV) subtype H3N8 continues to be the most important respiratory pathogen of horses in many countries around the world. The need to achieve a rapid diagnosis and to implement effective quarantine and movement restrictions is critical in controlling the spread of EIV. In this study, a novel, inexpensive and user-friendly assay based on an insulated isothermal RT-PCR (iiRT-PCR) method on the POCKIT™, a field-deployable device, was described and validated for point-of-need detection of EIV-H3N8 in clinical samples. The newly established iiRT-PCR assay targeting the EIV HA3 gene was evaluated for its sensitivity using in vitro transcribed (IVT) RNA, as well as ten-fold serial dilutions of RNA extracted from the prototype H3N8 strain A/equine/Miami/1/63. Inclusivity and exclusivity panels were tested for specificity evaluation. Published real-time RT-PCR (rRT-PCR) assays targeting the NP and HA3 genes were used as the reference standards for comparison of RNA extracted from field strains and from nasal swab samples collected from experimentally infected horses, respectively. Limit of detection with a 95% probability (LoD95%) was estimated to be 11copies of IVT RNA. Clinical sensitivity analysis using RNA prepared from serial dilutions of a prototype EIV (Miami 1/63/H3N8) showed that the iiRT-PCR assay was about 100-fold more sensitive than the rRT-PCR assay targeting the NP gene of EIV subtype H3N8. The iiRT-PCR assay identified accurately fifteen EIV H3N8 strains and two canine influenza virus (CIV) H3N8 strains, and did not cross-react with H6N2, H7N7, H1N1 subtypes or any other equine respiratory viral pathogens. Finally, 100% agreement was found between the iiRT-PCR assay and the universal influenza virus type A rRT-PCR assay in detecting the EIV A/equine/Kentucky/7/07 strain in 56 nasal swab samples collected from experimentally inoculated

  5. Detection of 22 common leukemic fusion genes using a single-step multiplex qRT-PCR-based assay.

    Science.gov (United States)

    Lyu, Xiaodong; Wang, Xianwei; Zhang, Lina; Chen, Zhenzhu; Zhao, Yu; Hu, Jieying; Fan, Ruihua; Song, Yongping

    2017-07-25

    Fusion genes generated from chromosomal translocation play an important role in hematological malignancies. Detection of fusion genes currently employ use of either conventional RT-PCR methods or fluorescent in situ hybridization (FISH), where both methods involve tedious methodologies and require prior characterization of chromosomal translocation events as determined by cytogenetic analysis. In this study, we describe a real-time quantitative reverse transcription PCR (qRT-PCR)-based multi-fusion gene screening method with the capacity to detect 22 fusion genes commonly found in leukemia. This method does not require pre-characterization of gene translocation events, thereby facilitating immediate diagnosis and therapeutic management. We performed fluorescent qRT-PCR (F-qRT-PCR) using a commercially-available multi-fusion gene detection kit on a patient cohort of 345 individuals comprising 108 cases diagnosed with acute myeloid leukemia (AML) for initial evaluation; remaining patients within the cohort were assayed for confirmatory diagnosis. Results obtained by F-qRT-PCR were compared alongside patient analysis by cytogenetic characterization. Gene translocations detected by F-qRT-PCR in AML cases were diagnosed in 69.4% of the patient cohort, which was comparatively similar to 68.5% as diagnosed by cytogenetic analysis, thereby demonstrating 99.1% concordance. Overall gene fusion was detected in 53.7% of the overall patient population by F-qRT-PCR, 52.9% by cytogenetic prediction in leukemia, and 9.1% in non-leukemia patients by both methods. The overall concordance rate was calculated to be 99.0%. Fusion genes were detected by F-qRT-PCR in 97.3% of patients with CML, followed by 69.4% with AML, 33.3% with acute lymphoblastic leukemia (ALL), 9.1% with myelodysplastic syndromes (MDS), and 0% with chronic lymphocytic leukemia (CLL). We describe the use of a F-qRT-PCR-based multi-fusion gene screening method as an efficient one-step diagnostic procedure as an

  6. Rapid Detection Co-infections of Classical Swine Fever Virus and Porcine Reproductive and Respiratory Syndrome Virus by One-step Multiplex RT-PCR

    Institute of Scientific and Technical Information of China (English)

    TIAN Hong; WU Jinyan; YAN Chen; SHANG Youjun; YIN Shuanghui; LIU Xiangtao

    2011-01-01

    Classical swine fever virus (CSFV) and porcine reproductive and respiratory syndrome virus (PRRSV) have caused immense economic loss in the pig industry and are considered to be the two most important infectious diseases of pigs in the world A multiplex reverse transcription polymerase chain reaction (multiplex RT-PCR) was developed for CSFV and PRRSV co-infections or infections, respectively. A set of two pairs of primer was designed based on the sequence of nonstructural protein NS54B of CSFV and ORF7 gene of PRRSV. The diagnostic accuracy of multiplex RT-PCR assay was evaluated by using 56 field clinical samples by multiplex RT-PCR, single RT-PCR and sequence analysis; and the specificity of multiplex PCR was verified by using constructed plasmids containing the specific viral target fragments of PRRSV and CSFV, respectively. The results indicated that this assay could reliably differentiate PRRSV and CSFV in co-infection samples. The multiplex RT-PCR developed in this study might provide a new avenue to the rapid the detection of CSFV and PRRSV in one reaction.

  7. Development and evaluation of one-step rRT-PCR and immunohistochemical methods for detection of Rift Valley fever virus in biosafety level 2 diagnostic laboratories.

    Science.gov (United States)

    Drolet, Barbara S; Weingartl, Hana M; Jiang, Jieyuan; Neufeld, James; Marszal, Peter; Lindsay, Robbin; Miller, Myrna M; Czub, Markus; Wilson, William C

    2012-02-01

    Rift Valley fever virus (RVFV) is a zoonotic insect transmitted virus endemic to Africa and the Arabian Peninsula. Infection causes abortions and high mortality in newborn ruminants. The overall human infection rate is BSL-4 conditions and require inactivation and safety testing for use outside of containment. In this study, antiserum against recombinant RVFV-nucleocapsid (N) was produced to develop an immunohistochemical (IHC) assay which was subsequently evaluated on formalin fixed lamb and calf tissues at BSL-2 laboratory conditions. Antigen was detected by IHC in 79% of rRT-PCR-positive sheep and 70% of rRT-PCR-positive calf tissues tested. Once validated and approved by national regulatory agencies, these assays can be safely produced and distributed to regional diagnostic laboratories, providing capacity for early detection of RVFV in suspected ruminant samples.

  8. DEVELOPMENT OF HOMOLOGOUS VIRAL INTERNAL CONTROLS FOR USE IN RT-PCR ASSAYS OF WATERBORNE ENTERIC VIRUSES

    Science.gov (United States)

    Enteric viruses often contaminate water sources causing frequent outbreaks of gastroenteritis. Reverse transcription-polymerase chain reaction (RT-PCR) assays are commonly used for detection of human enteric viruses in environmental and drinking water samples. RT-PCR provides ...

  9. DEVELOPMENT OF HOMOLOGOUS VIRAL INTERNAL CONTROLS FOR USE IN RT-PCR ASSAYS OF WATERBORNE ENTERIC VIRUSES

    Science.gov (United States)

    Enteric viruses often contaminate water sources causing frequent outbreaks of gastroenteritis. Reverse transcription-polymerase chain reaction (RT-PCR) assays are commonly used for detection of human enteric viruses in environmental and drinking water samples. RT-PCR provides ...

  10. Real-Time PCR (RT-PCR) Assays for Burkholderia mallei and B. pseudomallei

    Science.gov (United States)

    2005-10-01

    1 Real - time PCR (RT-PCR) Assays for Burkholderia mallei and B. pseudomallei Vipin K. Rastogi1, Tu-chen Cheng1, Lisa Collins1 and Jennifer Bagley2 1...A 3. DATES COVERED - 4. TITLE AND SUBTITLE Real - time PCR (RT-PCR) Assays for Burkholderia mallei and B.pseudomallei 5a. CONTRACT NUMBER 5b...risk. There is currently no real - time PCR assay for detection of both of these pathogens. Primers and probes corresponding to specific genomic regions

  11. Sensitive Detection of Measles Virus Infection in the Blood and Tissues of Humanized Mouse by One-step Quantitative RT-PCR

    Directory of Open Access Journals (Sweden)

    Shota eIkeno

    2013-10-01

    Full Text Available Live attenuated measles virus (MV has long been recognized as a safe and effective vaccine, and it has served as the basis for development of various MV-based vaccines. However, because MV is a human-tropic virus, the evaluation of MV-based vaccines has been hampered by the lack of a small-animal model. The humanized mouse, a recently developed system in which an immunodeficient mouse is transplanted with human fetal tissues or hematopoietic stem cells, may represent a suitable model. Here, we developed a sensitive one-step quantitative reverse transcription (qRT PCR that simultaneously measures nucleocapsid (N and human RNase P mRNA levels. The results can be used to monitor MV infection in a humanized mouse model. Using this method, we elucidated the replication kinetics of MV expressing EGFP both in vitro and in humanized mice in parallel with flow-cytometric analysis. Because our qRT-PCR system was sensitive enough to detect MV expression using RNA extracted from a small number of cells, it can be used to monitor MV infection in humanized mice by sequential blood sampling.

  12. A quantitative real-time RT-PCR assay for mature C. albicans biofilms

    Directory of Open Access Journals (Sweden)

    Dongari-Bagtzoglou Anna

    2011-05-01

    Full Text Available Abstract Background Fungal biofilms are more resistant to anti-fungal drugs than organisms in planktonic form. Traditionally, susceptibility of biofilms to anti-fungal agents has been measured using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl-2H-tetrazolium-5-carboxyanilide (XTT assay, which measures the ability of metabolically active cells to convert tetrazolium dyes into colored formazan derivatives. However, this assay has limitations when applied to high C. albicans cell densities because substrate concentration and solubility are limiting factors in the reaction. Because mature biofilms are composed of high cell density populations we sought to develop a quantitative real-time RT-PCR assay (qRT-PCR that could accurately assess mature biofilm changes in response to a wide variety of anti-fungal agents, including host immune cells. Results The XTT and qRT-PCR assays were in good agreement when biofilm changes were measured in planktonic cultures or in early biofilms which contain lower cell densities. However, the real-time qRT-PCR assay could also accurately quantify small-medium size changes in mature biofilms caused by mechanical biomass reduction, antifungal drugs or immune effector cells, that were not accurately quantifiable with the XTT assay. Conclusions We conclude that the qRT-PCR assay is more accurate than the XTT assay when measuring small-medium size effects of anti-fungal agents against mature biofilms. This assay is also more appropriate when mature biofilm susceptibility to anti-fungal agents is tested on complex biological surfaces, such as organotypic cultures.

  13. Development of a One-Step Duplex RT-PCR Method for the Simultaneous Detection of VP3/VP1 and VP1/P2B Regions of the Hepatitis A Virus.

    Science.gov (United States)

    Kim, Mi-Ju; Lee, Shin-Young; Kim, Hyun-Joong; Lee, Jeong Su; Joo, In Sun; Kwak, Hyo Sun; Kim, Hae-Yeong

    2016-08-28

    The simultaneous detection and accurate identification of hepatitis A virus (HAV) is critical in food safety and epidemiological studies to prevent the spread of HAV outbreaks. Towards this goal, a one-step duplex reverse-transcription (RT)-PCR method was developed targeting the VP1/P2B and VP3/VP1 regions of the HAV genome for the qualitative detection of HAV. An HAV RT-qPCR standard curve was produced for the quantification of HAV RNA. The detection limit of the duplex RT-PCR method was 2.8 × 10(1) copies of HAV. The PCR products enabled HAV genotyping analysis through DNA sequencing, which can be applied for epidemiological investigations. The ability of this duplex RT-PCR method to detect HAV was evaluated with HAV-spiked samples of fresh lettuce, frozen strawberries, and oysters. The limit of detection of the one-step duplex RT-PCR for each food model was 9.4 × 10(2) copies/20 g fresh lettuce, 9.7 × 10(3) copies/20 g frozen strawberries, and 4.1 × 10(3) copies/1.5 g oysters. Use of a one-step duplex RT-PCR method has advantages such as shorter time, decreased cost, and decreased labor owing to the single amplification reaction instead of four amplifications necessary for nested RT-PCR.

  14. Sequence Optimized Real-Time RT-PCR Assay for Detection of Crimean-Congo Hemorrhagic Fever Virus

    Science.gov (United States)

    2017-03-21

    density macroarrays [15], high density resequencing arrays [16], 55 padlock probes with colorimetric readout [17], LAMP [18], and polymerase chain reaction ...Announcement). Briefly, the S segment of each virus was amplified using the 83 SuperScript III One-Step RT- PCR system with Platinum Taq DNA Polymerase High...Platinum One-Step Quantitative RT- PCR System) # Rxns = Reagents Stock [Final] 1rxn 28 2X Reaction Mix 2 1 10 280 Nuclease-free water 1.7 47.6 100 µM F

  15. A duplex real-time RT-PCR assay for profiling inhibitors of four dengue serotypes.

    Science.gov (United States)

    Gong, Edwin Yunhao; Smets, Alexandra; Verheyen, Nick; Clynhens, Marleen; Gustin, Emmanuel; Lory, Pedro; Kraus, Guenter

    2013-01-01

    We have developed a duplex real-time RT-PCR assay for profiling antiviral inhibitors of four dengue virus (DENV) serotypes. In this assay, the primers and the probe for amplifying DENV were designed in the conserved regions of the genome after aligned more than 300 nucleotide sequences of four dengue serotypes deposited in the GeneBank. To discriminate the antiviral activity from the cytotoxicity of compounds, a housekeeping gene of the Vero cells, β-actin, was used to design the primers and the probe for the second set of PCR as an internal control, which is used to normalize the RNA levels of dengue-specific PCR due to the cellular toxicity of test compounds. For compound profiling, the duplex PCR is performed using LightCycler(®) in a single tube to simultaneously amplify both the dengue target gene and the Vero cell housekeeping gene from the compound-treated Vero cell lysates. This assay was validated against a panel of reference compounds. The results show that the universal primers and probe in this duplex RT-PCR assay can efficiently amplify all four dengue serotypes and that the PCR efficiency for both the dengue target gene and the Vero cells β-actin gene is 100%.

  16. Design and analysis of Q-RT-PCR assays for haematological malignancies using mixed effects models

    DEFF Research Database (Denmark)

    Bøgsted, Martin; Mandrup, Charlotte; Petersen, Anders;

    research use and needs qualit control for accuracy and precision. Especially the identification of experimental variations and statistical analysis has recently created discussions. The standard analytical technique is to use the Delta-Delta-Ct method. Although this method accounts for sample specific...... variations such as RNA purification, it does not account for other experimental effects as variations in cDNA synthesis, amplification efficiency and assay variations. To obtain an assessment of the accuracy and precision of the assays a novel approach for the statistical analysis of Q-RT-PCR has been...... developed based on a linear mixed effects model for factorial designs. The model consists of an analysis of variance where the variation of each fixed effect of interest and identified experimental and biological nuisance variations are split. Hereby it accounts for varying efficiency, inhomogeneous...

  17. Comparison and evaluation of conventional RT-PCR, SYBR green I and TaqMan real-time RT-PCR assays for the detection of porcine epidemic diarrhea virus.

    Science.gov (United States)

    Zhou, Xinrong; Zhang, Tiansheng; Song, Deping; Huang, Tao; Peng, Qi; Chen, Yanjun; Li, Anqi; Zhang, Fanfan; Wu, Qiong; Ye, Yu; Tang, Yuxin

    2017-06-01

    Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV) is a highly contagious intestinal disease, resulting in substantial economic losses to the swine industry worldwide. In this study, three assays, namely a conventional reverse transcription-polymerase chain reaction (RT-PCR), a SYBR Green I real-time RT-PCR and a TaqMan real-time RT-PCR targeting the highly conserved M gene of PEDV, were developed and evaluated. Then, the analytical specificity, sensitivity and reproducibility of these assays were determined and compared. The TaqMan real-time RT-PCR was 100-fold and 10,000-fold more sensitive than that of the SYBR Green I real-time RT-PCR and the conventional RT-PCR, respectively. The analytical sensitivity of TaqMan real-time RT-PCR was 10 copies/μl of target gene and no cross amplification with other viruses tested was observed. With the features of high specificity, sensitivity, and reproducibility, the TaqMan real-time RT-PCR established in this study could be a useful tool for clinical diagnosis, epidemiological surveys and outbreak investigations of PED. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Evaluation of real-time RT-PCR assays for detection and quantification of norovirus genogroups I and II.

    Science.gov (United States)

    Rupprom, Kitwadee; Chavalitshewinkoon-Petmitr, Porntip; Diraphat, Pornphan; Kittigul, Leera

    2017-02-20

    Noroviruses are the leading cause of acute gastroenteritis in humans. Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) is a promising molecular method for the detection of noroviruses. In this study, the performance of three TaqMan real-time RT-PCR assays was assessed, which were one commercially available real-time RT-PCR kit (assay A: Norovirus Real Time RT-PCR kit) and two in-house real-time RT-PCR assays (assay B: LightCycler RNA Master Hybprobe and assay C: RealTime ready RNA Virus Master). Assays A and B showed higher sensitivity than assay C for norovirus GI, while they all had the same sensitivity (10(3) DNA copies/mL) for GII DNA standard controls. Assay B had the highest efficiency for both genogroups. No cross-reactivity was observed among GI and GII noroviruses, rotavirus, hepatitis A virus, and poliovirus. The detection rates of these assays in GI and GII norovirus-positive fecal samples were not significantly different. However, the mean quantification cycle (Cq) value of assay B for GII was lower than assays A and C with statistical significance (P-value, 0.000). All three real-time RT-PCR assays could detect a variety of noroviruses including GI.2, GII.2, GII.3, GII.4, GII.6, GII.12, GII.17, and GII.21. This study suggests assay B as a suitable assay for the detection and quantification of noroviruses GI and GII due to good analytical sensitivity and higher performance to amplify norovirus on DNA standard controls and clinical samples.

  19. Utility of Pooled HIV RNA RT-PCR Assay in Diagnosing Acute HIV Infections

    Institute of Scientific and Technical Information of China (English)

    张麒; 蒋岩; 刘全忠

    2004-01-01

    Abstract: The P24 antigen test, HIV RNA PCR test,HIV isolation/culture and fourth-generation HIV uniform Ag/Ab assay are being utilized in diagnosing acute HIV infection in different labs. Many factors limit the use of screening for acute HIV in high-risk populations, in blood donors and during voluntary HIV testing, including, cost, technique, sensitivity and specificity. In this review we explore a new NAAT method which involves HIV RNA RT-PCR on pooled samples. This technique is able to screen for acute infections in a large testing volume and may he used as a screening method in high-risk populations and blood donors.

  20. The development and application of the two real-time RT-PCR assays to detect the pathogen of HFMD.

    Directory of Open Access Journals (Sweden)

    Aili Cui

    Full Text Available Large-scale Hand, Foot, and Mouth Disease (HFMD outbreaks have frequently occurred in China since 2008, affecting more than one million children and causing several hundred children deaths every year. The pathogens of HFMD are mainly human enteroviruses (HEVs. Among them, human enterovirus 71 (HEV71 and coxsackievirus A16 (CVA16 are the most common pathogens of HFMD. However, other HEVs could also cause HFMD. To rapidly detect HEV71 and CVA16, and ensure detection of all HEVs causing HFMD, two real-time hybridization probe-based RT-PCR assays were developed in this study. One is a multiplex real-time RT-PCR assay, which was developed to detect and differentiate HEV71 specifically from CVA16 directly from clinical specimens within 1-2 h, and the other is a broad-spectrum real-time RT-PCR assay, which targeted almost all HEVs. The experiments confirmed that the two assays have high sensitivity and specificity, and the sensitivity was up to 0.1 TCID50/ml for detection of HEVs, HEV71, and CVA16, respectively. A total of 213 clinical specimens were simultaneously detected by three kinds of assays, including the two real-time RT-PCR assays, direct conventional RT-PCR assay, and virus isolation assay on human rhabdomyosarcoma cells (RD cells. The total positive rate of both HEV71 and CVA16 was 69.48% with real-time RT-PCR assay, 47.42% with RT-PCR assay, and 34.58% with virus isolation assay. One HFMD clinical specimen was positive for HEV, but negative for HEV71 or CVA16, which was identified as Echovirus 11 (Echo11 by virus isolation, RT-PCR, and sequencing for the VP1 gene. The two real-time RT-PCR assays had been applied in 31 provincial HFMD labs to detect the pathogens of HFMD, which has contributed to the rapid identification of the pathogens in the early stages of HFMD outbreaks, and helped to clarify the etiologic agents of HFMD in China.

  1. A SYBR Green RT-PCR assay in single tube to detect human and bovine noroviruses and control for inhibition

    Directory of Open Access Journals (Sweden)

    Saegerman Claude

    2008-08-01

    Full Text Available Abstract Background Noroviruses are single-stranded RNA viruses belonging to the family Caliciviridae. They are a major cause of epidemic and sporadic gastroenteritis in humans and clinical signs and lesions of gastroenteritis were reported in bovines. Due to their genetic proximity, potential zoonotic transmission or animal reservoir can be hypothesized for noroviruses. RT-PCR has become the "gold standard" for the detection of noroviruses in faecal and environmental samples. With such samples, the control for inhibition of the reaction during amplification and detection is crucial to avoid false negative results, which might otherwise not be detected. The aim of the reported method is to detect, with a SYBR Green technology, a broad range of noroviruses with a control for inhibition. Results A SYBR Green real-time RT-PCR assay was developed making use of a foreign internal RNA control added in the same tube. This assay is able to detect human and bovine noroviruses belonging to genogroups I, II and III and to distinguish between norovirus and internal control amplicons using melting curve analysis. A 10-fold dilution of samples appears to be the method of choice to remove inhibition. This assay was validated with human and bovine stool samples previously tested for norovirus by conventional RT-PCR. Conclusion This SYBR Green real-time RT-PCR assay allows the detection of the most important human and bovine noroviruses in the same assay, and avoids false negative results making use of an internal control. Melting curves allow the discrimination between the internal control and norovirus amplicons. It gives preliminary information about the species of origin. The sensitivity of the developed assay is higher than conventional RT-PCR and a 10-fold dilution of samples showed a better efficiency and reproducibility to remove RT-PCR inhibition than addition of bovine serum albumin.

  2. One-step multiplex real time RT-PCR for the detection of bovine respiratory syncytial virus, bovine herpesvirus 1 and bovine parainfluenza virus 3

    Directory of Open Access Journals (Sweden)

    Thonur Leenadevi

    2012-03-01

    Full Text Available Abstract Background Detection of respiratory viruses in veterinary species has traditionally relied on virus detection by isolation or immunofluorescence and/or detection of circulating antibody using ELISA or serum neutralising antibody tests. Multiplex real time PCR is increasingly used to diagnose respiratory viruses in humans and has proved to be superior to traditional methods. Bovine respiratory disease (BRD is one of the most common causes of morbidity and mortality in housed cattle and virus infections can play a major role. We describe here a one step multiplex reverse transcriptase quantitative polymerase chain reaction (mRT-qPCR to detect the viruses commonly implicated in BRD. Results A mRT-qPCR assay was developed and optimised for the simultaneous detection of bovine respiratory syncytial virus (BRSV, bovine herpes virus type 1 (BoHV-1 and bovine parainfluenza virus type 3 (BPI3 i & ii nucleic acids in clinical samples from cattle. The assay targets the highly conserved glycoprotein B gene of BoHV-1, nucleocapsid gene of BRSV and nucleoprotein gene of BPI3. This mRT-qPCR assay was assessed for sensitivity, specificity and repeatability using in vitro transcribed RNA and recent field isolates. For clinical validation, 541 samples from clinically affected animals were tested and mRT-qPCR result compared to those obtained by conventional testing using virus isolation (VI and/or indirect fluorescent antibody test (IFAT. Conclusions The mRT-qPCR assay was rapid, highly repeatable, specific and had a sensitivity of 97% in detecting 102 copies of BRSV, BoHV-1 and BPI3 i & ii. This is the first mRT-qPCR developed to detect the three primary viral agents of BRD and the first multiplex designed using locked nucleic acid (LNA, minor groove binding (MGB and TaqMan probes in one reaction mix. This test was more sensitive than both VI and IFAT and can replace the aforesaid methods for virus detection during outbreaks of BRD.

  3. Analytical and clinical performance of the CDC real time RT-PCR assay for detection and typing of dengue virus.

    Directory of Open Access Journals (Sweden)

    Gilberto A Santiago

    Full Text Available Dengue is an acute illness caused by the positive-strand RNA dengue virus (DENV. There are four genetically distinct DENVs (DENV-1-4 that cause disease in tropical and subtropical countries. Most patients are viremic when they present with symptoms; therefore, RT-PCR has been increasingly used in dengue diagnosis. The CDC DENV-1-4 RT-PCR Assay has been developed as an in-vitro diagnostic platform and was recently approved by the US Food and Drug Administration (FDA for detection of dengue in patients with signs or symptoms of mild or severe dengue. The primers and probes of this test have been designed to detect currently circulating strains of DENV-1-4 from around the world at comparable sensitivity. In a retrospective study with 102 dengue cases confirmed by IgM anti-DENV seroconversion in the convalescent sample, the RT-PCR Assay detected DENV RNA in 98.04% of the paired acute samples. Using sequencing as a positive indicator, the RT-PCR Assay had a 97.92% positive agreement in 86 suspected dengue patients with a single acute serum sample. After extensive validations, the RT-PCR Assay performance was highly reproducible when evaluated across three independent testing sites, did not produce false positive results for etiologic agents of other febrile illnesses, and was not affected by pathological levels of potentially interfering biomolecules. These results indicate that the CDC DENV-1-4 RT-PCR Assay provides a reliable diagnostic platform capable for confirming dengue in suspected cases.

  4. The use of a one-step real-time reverse transcription polymerase chain reaction (rRT-PCR) for the surveillance of viral hemorrhagic septicemia virus (VHSV) in Minnesota.

    Science.gov (United States)

    Phelps, Nicholas B D; Patnayak, Devi P; Jiang, Yin; Goyal, Sagar M

    2012-12-01

    Viral hemorrhagic septicemia virus (VHSV) is a highly contagious and pathogenic virus of fish. The virus infects more than 70 fish species worldwide, in both fresh and salt water. A new viral strain (VHSV-IVb) has proven both virulent and persistent, spreading throughout the Great Lakes of North America and to inland water bodies in the region. To better understand the geographic distribution of the virus, we used a modified real-time reverse transcription polymerase chain reaction (rRT-PCR) assay for high-throughput testing of fish for VHSV. The assay was shown to be twice as sensitive as the gold standard, virus isolation, and did not cross react with other viruses found in fish. In addition, the diagnostic turnaround time was reduced from 28 to 30 d for virus isolation to 2-4 d for rRT-PCR. To demonstrate the usefulness of the rRT-PCR assay, 115 high-priority water bodies in Minnesota were tested by both methods from April 2010 to June 2011. All survey sites tested negative for VHSV by both methods. The survey results have informed fisheries managers on the absence of VHSV in Minnesota and have better prepared them for the eventual arrival of the disease. In addition, the results demonstrate the value of this rRT-PCR as a surveillance tool to rapidly identify an outbreak so that it can be controlled in a timely manner.

  5. The development of a qualitative real-time RT-PCR assay for the detection of hepatitis C virus

    NARCIS (Netherlands)

    Clancy, A.; Crowley, B.; Niesters, H.; Herra, C.

    2008-01-01

    Real-time polymerase chain reaction (PCR) represents a favourable option for the detection of hepatitis C virus (HCV). A real-time reverse transcriptase PCR (RT-PCR) assay was developed as a qualitative diagnostic screening method for the detection of HCV using the ABI PRISM 7500 Sequence Detection

  6. The development of a qualitative real-time RT-PCR assay for the detection of hepatitis C virus

    NARCIS (Netherlands)

    Clancy, A.; Crowley, B.; Niesters, H.; Herra, C.

    2008-01-01

    Real-time polymerase chain reaction (PCR) represents a favourable option for the detection of hepatitis C virus (HCV). A real-time reverse transcriptase PCR (RT-PCR) assay was developed as a qualitative diagnostic screening method for the detection of HCV using the ABI PRISM 7500 Sequence Detection

  7. Duplex Real-Time RT-PCR Assays for the Detection and Typing of Epizootic Haemorrhagic Disease Virus

    Science.gov (United States)

    Viarouge, Cyril; Breard, Emmanuel; Zientara, Stephan; Vitour, Damien; Sailleau, Corinne

    2015-01-01

    Epizootic haemorrhagic disease virus (EHDV) may cause severe clinical episodes in some species of deer and sometimes in cattle. Laboratory diagnosis provides a basis for the design and timely implementation of disease control measures. There are seven distinct EHDV serotypes, VP2 coding segment 2 being the target for serotype specificity. This paper reports the development and validation of eight duplex real-time RT-PCR assays to simultaneously amplify the EHDV target (S9 for the pan-EHDV real-time RT-PCR assay and S2 for the serotyping assays) and endogenous control gene Beta-actin. Analytical and diagnostic sensitivity and specificity, inter- and intra-assay variation and efficiency were evaluated for each assay. All were shown to be highly specific and sensitive. PMID:26161784

  8. Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth disease virus and look-alike disease viruses

    Energy Technology Data Exchange (ETDEWEB)

    Hindson, B J; Reid, S M; Baker, B R; Ebert, K; Ferris, N P; Bentley Tammero, L F; Lenhoff, R J; Naraghi-Arani, P; Vitalis, E A; Slezak, T R; Hullinger, P J; King, D P

    2007-07-26

    A high-throughput multiplexed assay was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspect cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRT-PCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays.

  9. Real time RT-PCR assay for detection of different serotypes of FMDV in Egypt

    Directory of Open Access Journals (Sweden)

    Laila El-Shehawy

    Full Text Available Aim: The present study indicated that rRT-PCR could be provided for the detection of FMDV in infected, contact and carrier cattle and also provide a rapid sensitive tool aiming to aid in rapid disease detection and control. Foot and Mouth disease virus serotypes O and A still existing in Egypt. In January 2012, sever outbreaks struck the animal population in most Egyptian 1 governorates. The causative virus was identified as FMDV SAT2. Material and Methods: Five samples of tongue epithelium (ET and five oesophageal-pharyngeal (OP fluid samples were collected from FMD suspected cattle in infected farm at El-Fayoum and 20 OP samples from in-contact cattle at the same farm in addition to 30 OP samples from apparently healthy cattle at three different localities in El-Fayoum governorate (12 from Fayoum; 9 from Sinoras and 9 from Edsa in order to detect carrier cattle. All of these samples were collected during November and December 2011 and January 2012. Results: All the ET and OP samples were inoculated on BHK cell culture and baby mice. The obtained results were identified using complement fixation test in addition to real-time reverse transcriptase polymerase chain reaction (rRT-PCR. In the infected farm at El-Fayoum FMDV type SAT2 was detected in cattle which are considered as the first introduction of this type while FMDV type O and SAT2 were detected in the in-contact cattle in the same farm. The sensitivity of rRT-PCR was cleared in the in-contact cattle as 13 out of 20 OP samples were positive to FMDV by rRT-PCR while 11 out of 20 OP samples were positive to FMDV by CFT. The OP samples collected from apparently healthy cattle from Fayoum, Sinoras and Edsa localities in Fayoum governorate demonstrate the circulation of the FMDV type A, O and the recent SAT2 in carrier cattle which threaten cattle population in Fayoum governorate. Also the sensitivity of real time RT-PCR over the CFT in detection of FMDV carrier cattle was clearly noticed in

  10. Development of a multiplex real-time RT-PCR assay for simultaneous detection of dengue and chikungunya viruses.

    Science.gov (United States)

    Cecilia, D; Kakade, M; Alagarasu, K; Patil, J; Salunke, A; Parashar, D; Shah, P S

    2015-01-01

    Dengue and chikungunya viruses co-circulate and cause infections that start with similar symptoms but progress to radically different outcomes. Therefore, an early diagnostic test that can differentiate between the two is needed. A single-step multiplex real-time RT-PCR assay was developed that can simultaneously detect and quantitate RNA of all dengue virus (DENV) serotypes and chikungunya virus (CHIKV). The sensitivity was 100 % for DENV and 95.8 % for CHIKV, whilst the specificity was 100 % for both viruses when compared with conventional RT-PCR. The detection limit ranged from 1 to 50 plaque-forming units. The assay was successfully used for differential diagnosis of dengue and chikungunya in Pune, where the viruses co-circulate.

  11. Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth and look-alike disease viruses

    Energy Technology Data Exchange (ETDEWEB)

    Hindson, B J; Baker, B R; Bentley Tammero, L F; Lenhoff, R J; Naraghi-Arani, P; Vitalis, E A; Slezak, T R; Hullinger, P J; Reid, S M; Ebert, K; Ferris, N P; King, D P

    2007-09-18

    A high-throughput multiplexed assay (Multiplex Version 1.0) was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspect cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRTPCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays.

  12. Respiratory virus multiplex RT-PCR assay sensitivities and influence factors in hospitalized children with lower respiratory tract infections.

    Science.gov (United States)

    Deng, Jikui; Ma, Zhuoya; Huang, Wenbo; Li, Chengrong; Wang, Heping; Zheng, Yuejie; Zhou, Rong; Tang, Yi-Wei

    2013-04-01

    Multiplex RT-PCR assays have been widely used tools for detection and differentiation of a panel of respiratory viral pathogens. In this study, we evaluated the Qiagen ResPlex II V2.0 kit and explored factors influencing its sensitivity. Nasopharyngeal swab (NPS) specimens were prospectively collected from pediatric inpatients with lower respiratory tract infections at the time of admission in the Shenzhen Children's Hospital from May 2009 to April 2010. Total nucleic acids were extracted using the EZ1 system (Qiagen, Germany) and 17 respiratory viruses and genotypes including influenza A virus (FluA), FluB, parainfluenza virus 1 (PIV1), PIV2, PIV3, PIV4, respiratory syncytial virus (RSV), human metapneumovirus (hMPV), rhinoviruses (RhV), enteroviruses (EnV), human bocaviruses (hBoV), adenoviruses (AdV), four coronaviruses (229E, OC43, NL63 and HKU1), and FluA 2009 pandemic H1N1(H1N1-p) were detected and identified by the ResPlex II kit. In parallel, 16 real-time TaqMan quantitative RT-PCR assays were used to quantitatively detect each virus except for RhV. Influenza and parainfluenza viral cultures were also performed. Among the total 438 NPS specimens collected during the study period, one or more viral pathogens were detected in 274 (62.6%) and 201(45.9%) specimens by monoplex TaqMan RT-PCR and multiplex ResPlex, respectively. When results from monoplex PCR or cell culture were used as the reference standard, the multiplex PCR possessed specificities of 92.9-100.0%. The sensitivity of multiplex PCR for PIV3, hMPV, PIV1 and BoV were 73.1%, 70%, 66.7% and 55.6%, respectively, while low sensitivities (11.1%-40.0%) were observed for FluA, EnV, OC43, RSV and H1N1. Among the seven viruses/genotypes detected with higher frequencies, multiplex PCR sensitivities were correlated significantly with viral loads determined by the TaqMan RT-PCR in FluA, H1N1-p and RSV (p=0.011-0.000). The Qiagen ResPlex II multiplex RT-PCR kit possesses excellent specificity for simultaneous

  13. Simultaneous detection and quantitation of Chikungunya, dengue and West Nile viruses by multiplex RT-PCR assays and dengue virus typing using high resolution melting.

    Science.gov (United States)

    Naze, F; Le Roux, K; Schuffenecker, I; Zeller, H; Staikowsky, F; Grivard, P; Michault, A; Laurent, P

    2009-12-01

    Chikungunya (CHIKV), Dengue (DENV) and West Nile (WNV) viruses are arthropod-borne viruses that are able to emerge or re-emerge in many regions due to climatic changes and increase in travel. Since these viruses produce similar clinical signs it is important for physicians and epidemiologists to differentiate them rapidly. A molecular method was developed for their detection and quantitation in plasma samples and a DENV typing technique were developed. The method consisted in performing two multiplex real-time one-step RT-PCR assays, to detect and quantify the three viruses. Both assays were conducted in a single run, from a single RNA extract containing a unique coextracted and coamplified composite internal control. The quantitation results were close to the best detection thresholds obtained with simplex RT-PCR techniques. The differentiation of DENV types was performed using a High Resolution Melting technique. The assays enable the early diagnosis of the three arboviruses during viremia, including cases of coinfection. The method is rapid, specific and highly sensitive with a potential for clinical diagnosis and epidemiological surveillance. A DENV positive sample can be typed conveniently using the High Resolution Melting technique using the same apparatus.

  14. Development and Preliminary Evaluation of a New Real-Time RT-PCR Assay For Detection of Peste des petits Ruminants Virus Genome.

    Science.gov (United States)

    Polci, A; Cosseddu, G M; Ancora, M; Pinoni, C; El Harrak, M; Sebhatu, T T; Ghebremeskel, E; Sghaier, S; Lelli, R; Monaco, F

    2015-06-01

    A duplex real-time reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed for a simple and rapid diagnosis of Peste des petits ruminants (PPR). qRT-PCR primers and TaqMan probe were designed on a conserved region of nucleocapsid protein (Np) of PPR virus (PPRV) genome. An in vitro transcript of the target region was constructed and tested to determine analytical sensitivity. Commercial heterologous Armored RNA(®) was used as an internal positive control (IPC) for either RNA isolation or RT-PCR steps. The detection limit of the newly designed duplex real-time RT-PCR (qRT-PCR PPR_Np) was approximately 20 copies/μl with a 95% probability. No amplification signals were recorded when the qRT-PCR PPR_Np was applied to viruses closely related or clinically similar to PPRV- or to PPR-negative blood samples. A preliminary evaluation of the diagnostic performance was carried out by testing a group of 43 clinical specimens collected from distinct geographic areas of Africa and Middle East. qRT-PCR PPR_Np showed higher sensitivity than the conventional gel-based RT-PCR assays, which have been used as reference standards. Internal positive control made it possible to identify the occurrence of 5 false-negative results caused by the amplification failure, thus improving the accuracy of PPRV detection. © 2013 Blackwell Verlag GmbH.

  15. Respiratory Virus Multiplex RT-PCR Assay Sensitivities and Influence Factors in Hospitalized Children with Lower Respiratory Tract Infections

    Institute of Scientific and Technical Information of China (English)

    Jikui Deng; Zhuoya Ma; Wenbo Huang; Chengrong Li; Heping Wang; Yuejie Zheng; Rong Zhou

    2013-01-01

    Multiplex RT-PCR assays have been widely used tools for detection and differentiation of a panel of respiratory viral pathogens.In this study,we evaluated the Qiagen ResPlex Ⅱ V2.0 kit and explored factors influencing its sensitivity.Nasopharyngeal swab (NPS) specimens were prospectively collected from pediatric inpatients with lower respiratory tract infections at the time of admission in the Shenzhen Children's Hospital from May 2009 to April 2010.Total nucleic acids were extracted using the EZ1 system (Qiagen,Germany) and 17 respiratory viruses and genotypes including influenza A virus (FluA),FluB,parainfluenza virus 1 (PIV1),PIV2,PIV3,PIV4,respiratory syncytial virus (RSV),human metapneumovirus (hMPV),rhinoviruses (RhV),enteroviruses (EnV),human bocaviruses (hBoV),adenoviruses (AdV),four coronaviruses (229E,OC43,NL63 and HKU1),and FluA 2009 pandemic H1N1(H1N1-p) were detected and identified by the ResPlex Ⅱ kit.In parallel,16 real-time TaqMan quantitative RT-PCR assays were used to quantitatively detect each virus except for RhV.Influenza and parainfluenza viral cultures were also performed.Among the total 438 NPS specimens collected during the study period,one or more viral pathogens were detected in 274 (62.6%) and 201(45.9%) specimens by monoplex TaqMan RT-PCR and multiplex ResPlex,respectively.When results from monoplex PCR or cell culture were used as the reference standard,the multiplex PCR possessed specificities of 92.9-100.0%.The sensitivity of multiplex PCR for PIV3,hMPV,PIV1 and BoV were 73.1%,70%,66.7% and 55.6%,respectively,while low sensitivities (11.1%-40.0%) were observed for FluA,EnV,OC43,RSV and H1N1.Among the seven viruses/genotypes detected with higher frequencies,multiplex PCR sensitivities were correlated significantly with viral loads determined by the TaqMan RT-PCR in F luA,H 1N 1-p and RSV (p=0.011-0.000).The Qiagen ResPlex Ⅱ multiplex RT-PCR kit possesses excellent specificity for simultaneous detection of 17

  16. Establishment of realtime RT-PCR assay to detect polio virus in the Acute Flaccid Paralysis laboratory surveillance

    Directory of Open Access Journals (Sweden)

    Nike Susanti

    2016-07-01

    Polio Vaccine into Vaccine-Derived Poliovirus still continue. Since 1991, WHO has developedAcute Flaccid Paralysis (AFP laboratory based surveillance. In 2014, the polioviruses identification by real-timeReverse Transcriptase Polymerase Chain Reaction (rRT-PCR, has been introduced to National Polio Laboratory(NPL Center for Biomedical and Basic Technology of Health. The objective of the rRT-PCR application is to havefaster and better diagnostic methods to monitor the circulation and mutation of polio viruses.Methods: Isolate tested by rRT-PCR using a combination of primers and probe mentioned by WHO manual.The viral RNA is converted to cDNA using reverse transcriptase and amplified in a PCR reaction using Taqpolymerase. The PCR products are detected and identified by hybridization with specific probes. The combinationof primers and probes will result in the serotype identification and intratypic differentiation of poliovirus isolates.Results: In 2014 NPL Jakarta received 604 AFP cases through the surveillance system, five cases foundpositive for polio viruses by culture. All of the specimens were positive for polio vaccine viruses. Twocases were polio virus type P2 (40%, one cases polio virus type P1 (20%, 1 case polio virus type P3(20% and one case mix polio viruses type P1+P2 (20%.Conclusion: The real-time PCR assay was able to help the identification of polio viruses rapidly in Jakartalab. The test can be utilized for monitoring the population routinely immunized with OPV. (Health ScienceJournal of Indonesia 2016;7:27-31Keywords: ITD, Poliovirus, Identification, rRT-PCR

  17. Development and evaluation of a SYBR green-based real time RT-PCR assay for detection of the emerging avian influenza A (H7N9 virus.

    Directory of Open Access Journals (Sweden)

    Zheng Zhu

    Full Text Available Most recently a novel avian-origin influenza A (H7N9 virus emerged in China and has been associated with lots of human infection and fatal cases. Genetic analysis of the viral genome revealed that this reassortant virus might be better adapted to humans than other avian influenza viruses. Molecular diagnostic methods are thus urgently needed in public health laboratories. In this study, a SYBR green-based one-step real time reverse transcription-PCR (RT-PCR was developed to detect the novel H7N9 virus. The primer pairs on the basis of the hemagglutinin and neuraminidase gene sequences of H7N9 viruses amplified subtype-specific fragments with Tm values of 80.77±0.06°C for H7 and 81.20±0.17°C for N9 respectively. The standard curves showed a dynamic linear range across 6 log units of RNA copy number (10(6 to 10(1 copies/ µl with a detection limit of 10 copies per reaction for both H7 and N9 assays by using serial ten-fold diluted in-vitro transcribed viral RNA. In addition, no cross-reactivity was observed with seasonal H1N1, H1N1 pdm09, H3N2, H5N1 and H9N2 viruses as well as other human respiratory viruses. When the assay was further evaluated in H7N9 virus infected clinical samples, positive amplification signals were obtained in all of the specimens with the accordance between H7 and N9 assays. Therefore, the established SYBR green-based real time RT-PCR assay could provide a rapid, sensitive, specific and reliable alternative approach with lower costs for high throughput screening of suspected samples from humans, animals and environments in first line public health laboratories.

  18. A multiplex endpoint RT-PCR assay for quality assessment of RNA extracted from formalin-fixed paraffin-embedded tissues

    Directory of Open Access Journals (Sweden)

    Dobrovic Alexander

    2010-12-01

    Full Text Available Abstract Background RNA extracted from formalin-fixed paraffin-embedded (FFPE samples is chemically modified and degraded, which compromises its use in gene expression studies. Most of the current approaches for RNA quality assessment are not suitable for FFPE derived RNA. Results We have developed a single-tube multiplex endpoint RT-PCR assay specifically designed to evaluate RNA extracted from FFPE tissues for mRNA integrity and performance in reverse transcription - quantitative real-time PCR (RT-qPCR assays. This single-tube quality control (QC assay minimises the amount of RNA used in quality control. mRNA integrity and the suitability of RNA for RT-PCR is evaluated by the multiplex endpoint RT-PCR assay using the TBP gene mRNA as the target sequence. The RT-PCR amplicon sizes, 92, 161, 252 and 300 bp, cover a range of amplicon sizes suitable for a wide range of RT-qPCR assays. The QC assay was used to evaluate RNA prepared by two different protocols for extracting total RNA from needle microdissected FFPE breast tumour samples. The amplification products were analysed by gel electrophoresis where the spectrum of amplicon sizes indicated the level of RNA degradation and thus the suitability of the RNA for PCR. The ability of the multiplex endpoint RT-PCR QC assay to identify FFPE samples with an adequate RNA quality was validated by examining the Cq values of an RT-qPCR assay with an 87 bp amplicon. Conclusions The multiplex endpoint RT-PCR assay is well suited for the determination of the quality of FFPE derived RNAs, to identify which RT-PCR assays they are suitable for, and is also applicable to assess non-FFPE RNA for gene expression studies. Furthermore, the assay can also be used for the evaluation of RNA extraction protocols from FFPE samples.

  19. Evaluation of a broad range real-time polymerase chain reaction (RT-PCR) assay for the diagnosis of septic synovitis in horses.

    Science.gov (United States)

    Elmas, Colette R; Koenig, Judith B; Bienzle, Dorothee; Cribb, Nicola C; Cernicchiaro, Natalia; Coté, Nathalie M; Weese, J Scott

    2013-07-01

    Septic synovitis is a potentially debilitating and life-threatening disorder in horses. We hypothesized that a universal bacterial real-time PCR (RT-PCR) assay would have improved sensitivity and decreased turn-around time for detection of bacteria in synovial fluid (SF) samples. Forty-eight SF samples were collected from 36 horses that presented to two referral institutions with suspected septic synovitis. Universal RT-PCR, bacterial culture and SF analysis were performed on all samples, and an interpretation on the sample being septic or not was derived by three board certified specialists from the history, clinical assessment and SF characteristics. RT-PCR results were compared to a composite standard comprised of positive culture and interpretation by all three specialists of samples as "septic". For 41 of 48 samples (85%), culture and RT-PCR results were concordant. Compared to the composite standard, 83% of samples were correctly classified by RT-PCR (turn-around time of approximately 4 hours). Relative sensitivity and specificity of RT-PCR were 87% and 72% respectively, and 56% and 86% for culture. Hence, universal RT-PCR was a rapid and highly sensitive test, which may accelerate diagnosis and improve outcome for horses with septic synovitis.

  20. Development and Application of One Step RT-PCR Method for Detection of Bovine Viral Diarrhea Virus%牛病毒性腹泻病毒一步法RT-PCR检测方法的建立与应用

    Institute of Scientific and Technical Information of China (English)

    王春庆

    2013-01-01

    To establish a rapid bovine viral diarrhea virus (BVDV) pathogen detection methods, based on BVDV gene sequence in GenBank, we synthesized one pair of primers, established one step RT-PCR for BVDV detection. The method was used for infectious bovine rhinotracheitis virus (IBRV), classical swine fever virus (CSFV), bovine para influenza virus 3 (BPIV3) , all of the PCR results were negative, the sensitivity was 1 ng RNA. The established one step PCR method had good specificity, sensitivity, reproducibility, it could detect very low levels of BVDV quickly and accurately, it provided a kind of rapid, sensitive, specific and precise molecular biology detection method for pathogen detection and molecular epidemiology of material such as BVDV.%为建立一种快速检测牛病毒性腹泻病毒病原的方法,本研究根据GenBank上登录的牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)基因组序列,设计1对引物,建立了检测BVDV的一步法RT-PCR方法.该方法对牛传染性鼻气管炎病毒、猪瘟病毒、牛副流感病毒3型的扩增结果均为阴性,检测的敏感性达1 ng RNA.该一步法RT-PCR方法具有良好的特异性、敏感性、重复性,可以准确快速检测出极低含量的BVDV,将为BVDV的病原检测及分子流行病学调查等提供一种快速、灵敏、特异、准确的分子生物学检测方法.

  1. Development of a two-step SYBR Green I based real time RT-PCR assay for detecting and quantifying peste des petits ruminants virus in clinical samples.

    Science.gov (United States)

    Abera, Tsegalem; Thangavelu, Ardhanary

    2014-12-01

    A two-step SYBR Green I based real time RT-PCR targeting the matrix (M) gene of Peste des petits ruminants virus (PPRV) was developed. The specificity of the assay was assessed against viral nucleic acid extracted from a range of animal viruses of clinical and structural similarities to PPRV including canine distemper virus, measles virus, bluetongue virus and Newcastle disease virus. But none of the viruses and no template control showed an amplification signal. Sensitivity of the same assay was assessed based on plasmid DNA copy number and with respect to infectivity titre. The lower detection limit achieved was 2.88 plasmid DNA copies/μl with corresponding Ct value of 35.93. Based on tissue culture infectivity titre the lower detection limits were 0.0001TCID50/ml and 1TCID50/ml for the SYBR green I based real time RT-PCR and conventional RT-PCR, respectively. The calculated coefficient of variations values for intra- and inter-assay variability were low, ranging from 0.21% to 1.83% and 0.44% to 1.97%, respectively. The performance of newly developed assay was evaluated on a total of 36 clinical samples suspected of PPR and compared with conventional RT-PCR. The SYBR Green I based real time RT-PCR assay detected PPRV in 32 (88.8%) of clinical samples compared to 19 (52.7%) by conventional RT-PCR. Thus, the two-step SYBR Green I based real time RT-PCR assay targeting the M gene of PPRV reported in this study was highly sensitive, specific and reproducible for detection and quantitation of PPRV nucleic acids. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Establishment of one-step SYBR green-based real time-PCR assay for rapid detection and quantification of chikungunya virus infection.

    Science.gov (United States)

    Ho, Phui San; Ng, Mary Mah Lee; Chu, Justin Jang Hann

    2010-01-21

    Chikungunya virus (CHIKV) is a mosquito-borne alphavirus and one of the prevalent re-emerging arbovirus in tropical and subtropical regions of Asia, Africa, and Central and South America. It produces a spectrum of illness ranging from inapparent infection to moderate febrile illness as well as severe arthralgia or arthritis affecting multiple joints. In this study, a quantitative, one-step real-time SYBR Green-based RT-PCR system for the non-structural protein 2 (nsP2) of CHIKV that can quantify a wide range of viral RNA concentrations was developed. Comparisons between the conventional semi-quantitative RT-PCR assay, immunofluorescence detection method and the one-step SYBR Green-based RT-PCR assay in the detection of CHIKV infection revealed much rapid and increase sensitivity of the latter method. Furthermore, this newly developed assay was validated by in vitro experiments in which ribavirin, a well-known RNA virus inhibitor, showed a dose-dependent inhibition of virus replication on cells that was assessed by viral infectivity and viral RNA production. Our results demonstrate the potential of this newly developed one-step SYBR Green I-based RT-PCR assay may be a useful tool in rapid detection of CHIKV and monitoring the extent of viral replication possibly in patients' samples.

  3. Establishment of one-step SYBR green-based real time-PCR assay for rapid detection and quantification of chikungunya virus infection

    Directory of Open Access Journals (Sweden)

    Chu Justin

    2010-01-01

    Full Text Available Abstract Chikungunya virus (CHIKV is a mosquito-borne alphavirus and one of the prevalent re-emerging arbovirus in tropical and subtropical regions of Asia, Africa, and Central and South America. It produces a spectrum of illness ranging from inapparent infection to moderate febrile illness as well as severe arthralgia or arthritis affecting multiple joints. In this study, a quantitative, one-step real-time SYBR Green-based RT-PCR system for the non-structural protein 2 (nsP2 of CHIKV that can quantify a wide range of viral RNA concentrations was developed. Comparisons between the conventional semi-quantitative RT-PCR assay, immunofluorescence detection method and the one-step SYBR Green-based RT-PCR assay in the detection of CHIKV infection revealed much rapid and increase sensitivity of the latter method. Furthermore, this newly developed assay was validated by in vitro experiments in which ribavirin, a well-known RNA virus inhibitor, showed a dose-dependent inhibition of virus replication on cells that was assessed by viral infectivity and viral RNA production. Our results demonstrate the potential of this newly developed one-step SYBR Green I-based RT-PCR assay may be a useful tool in rapid detection of CHIKV and monitoring the extent of viral replication possibly in patients' samples.

  4. RT-PCR assay for detection of Lassa virus and related Old World arenaviruses targeting the L gene.

    Science.gov (United States)

    Vieth, Simon; Drosten, Christian; Lenz, Oliver; Vincent, Martin; Omilabu, Sunday; Hass, Meike; Becker-Ziaja, Beate; ter Meulen, Jan; Nichol, Stuart T; Schmitz, Herbert; Günther, Stephan

    2007-12-01

    This study describes an RT-PCR assay targeting the L RNA segment of arenaviruses. Conserved regions were identified in the polymerase domain of the L gene on the basis of published sequences for Lassa virus, lymphocytic choriomeningitis virus (LCMV), Pichinde virus and Tacaribe virus, as well as 15 novel sequences for Lassa virus, LCMV, Ippy virus, Mobala virus and Mopeia virus determined in this study. Using these regions as target sites, a PCR assay for detection of all known Old World arenaviruses was developed and optimized. The concentration that yields 95% positive results in a set of replicate tests (95% detection limit) was determined to be 4290 copies of Lassa virus L RNA per ml of serum, corresponding to 30 copies per reaction. The ability of the assay to detect various Old World arenaviruses was demonstrated with in vitro transcribed RNA, material from infected cell cultures and samples from patients with Lassa fever and monkeys with LCMV-associated callitrichid hepatitis. The L gene PCR assay may be applicable: (i) as a complementary diagnostic test for Lassa virus and LCMV; (ii) to identify unknown Old World arenaviruses suspected as aetiological agents of disease; and (iii) for screening of potential reservoir hosts for unknown Old World arenaviruses.

  5. Evaluation of a single-tube fluorogenic RT-PCR assay for detection of bovine respiratory syncytial virus in clinical samples

    DEFF Research Database (Denmark)

    Hakhverdyan, Mikhayil; Hägglund, Sara; Larsen, Lars Erik

    2005-01-01

    , antigen ELISA (Ag-ELISA) and virus isolation (VI). Interspersed negative control samples, samples from healthy animals and eight symptomatically or genetically related viruses were all negative, confirming a high specificity of the assay. Taken together, the data indicated that the fRT-PCR assay can...... understanding of the virus. In this study, a BRSV fluorogenic reverse transcription PCR (fRT-PCR) assay, based on TaqMan principle, was developed and evaluated on a large number of clinical samples, representing various cases of natural and experimental BRSV infections. By using a single-step closed-tube format...

  6. Development of a real-time RT-PCR assay for the detection of Crimean-Congo hemorrhagic fever virus.

    Science.gov (United States)

    Atkinson, Barry; Chamberlain, John; Logue, Christopher H; Cook, Nicola; Bruce, Christine; Dowall, Stuart D; Hewson, Roger

    2012-09-01

    Crimean-Congo hemorrhagic fever (CCHF) is a virulent tick-borne disease with a case fatality rate ranging from 10-50% for tick-borne transmission, and up to 80% for nosocomial transmission. Human cases have been reported in over 30 countries across Europe, Asia, and Africa. It appears to be spreading to new areas with several countries reporting their first human cases of CCHF disease within the past 10 years. We report a novel real-time RT-PCR assay designed to amplify a conserved region of the CCHF virus S segment. It is capable of detecting strains from all 7 groups of CCHF, including the AP92 strain that until recently represented a lineage of strains that were not associated with human disease. The limit of detection of the assay is 5 copies of target RNA, and the assay shows no cross-reactivity with other viruses from within the same genus, or with viruses causing similar human disease.

  7. Detection of Citrus leprosis virus C using specific primers and TaqMan probe in one-step real-time reverse-transcription polymerase chain reaction assays.

    Science.gov (United States)

    Choudhary, Nandlal; Wei, G; Govindarajulu, A; Roy, Avijit; Li, Wenbin; Picton, Deric D; Nakhla, M K; Levy, L; Brlansky, R H

    2015-11-01

    Citrus leprosis virus C (CiLV-C), a causal agent of the leprosis disease in citrus, is mostly present in the South and Central America and spreading toward the North America. To enable better diagnosis and inhibit the further spread of this re-emerging virus a quantitative (q) real-time reverse transcription polymerase chain reaction (qRT-PCR) assay is needed for early detection of CiLV-C when the virus is present in low titer in citrus leprosis samples. Using the genomic sequence of CiLV-C, specific primers and probe were designed and synthesized to amplify a 73 nt amplicon from the movement protein (MP) gene. A standard curve of the 73 nt amplicon MP gene was developed using known 10(10)-10(1) copies of in vitro synthesized RNA transcript to estimate the copy number of RNA transcript in the citrus leprosis samples. The one-step qRT-PCR detection assays for CiLV-C were determined to be 1000 times more sensitive when compared to the one-step conventional reverse transcription polymerase chain reaction (RT-PCR) CiLV-C detection method. To evaluate the quality of the total RNA extracts, NADH dehydrogenase gene specific primers (nad5) and probe were included in reactions as an internal control. The one-step qRT-PCR specificity was successfully validated by testing for the presence of CiLV-C in the total RNA extracts of the citrus leprosis samples collected from Belize, Costa Rica, Mexico and Panama. Implementation of the one-step qRT-PCR assays for CiLV-C diagnosis should assist regulatory agencies in surveillance activities to monitor the distribution pattern of CiLV-C in countries where it is present and to prevent further dissemination into citrus growing countries where there is no report of CiLV-C presence.

  8. Generation of HIV-1 and Internal Control Transcripts as Standards for an In-House Quantitative Competitive RT-PCR Assay to Determine HIV-1 Viral Load

    Directory of Open Access Journals (Sweden)

    Anny Armas Cayarga

    2011-01-01

    Full Text Available Human immunodeficiency virus type-1 (HIV-1 viral load is useful for monitoring disease progression in HIV-infected individuals. We generated RNA standards of HIV-1 and internal control (IC by in vitro transcription and evaluated its performance in a quantitative reverse transcription polymerase chain reaction (qRT-PCR assay. HIV-1 and IC standards were obtained at high RNA concentrations, without DNA contamination. When these transcripts were included as standards in a qRT-PCR assay, it was obtained a good accuracy (±0.5 log10 unit of the expected results in the quantification of the HIV-1 RNA international standard and controls. The lower limit detection achieved using these standards was 511.0 IU/mL. A high correlation (=0.925 was obtained between the in-house qRT-PCR assay and the NucliSens easyQ HIV-1 test (bioMerieux for HIV-1 RNA quantitation with clinical samples (=14. HIV-1 and IC RNA transcripts, generated in this study, proved to be useful as standards in an in-house qRT-PCR assay for determination of HIV-1 viral load.

  9. Dataset of proinflammatory cytokine and cytokine receptor gene expression in rainbow trout (Oncorhynchus mykiss) measured using a novel GeXP multiplex, RT-PCR assay

    Science.gov (United States)

    A GeXP multiplex, RT-PCR assay was developed and optimized that simultaneously measures expression of a suite of immune-relevant genes in rainbow trout (Oncorhynchus mykiss), concentrating on tumor necrosis factor and interleukin-1 ligand/receptor systems and acute phase response genes. The dataset ...

  10. Development of a novel quantitative real-time RT-PCR assay for the simultaneous detection of all serotypes of Foot-and-mouth disease virus

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Uttenthal, Åse; de Stricker, K.;

    2003-01-01

    Foot-and-mouth disease virus (FMDV) spreads extremely fast and the need for rapid and robust diagnostic virus detection systems was obvious during the recent European epidemic. Using a novel real-time RT-PCR system based on primer-probe energy transfer (PriProET) we present here an assay targeting...

  11. Generation of HIV-1 and Internal Control Transcripts as Standards for an In-House Quantitative Competitive RT-PCR Assay to Determine HIV-1 Viral Load

    Science.gov (United States)

    Armas Cayarga, Anny; Perea Hernández, Yenitse; González González, Yaimé J.; Dueñas Carrera, Santiago; González Pérez, Idania; Robaina Álvarez, René

    2011-01-01

    Human immunodeficiency virus type-1 (HIV-1) viral load is useful for monitoring disease progression in HIV-infected individuals. We generated RNA standards of HIV-1 and internal control (IC) by in vitro transcription and evaluated its performance in a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay. HIV-1 and IC standards were obtained at high RNA concentrations, without DNA contamination. When these transcripts were included as standards in a qRT-PCR assay, it was obtained a good accuracy (±0.5 log10 unit of the expected results) in the quantification of the HIV-1 RNA international standard and controls. The lower limit detection achieved using these standards was 511.0 IU/mL. A high correlation (r = 0.925) was obtained between the in-house qRT-PCR assay and the NucliSens easyQ HIV-1 test (bioMerieux) for HIV-1 RNA quantitation with clinical samples (N = 14). HIV-1 and IC RNA transcripts, generated in this study, proved to be useful as standards in an in-house qRT-PCR assay for determination of HIV-1 viral load. PMID:21766036

  12. A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses.

    Directory of Open Access Journals (Sweden)

    Ashutosh Wadhwa

    2017-01-01

    Full Text Available Rabies, resulting from infection by Rabies virus (RABV and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses

  13. Improved RT-PCR Assay to Quantitate the Pri-, Pre-, and Mature microRNAs with Higher Efficiency and Accuracy.

    Science.gov (United States)

    Tong, Li; Xue, Huihui; Xiong, Li; Xiao, Junhua; Zhou, Yuxun

    2015-10-01

    Understanding of the functional significance of microRNAs (miRNAs) requires efficient and accurate detection method. In this study, we developed an improved miRNAs quantification system based on quantitative real-time polymerase chain reaction (qRT-PCR). This method showed higher efficiency and accuracy to survey the expression of primary miRNAs (pri-miRNAs), precursor miRNAs (pre-miRNAs), and mature miRNAs. Instead of relative quantification method, we quantified the pri-miRNAs and pre-miRNAs with absolute qRT-PCR based on SYBR Green I fluorescence. This improvement corrected for the inaccuracy caused by the differences in amplicon length and PCR efficiency. We also used SYBR Green method to quantify mature miRNAs based on the stem-loop qRT-PCR method. We extended the pairing part of the stem-loop reverse transcript (RT) primer from 6 to 11 bp, which greatly increased the efficiency of reverse transcription PCR (RT-PCR). The performance of the improved RT primer was tested using synthetic mature miRNAs and tissue RNA samples. Results showed that the improved RT primer demonstrated dynamic range of seven orders of magnitude and sensitivity of detection of hundreds of copies of miRNA molecules.

  14. Development and evaluation of tailored specific real-time RT-PCR assays for detection of foot-and-mouth disease virus serotypes circulating in East Africa

    DEFF Research Database (Denmark)

    Bachanek-Bankowska, Katarzyna; Mero, Herieth R.; Wadsworth, Jemma

    2016-01-01

    the VP1-coding region that share high intra-lineage identity, but do not cross-react with FMD viruses from other serotypes that circulate in the region. These serotype-specific assays operate with the same thermal profile as the pan-diagnostic tests making it possible to run them in parallel to produce...... methods mainly rely either on antigen detection ELISAs or nucleotide sequencing approaches. This report describes the development of a panel of serotype-specific real-time RT-PCR assays (rRT-PCR) tailored to detect FMDV lineages currently circulating in East Africa. These assays target sequences within...... CT values comparable to the pan-diagnostic test detecting the 3D-coding region. These assays were evaluated alongside the established pan-specific molecular test using field samples and virus isolates collected from Tanzania, Kenya and Ethiopia that had been previously characterised by nucleotide...

  15. THE IMPROVEMENT OF RT-PCR TECHNIQUE ON DETECTING ROTAVIRUS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To establish a speed and effective method to detect rotavirus. Methods Using ELISA and one step RT-PCR to detect 196 clinic samples from Xi'an area. Results Compared with ELISA method, one step RT PCR was more sensitive and specific (P <0.05). Conclusion One step RT-PCR is a simple, speed, sensitive and spe cific method for clinic and epidemic studies of rotavirus.

  16. Multiplex RT-PCR and indirect immunofluorescence assays for detection and subtyping of human influenza virus in Tunisia.

    Science.gov (United States)

    Ben M'hadheb, Manel; Harrabi, Myriam; Souii, Amira; Jrad-Battikh, Nadia; Gharbi, Jawhar

    2015-03-01

    Influenza viruses are negative stranded segmented RNA viruses belonging to Orthomyxoviridae family. They are classified into three types A, B, and C. Type A influenza viruses are classified into subtypes according to the antigenic characters of the surface glycoproteins: hemagglutinin (H) and neuraminidase (N). The aim of the present study is to develop a fast and reliable multiplex RT-PCR technique for detecting simultaneously the subtypes A/H1N1 and A/H3N2 of influenza virus. Our study included 398 patients (mean age 30.33 ± 19.92 years) with flu or flu-like syndromes, consulting physicians affiliated with collaborating teams. A multiplex RT-PCR detecting A/H1N1 and A/H3N2 influenza viruses and an examination by indirect immunofluorescence (IFI) were performed. In the optimized conditions, we diagnosed by IFI a viral infection in 90 patients (22.6 %): 85 cases of influenza type A, four cases of influenza type B, and only one case of coinfection with types A and B. An evaluation of the technique was performed on 19 clinical specimens positive in IFI, and we detected eight cases of A/H3N2, five cases of A/H1N1, one case of influenza virus type A which is not an H1N1 nor H3N2, and five negative cases. Multiplex RT-PCR is a sensitive technique allowing an effective and fast diagnosis of respiratory infections caused by influenza viruses in which the optimization often collides with problems of sensibility.

  17. Comparison of Luminex xTAG® RVP fast assay and real time RT-PCR for the detection of respiratory viruses in adults with community-acquired pneumonia.

    Science.gov (United States)

    Luchsinger, Vivian; Prades, Yara; Ruiz, Mauricio; Pizarro, Rolando; Rossi, Patricio; Lizama, Luis; Garmendia, María Luisa; Meza, Angela; Larrañaga, Carmen; Avendaño, Luis F

    2016-07-01

    Community-acquired pneumonia (CAP) is the third cause of death worldwide. Viruses are frequently detected in adult CAP. Highly sensitive diagnostic techniques should be used due to poor viral shedding. Different sampling methods can affect viral detection, being necessary to establish the optimal type of sample for identifying respiratory viruses in adults. The detection rates of respiratory viruses by Luminex xTAG® RVP fast assay, real time RT-PCR (rtRT-PCR) (Sacace®), and immunofluorescence assay (IFA) in adult CAP were performed in nasopharyngeal swabs (NPS) and aspirates (NPA) from 179 hospitalized adults. Positivity was 47.5% for Luminex®, 42.5% for rtRT-PCR (P = 0.3), and 2.7% for IFA (2.7%) (P viruses in 112 NPA and 35 (34.3%) and 31 (30.4%) in 102 NPS, respectively (P viruses in CAP adults. Both molecular techniques yielded better results with nasopharyngeal aspirate than swabs.

  18. Novel Molecular Beacon Probe-Based Real-Time RT-PCR Assay for Diagnosis of Crimean-Congo Hemorrhagic Fever Encountered in India

    Directory of Open Access Journals (Sweden)

    Aman Kamboj

    2014-01-01

    Full Text Available Crimean-Congo hemorrhagic fever (CCHF is an emerging zoonotic disease in India and requires immediate detection of infection both for preventing further transmission and for controlling the infection. The present study describes development, optimization, and evaluation of a novel molecular beacon-based real-time RT-PCR assay for rapid, sensitive, and specific diagnosis of Crimean-Congo hemorrhagic fever virus (CCHFV. The developed assay was found to be a better alternative to the reported TaqMan assay for routine diagnosis of CCHF.

  19. A family-wide RT-PCR assay for detection of paramyxoviruses and application to a large-scale surveillance study.

    Directory of Open Access Journals (Sweden)

    Sander van Boheemen

    Full Text Available Family-wide molecular diagnostic assays are valuable tools for initial identification of viruses during outbreaks and to limit costs of surveillance studies. Recent discoveries of paramyxoviruses have called for such assay that is able to detect all known and unknown paramyxoviruses in one round of PCR amplification. We have developed a RT-PCR assay consisting of a single degenerate primer set, able to detect all members of the Paramyxoviridae family including all virus genera within the subfamilies Paramyxovirinae and Pneumovirinae. Primers anneal to domain III of the polymerase gene, with the 3' end of the reverse primer annealing to the conserved motif GDNQ, which is proposed to be the active site for nucleotide polymerization. The assay was fully optimized and was shown to indeed detect all available paramyxoviruses tested. Clinical specimens from hospitalized patients that tested positive for known paramyxoviruses in conventional assays were also detected with the novel family-wide test. A high-throughput fluorescence-based RT-PCR version of the assay was developed for screening large numbers of specimens. A large number of samples collected from wild birds was tested, resulting in the detection of avian paramyxoviruses type 1 in both barnacle and white-fronted geese, and type 8 in barnacle geese. Avian metapneumovirus type C was found for the first time in Europe in mallards, greylag geese and common gulls. The single round family-wide RT-PCR assay described here is a useful tool for the detection of known and unknown paramyxoviruses, and screening of large sample collections from humans and animals.

  20. Development of a real-time RT-PCR and Reverse Line probe Hybridisation assay for the routine detection and genotyping of Noroviruses in Ireland.

    LENUS (Irish Health Repository)

    Menton, John F

    2007-01-01

    BACKGROUND: Noroviruses are the most common cause of non-bacterial gastroenteritis. Improved detection methods have seen a large increase in the number of human NoV genotypes in the last ten years. The objective of this study was to develop a fast method to detect, quantify and genotype positive NoV samples from Irish hospitals. RESULTS: A real-time RT-PCR assay and a Reverse Line Blot Hybridisation assay were developed based on the ORF1-ORF2 region. The sensitivity and reactivity of the two assays used was validated using a reference stool panel containing 14 NoV genotypes. The assays were then used to investigate two outbreaks of gastroenteritis in two Irish hospitals. 56 samples were screened for NoV using a real-time RT-PCR assay and 26 samples were found to be positive. Genotyping of these positive samples found that all positives belonged to the GII\\/4 variant of NoV. CONCLUSION: The combination of the Real-time assay and the reverse line blot hybridisation assay provided a fast and accurate method to investigate a NoV associated outbreak. It was concluded that the predominant genotype circulating in these Irish hospitals was GII\\/4 which has been associated with the majority of NoV outbreaks worldwide. The assays developed in this study are useful tools for investigating NoV infection.

  1. Design and development of an in-house multiplex RT-PCR assay for simultaneous detection of HIV-1 and HCV in plasma samples

    Directory of Open Access Journals (Sweden)

    M Paryan

    2012-03-01

    Full Text Available Background and Objectives: HIV-1 and HCV infections are life threatening problems in patients who receive blood products. Serological methods have proven useful in detecting these infections, but there are setbacks that make it challenging to detect these infectious agents. By the advent of Nucleic Acid Testing (NAT methods, especially in multiplex format, more precise detection is possible.Materials and Methods: We have developed a multiplex RT-PCR assay for simultaneous detection of HIV-1 and HCV. Primers were designed for highly conserved region of genome of each virus. Using these primers and standard plasmids, we determined the limit of detection, clinical and analytical specificity and sensitivity of the assay. Monoplex and multiplex RT-PCR were performed.Results: Analytical sensitivity was considered to be 100 and 200 copies/ml for HIV-1 and HCV, respectively. High concentration of one virus had no significant effect on the detection of the other one with low concentration. By analysis of 40 samples, clinical sensitivity of the assay was determined to be 97.5%. Using different viral and human genome samples, the specificity of the assay was evaluated to be 100%.Conclusions: The aim of this study was to develop a reliable, rapid and cost effective method to detect HIV-1 and HCV simultaneously. Results showed that this simple and rapid method is perfectly capable of detecting two viruses in clinical samples.

  2. Simultaneous Detection of CDC Category "A" DNA and RNA Bioterrorism Agents by Use of Multiplex PCR & RT-PCR Enzyme Hybridization Assays

    Directory of Open Access Journals (Sweden)

    Kelly J. Henrickson

    2009-10-01

    Full Text Available Assays to simultaneously detect multiple potential agents of bioterrorism are limited. Two multiplex PCR and RT-PCR enzyme hybridization assays (mPCR-EHA, mRT-PCR-EHA were developed to simultaneously detect many of the CDC category “A” bioterrorism agents. The “Bio T” DNA assay was developed to detect: Variola major (VM, Bacillus anthracis (BA, Yersinia pestis (YP, Francisella tularensis (FT and Varicella zoster virus (VZV. The “Bio T” RNA assay (mRT-PCR-EHA was developed to detect: Ebola virus (Ebola, Lassa fever virus (Lassa, Rift Valley fever (RVF, Hantavirus Sin Nombre species (HSN and dengue virus (serotypes 1-4. Sensitivity and specificity of the 2 assays were tested by using genomic DNA, recombinant plasmid positive controls, RNA transcripts controls, surrogate (spiked clinical samples and common respiratory pathogens. The analytical sensitivity (limit of detection (LOD of the DNA asssay for genomic DNA was 1×100~1×102 copies/mL for BA, FT and YP. The LOD for VZV whole organism was 1×10-2 TCID50/mL. The LOD for recombinant controls ranged from 1×102~1×103copies/mL for BA, FT, YP and VM. The RNA assay demonstrated LOD for RNA transcript controls of 1×104~1×106 copies/mL without extraction and 1×105~1×106 copies/mL with extraction for Ebola, RVF, Lassa and HSN. The LOD for dengue whole organisms was ~1×10-4 dilution for dengue 1 and 2, 1×104 LD50/mL and 1×102 LD50/mL for dengue 3 and 4. The LOD without extraction for recombinant plasmid DNA controls was ~1×103 copies/mL (1.5 input copies/reaction for Ebola, RVF, Lassa and HSN. No cross-reactivity of primers and probes used in both assays was detected with common respiratory pathogens or between targeted analytes. Clinical sensitivity was estimated using 264 surrogate clinical samples tested with the BioT DNA assay and 549 samples tested with the BioT RNA assay. The clinical specificity is 99.6% and 99.8% for BioT DNA assay and BioT RNA assay, respectively. The

  3. Production of mono- and polyclonal antibodies to Citrus leprosis virus C2 and their application in triple antibody sandwich ELISA and immunocapture RT-PCR diagnostic assays.

    Science.gov (United States)

    Choudhary, Nandlal; Roy, Avijit; Leon, M G; Wei, G; Nakhla, M K; Levy, L; Brlansky, R H

    2017-05-01

    The newly discovered Citrus leprosis virus cytoplasmic type 2 (CiLV-C2) is one of the causal virus of citrus leprosis disease complex; which leads to substantial loss of citrus production in the states of Meta and Casanare of Colombia. Specific and sensitive detection methods are needed to monitor the dissemination of CiLV-C2 in Colombia, and to prevent introduction of CiLV-C2 to other citrus growing countries. Toward this end, putative coat protein gene (CPG) of CiLV-C2 was amplified from CiLV-C2 infected citrus tissues. The CPG was cloned, expressed and purified a recombinant coat protein of ∼31kDa which used to generate monoclonal antibodies and polyclonal antisera. Four monoclonal antibodies and two polyclonal antisera were selected as being specific following Western blotting. The monoclonal antibody MAb E5 and polyclonal antiserum PAb UF715 were selected testing with an extract of CiLV-C2 infected leaves using triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). In addition, an immunocapture RT-PCR was standardized using MAb E5 for specific and sensitive detection of CiLV-C2. The standardized TAS-ELISA and IC-RT-PCR were able to detect CiLV-C2 in the extracts of symptomatic citrus leprosis tissues up to the dilutions of 1:160 and 1:2580, respectively. Result demonstrated that CiLV-C2 is present in citrus orchards in Meta and Casanare citrus growing areas of Colombia. TAS-ELISA could be used for routine detection of CiLV-C2, epidemiological studies, and for border inspections for quarantine purposes. IC-RT-PCR could be valuable for CiLV-C2 validation and viral genome analysis.

  4. 一步法RT-PCR检测血清混合样品在BVDV清除计划中的应用%The Application of the One-Step RT-PCR Method to Detect the Serum Mixed Samples in BVDV Eradication Programme

    Institute of Scientific and Technical Information of China (English)

    黄凯; 邵晓磊; 马翀

    2011-01-01

    Total 58 of BVDV (Bovine Viral diarrhea virus) antigen positive sera were detected by one-step RT-PCR method. The results revealed that the minimum detection limit was 5μL/per sample, and the standard sera value of OD was 0.3. In addition, the mathematical deduction proved that the test cost of BVDV eradication programme was the lowest, when the number of the serum mixed samples were 10 to 30. Compared with only using ELISA-Ag, the testing cost of RT-PCR and ELISA-Ag conjunctive use reduced 70%. In one words, the one-step RT-PCR which was used in our study has high sensitivity and specificity. When taking ELISA-Ag conjunotive use in BVDV eradication programme, the test cost could be reduced greatly, so this method was worth to use.%利用一步法RT-PCR对58份BVDV抗原阳性血清进行检测.试验证明,对OD450=0.3的标准血清,该方法的最低检出限为5μL/头,血清混合检测时至少可将60份OD450>0.3的阳性血清等量混合.此外,RT-PCR与ELISA-Ag联合使用时,数学推导证明当混合样品为10~30个/份时,BVDV清除计划检测成本最低,相比单独使用ELISA-Ag,成本至少下降70%.综上,本试验确立的一步法RT-PCR灵敏度高,特异性好,联合ELISA-Ag使用,可大幅降低BVDV清除计划的检测成本,故值得推广使用.

  5. Comparison of two real-time RT-PCR assays for differentiation of C-strain vaccinated from classical swine fever infected pigs and wild boars.

    Science.gov (United States)

    Widén, F; Everett, H; Blome, S; Fernandez Pinero, J; Uttenthal, A; Cortey, M; von Rosen, T; Tignon, M; Liu, L

    2014-10-01

    Classical swine fever is one of the most important infectious diseases for the pig industry worldwide due to its economic impact. Vaccination is an effective means to control disease, however within the EU its regular use is banned owing to the inability to differentiate infected and vaccinated animals, the so called DIVA principle. This inability complicates monitoring of disease and stops international trade thereby limiting use of the vaccine in many regions. The C-strain vaccine is safe to use and gives good protection. It is licensed for emergency vaccination in the EU in event of an outbreak. Two genetic assays that can distinguish between wild type virus and C-strain vaccines have recently been developed. Here the results from a comparison of these two real-time RT-PCR assays in an interlaboratory exercise are presented. Both assays showed similar performance.

  6. Development of a Quantitative Real-Time RT-PCR Assay for the Detection of MAGE-A3-Positive Tumors.

    Science.gov (United States)

    Gruselle, Olivier; Coche, Thierry; Louahed, Jamila

    2015-07-01

    Melanoma antigen A3 (MAGE-A3) is a member of the MAGE family of tumor antigens and a relevant candidate for use in cancer immunotherapy. However, not all tumors express MAGE-A3, and closely related members of the MAGE family can be co-expressed with MAGE-A3 in the same tumor. Therefore, in the frame of MAGE-A3 clinical trials, it appeared necessary to evaluate tumors for MAGE-A3 expression with a highly specific quantitative assay to select patients who are eligible for anti-MAGE-A3 immunotherapy treatment. Herein, we describe the development and validation of a quantitative real-time RT-PCR (RT-qPCR) assay for the determination of MAGEA3 gene expression in tumor tissues. In the early phases of development, the designed primers and probe were not able to distinguish between MAGE-A3 and MAGE-A6. To ensure the specificity for MAGE-A3 over MAGE-A6, our strategy was to use a 5'-nuclease probe (or hydrolysis probe). The final assay was shown to be specific and linear within the analytical range, with an acceptable CV for repeatability and intermediate precision. When compared with a reference semiquantitative RT-PCR assay, the two methods were in good agreement, with only 4.23% of the samples giving discordant results. In conclusion, we have developed a MAGE-A3-specific RT-qPCR assay, compatible with a high-throughput setting for the estimation of MAGEA3 gene expression in present and future clinical trials.

  7. Development and validation of a multiplex, real-time RT PCR assay for the simultaneous detection of classical and African swine fever viruses.

    Science.gov (United States)

    Haines, Felicity J; Hofmann, Martin A; King, Donald P; Drew, Trevor W; Crooke, Helen R

    2013-01-01

    A single-step, multiplex, real-time polymerase chain reaction (RT-PCR) was developed for the simultaneous and differential laboratory diagnosis of Classical swine fever virus (CSFV) and African swine fever virus (ASFV) alongside an exogenous internal control RNA (IC-RNA). Combining a single extraction methodology and primer and probe sets for detection of the three target nucleic acids CSFV, ASFV and IC-RNA, had no effect on the analytical sensitivity of the assay and the new triplex RT-PCR was comparable to standard PCR techniques for CSFV and ASFV diagnosis. After optimisation the assay had a detection limit of 5 CSFV genome copies and 22 ASFV genome copies. Analytical specificity of the triplex assay was validated using a panel of viruses representing 9 of the 11 CSFV subgenotypes, at least 8 of the 22 ASFV genotypes as well as non-CSFV pestiviruses. Positive and negative clinical samples from animals infected experimentally, due to field exposure or collected from the UK which is free from both swine diseases, were used to evaluate the diagnostic sensitivity and specificity for detection of both viruses. The diagnostic sensitivity was 100% for both viruses whilst diagnostic specificity estimates were 100% for CSFV detection and 97.3% for ASFV detection. The inclusion of a heterologous internal control allowed identification of false negative results, which occurred at a higher level than expected. The triplex assay described here offers a valuable new tool for the differential detection of the causative viruses of two clinically indistinguishable porcine diseases, whose geographical occurrence is increasingly overlapping.

  8. Development and validation of a multiplex, real-time RT PCR assay for the simultaneous detection of classical and African swine fever viruses.

    Directory of Open Access Journals (Sweden)

    Felicity J Haines

    Full Text Available A single-step, multiplex, real-time polymerase chain reaction (RT-PCR was developed for the simultaneous and differential laboratory diagnosis of Classical swine fever virus (CSFV and African swine fever virus (ASFV alongside an exogenous internal control RNA (IC-RNA. Combining a single extraction methodology and primer and probe sets for detection of the three target nucleic acids CSFV, ASFV and IC-RNA, had no effect on the analytical sensitivity of the assay and the new triplex RT-PCR was comparable to standard PCR techniques for CSFV and ASFV diagnosis. After optimisation the assay had a detection limit of 5 CSFV genome copies and 22 ASFV genome copies. Analytical specificity of the triplex assay was validated using a panel of viruses representing 9 of the 11 CSFV subgenotypes, at least 8 of the 22 ASFV genotypes as well as non-CSFV pestiviruses. Positive and negative clinical samples from animals infected experimentally, due to field exposure or collected from the UK which is free from both swine diseases, were used to evaluate the diagnostic sensitivity and specificity for detection of both viruses. The diagnostic sensitivity was 100% for both viruses whilst diagnostic specificity estimates were 100% for CSFV detection and 97.3% for ASFV detection. The inclusion of a heterologous internal control allowed identification of false negative results, which occurred at a higher level than expected. The triplex assay described here offers a valuable new tool for the differential detection of the causative viruses of two clinically indistinguishable porcine diseases, whose geographical occurrence is increasingly overlapping.

  9. Serotype specific primers and gel-based RT-PCR assays for 'typing' African horse sickness virus: identification of strains from Africa.

    Directory of Open Access Journals (Sweden)

    Narender S Maan

    Full Text Available African horse sickness is a devastating, transboundary animal disease, that is 'listed' by the Office International des Epizooties (OIE. Although attenuated, inactivated and subunit vaccines have been developed for African horse sickness virus (AHSV, these are serotype-specific and their effective deployment therefore relies on rapid and reliable identification of virus type. AHSV serotype is controlled by the specificity of interactions between neutralising antibodies, and components of the outer-capsid, particularly protein VP2 (encoded by AHSV genome segment 2 (Seg-2. We report the development and evaluation of novel gel based reverse transcription-PCR (RT-PCR assays targeting AHSV Seg-2, which can be used to very significantly increase the speed and reliability of detection and identification (compared to virus neutralisation tests of the nine serotypes of AHSV. Primer sets were designed targeting regions of Seg-2 that are conserved between strains within each of the AHSV serotype (types 1 to 9. These assays were evaluated using multiple AHSV strains from the orbivirus reference collection at IAH (www.reoviridae.org/dsRNA_virus_proteins/ReoID/AHSV-isolates.htm. In each case the Seg-2 primers showed a high level of specificity and failed to cross-amplify the most closely related heterologous AHSV types, or other related orbiviruses (such as bluetongue virus (BTV, or equine encephalosis virus (EEV. The assays are rapid and sensitive, and can be used to detect and type viral RNA in blood, tissue samples, or cultivated viral suspensions within 24 h. They were used to identify AHSV strains from recent outbreaks in sub-Saharan African countries. These methods also generate cDNAs suitable for sequencing and phylogenetic analyses of Seg-2, identifying distinct virus lineages within each virus-type and helping to identify strain movements/origins. The RT-PCR methods described here provide a robust and versatile tool for rapid and specific detection

  10. Perioperative cancer cell dissemination detected with a real-time RT-PCR assay for EpCAM is not associated with worse prognosis in pancreatic ductal adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Houtmeyers François

    2011-01-01

    Full Text Available Abstract Background Epithelial cell adhesion molecule (EpCAM has been used as surrogate marker for the quantification of circulating tumour cells (CTC. Our aim was to prospectively study the value of a real-time RT-PCR assay for EpCAM detection in the peripheral blood and peritoneal cavity of patients undergoing pancreatectomy for pancreatic ductal adenocarcinoma (PDAC. Methods From 48 patients with PDAC (40 resectable, 8 unresectable and 10 patients with chronic pancreatitis undergoing pancreatectomy 10 ml of venous blood was drawn preoperatively (PB and postoperatively (POB, day 1 (D1B, day 7 (D7B and after 6 weeks (6WB. Of all patients undergoing pancreatectomy, 40 ml peritoneal lavage fluid was taken preoperatively and postoperatively. A real-time RT-PCR assay (TaqMan, ABI Prism 7700 was developed for the detection of EpCAM mRNA. To discriminate between EpCAM-positive and negative samples a cut-off was applied. Median postoperative follow-up was 24.0 months (range: 0.7 - 41.3. Results PB was EpCAM-positive (+ in 25% of patients versus 65% of patients in POB (p At none of the time-points, an association was found between EpCAM positivity in blood and/or peritoneal cavity and cancer-specific or disease-free survival. Also, no significant associations were found between clinicopathological variables and perioperative EpCAM positivity. Conclusions Despite a significant increase in EpCAM counts in postoperative blood and peritoneal lavage fluid this was not associated with worse prognosis after pancreatectomy for PDAC. Trial registration Clinicaltrials.gov NCT00495924

  11. Field Evaluation of a Deployable RT-PCR Assay System for Real-Time Identification of Dengue Virus

    Science.gov (United States)

    2004-06-01

    strains of dengue serotypes 1-4, yellow fever, Japanese encephalitis, West Nile, and St. Louis encephalitis viruses as well as dengue virus infected...JA, Pyke A, Smith GA. Single rapid TaqMan fluorogenic probe based PCR assay that detects all four dengue serotypes . J Med Virol. 2002 April; 66(4

  12. Development and Characterization of A Multiplexed RT-PCR Species Specific Assay for Bovine and one for Porcine Foot-and-Mouth Disease Virus Rule-Out

    Energy Technology Data Exchange (ETDEWEB)

    Smith, S M; Danganan, L; Tammero, L; Vitalis, B; Lenhoff, R; Naraghi-arani, P; Hindson, B

    2007-08-06

    Lawrence Livermore National Laboratory (LLNL), in collaboration with the Department of Homeland Security (DHS) and the United States Department of Agriculture (USDA), Animal and Plant Health Inspection Services (APHIS) has developed candidate multiplexed assays that may potentially be used within the National Animal Health Laboratory Network (NAHLN), the National Veterinary Services Laboratory (Ames, Iowa) and the Plum Island Animal Disease Center (PIADC). This effort has the ability to improve our nation's capability to discriminate between foreign animal diseases and those that are endemic using a single assay, thereby increasing our ability to protect food and agricultural resources with a diagnostic test which could enhance the nation's capabilities for early detection of a foreign animal disease. In FY2005 with funding from the DHS, LLNL developed the first version (Version 1.0) of a multiplexed (MUX) nucleic-acid-based RT-PCR assay that included signatures for foot-and-mouth disease virus (FMDV) detection with rule-out tests for two other foreign animal diseases (FADs) of swine, Vesicular Exanthema of Swine (VESV) and Swine Vesicular Disease Virus (SVDV), and four other domestic viral diseases Bovine Viral Diarrhea Virus (BVDV), Bovine Herpes Virus 1 (BHV-1), Bluetongue virus (BTV) and Parapox virus complex (which includes Bovine Papular Stomatitis Virus [BPSV], Orf of sheep, and Pseudocowpox). In FY06, LLNL has developed Bovine and Porcine species-specific panel which included existing signatures from Version 1.0 panel as well as new signatures. The MUX RT-PCR porcine assay for detection of FMDV includes the FADs, VESV and SVD in addition to vesicular stomatitis virus (VSV) and porcine reproductive and respiratory syndrome (PRRS). LLNL has also developed a MUX RT-PCR bovine assay for detection of FMDV with rule out tests for the two bovine FADs malignant catarrhal fever (MCF), rinderpest virus (RPV) and the domestic diseases vesicular stomatitis

  13. Development of novel AllGlo-probe-based one-step multiplex qRT-PCR assay for rapid identification of avian influenza virus H7N9.

    NARCIS (Netherlands)

    Zhang, Y.; Mao, H.; Yan, J.; Wang, X.; Zhang, L.; Koch, G.; Li, H.; Li, Z.; Chen, Y.; Gong, L.; Chen, Z.; Xia, S.

    2014-01-01

    Recently, human deaths have resulted from infection with low-pathogenicity avian influenza virus H7N9 strains that have emerged recently in China. To strengthen H7N9 surveillance and outbreak control, rapid and reliable diagnostic methods are needed. To develop a sensitive quantitative real-time RT-

  14. Detection of circulating tumor cell-specific markers in breast cancer patients using the quantitative RT-PCR assay.

    Science.gov (United States)

    Wang, Hye-Young; Ahn, Sungwoo; Kim, Sunghyun; Park, Sunyoung; Jung, Dongju; Park, Sangjung; Han, Hyunju; Sohn, JooHyuk; Kim, SeungIl; Lee, Hyeyoung

    2015-10-01

    Breast cancer is a highly prevalent disease among women worldwide. While the expression of certain proteins within breast cancer tumors is used to determine the prognosis and select therapies, additional markers need to be identified. Circulating tumor cells (CTCs) are constituent cells that have detached from a primary tumor to circulate in the bloodstream. CTCs are considered the main source of breast cancer metastases; therefore, detection of CTCs could be a promising diagnostic method for metastatic breast cancer. In this study, the CircleGen CTC RT-qDx assay was used to analyze the mRNA expression levels of six CTC-specific markers including EpCAM, CK19, HER2, Ki67, hTERT, and vimentin with a total of 692 peripheral whole blood samples from 221 breast cancer patients and 376 healthy individuals. This assay showed high specificity with multiple markers; none of the healthy controls were detected positive, whereas 21.7 and 14 % of breast cancer patients were positive for EpCAM and CK19, respectively. Of the 221 breast cancer patients, 84 (38 %), 46 (20.8 %), 83 (37.6 %), and 39 (17.6 %) were positively for HER2, Ki67, hTERT, and vimentin mRNA, respectively. Of the 84 patients who were HER2 positive, nine (4 %) were also positive for EpCAM, CK19, Ki67, hTERT, and vimentin. Of the 139 breast cancer patients who were HER2 negative, 65 (29.1 %) were negative for EpCAM, CK19, Ki67, hTERT, and vimentin. Furthermore, the EpCAM-positive population decreased from 21.5 to 8.3 % after completion of anti-tumor treatment (TP4). Similarly, the CK19, HER2, hTERT, and vimentin positives also decreased from 13.9 to 9.5 %, from 37.7 to 21.4 %, from 37.2 to 33.3 %, and from 17.5 to 14.3 %, respectively, after completion of anti-tumor treatment. In contrast, the Ki67 positives increased from 20.6 to 41.7 % after completion of anti-tumor treatment. mRNA overexpression of six CTC-specific markers was detected by the CircleGen CTC RT-qDx assay with high specificity, and the obtained m

  15. Real-time RT-PCR assay to differentiate clades of H5N1 avian influenza viruses circulating in Vietnam.

    Science.gov (United States)

    Kis, Z; Jones, J; Creanga, A; Ferdinand, K; Inui, K; Gerloff, N; Davis, C T; Nguyen, T; Donis, R O

    2013-11-01

    Continued circulation and geographical expansion of highly pathogenic avian influenza H5N1 virus have led to the emergence of numerous clades in Vietnam. Although viral RNA sequencing and phylogenetic analysis are the gold standard for H5N1 HA clade designation, limited sequencing capacity in many laboratories precludes rapid H5N1 clade identification and detection of novel viruses. Therefore, a Taqman real-time RT-PCR assay for rapid differentiation of the four major H5N1 clades detected in Vietnam was developed. Using HA sequence alignments of clades 1.1, 2.3.2.1, 2.3.4, and 7 viruses, primers and FAM-labeled probes were designed to target conserved regions characteristic of each clade. The assay was optimized and evaluated using circulating clades of H5N1 collected in Vietnam from 2007 to 2012 and shown to be both sensitive and specific for the differentiation of the four H5N1 clades. The assay provides a useful tool for screening of large specimen collections for HA gene sequencing and phylogenetic analysis and for the rapid identification of molecular clade signatures to support outbreak investigations and surveillance activities. Finally, this assay may be useful to monitor for the emergence of novel or variant clades of H5N1 in Vietnam in the future or in other countries where these particular clades may circulate.

  16. Clinical Evaluation of a Single-Tube Multiple RT-PCR Assay for the Detection of 13 Common Virus Types/Subtypes Associated with Acute Respiratory Infection.

    Directory of Open Access Journals (Sweden)

    Dan Zhang

    Full Text Available Respiratory viruses are among the most important causes of human morbidity and mortality worldwide, especially for infants and young children. In the past years, a few commercial multiplex RT-PCR assays have been used to detect respiratory viruses in spite of the high cost. In the present study, an improved single-tube multiplex reverse transcription PCR assay for simultaneous detection of 13 respiratory viruses was evaluated and compared with a previously reported two-tube assay as the reference method using clinical nasopharyngeal aspirates samples. Of 310 prospectively tested respiratory specimens selected from children hospitalized with acute respiratory illness, 226 (72.90%, 226/310 and 214 (69.03%, 214/310 positive for one or more viruses were identified by the single-tube and the two-tube assays, respectively, with combined test results showing good concordance (Kappa value = 0.874. Individually, the single-tube assay for adenovirus (Adv, human metapneumovirus (HMPV, human rhinovirus (HRV, parainfluenza virus type 1 (PIV1, parainfluenza virus type 3 (PIV3 and parainfluenza virus type 4 (PIV4 showed the significantly superior sensitivities to those of the two-tube assay. No false positives were found. In conclusion, our results demonstrates the one-tube assay revealed significant improvements over the two-tube assay in terms of the better sensitivity, more accurate quality control, less nonspecific amplification, more cost-effective and shorter turn-around time and will be a valuable tool for routine surveillance of respiratory virus infection in China.

  17. In-house HIV-1 RNA real-time RT-PCR assays: principle, available tests and usefulness in developing countries.

    Science.gov (United States)

    Rouet, François; Ménan, Hervé; Viljoen, Johannes; Ngo-Giang-Huong, Nicole; Mandaliya, Kishor; Valéa, Diane; Lien, Truong Xuan; Danaviah, Sivapragashini; Rousset, Dominique; Ganon, Amandine; Nerrienet, Eric

    2008-09-01

    The principle of currently available licensed HIV-1 RNA assays is based on real-time technologies that continuously monitor the fluorescence emitted by the amplification products. Besides these assays, in-house quantitative (q) real-time reverse transcription (RT)-PCR (RT-qPCR) tests have been developed and evaluated particularly in developing countries, for two main reasons. First, affordable and generalized access to HIV-1 RNA viral load is urgently needed in the context of expected universal access to prevention and antiretroviral treatment programs in these settings. Second, since many non-B subtypes, circulating recombinant forms and unique recombinant forms circulate in these areas, in-house HIV-1 RNA RT-qPCR assays are ideal academic tools to thoroughly evaluate the impact of HIV-1 genetic diversity on the accuracy of HIV-1 RNA quantification, as compared with licensed techniques. To date, at least 15 distinct in-house assays have been designed. They differ by their chemistry and the HIV-1 target sequence (located in gag, Pol-IN or LTR gene). Analytical performances of the tests that have been extensively evaluated appear at least as good as (or even better than) those of approved assays, with regard to HIV-1 strain diversity. Their clinical usefulness has been clearly demonstrated for early diagnosis of pediatric HIV-1 infection and monitoring of highly active antiretroviral therapy efficacy. The LTR-based HIV-1 RNA RT-qPCR assay has been evaluated by several groups under the auspices of the Agence Nationale de Recherches sur le SIDA et les hépatites virales B et C. It exists now as a complete standardized commercial test.

  18. Characterization of human coronavirus etiology in Chinese adults with acute upper respiratory tract infection by real-time RT-PCR assays.

    Directory of Open Access Journals (Sweden)

    Roujian Lu

    Full Text Available BACKGROUND: In addition to SARS associated coronaviruses, 4 non-SARS related human coronaviruses (HCoVs are recognized as common respiratory pathogens. The etiology and clinical impact of HCoVs in Chinese adults with acute upper respiratory tract infection (URTI needs to be characterized systematically by molecular detection with excellent sensitivity. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we detected 4 non-SARS related HCoV species by real-time RT-PCR in 981 nasopharyngeal swabs collected from March 2009 to February 2011. All specimens were also tested for the presence of other common respiratory viruses and newly identified viruses, human metapneumovirus (hMPV and human bocavirus (HBoV. 157 of the 981 (16.0% nasopharyngeal swabs were positive for HCoVs. The species detected were 229E (96 cases, 9.8%, OC43 (42 cases, 4.3%, HKU1 (16 cases, 1.6% and NL63 (11 cases, 1.1%. HCoV-229E was circulated in 21 of the 24 months of surveillance. The detection rates for both OC43 and NL63 were showed significantly year-to-year variation between 2009/10 and 2010/11, respectively (P<0.001 and P = 0.003, and there was a higher detection frequency of HKU1 in patients aged over 60 years (P = 0.03. 48 of 157(30.57% HCoV positive patients were co-infected. Undifferentiated human rhinoviruses and influenza (Flu A were the most common viruses detected (more than 35% in HCoV co-infections. Respiratory syncytial virus (RSV, human parainfluenza virus (PIV and HBoV were detected in very low rate (less than 1% among adult patients with URTI. CONCLUSIONS/SIGNIFICANCE: All 4 non-SARS-associated HCoVs were more frequently detected by real-time RT-PCR assay in adults with URTI in Beijing and HCoV-229E led to the most prevalent infection. Our study also suggested that all non-SARS-associated HCoVs contribute significantly to URTI in adult patients in China.

  19. Incidence in diverse pig populations of an IGF2 mutation with potential influence on meat quality and quantity: An assay based on real time PCR (RT-PCR).

    Science.gov (United States)

    Carrodeguas, José Alberto; Burgos, Carmen; Moreno, Carlos; Sánchez, Ana Cristina; Ventanas, Sonia; Tarrafeta, Luis; Barcelona, José Antonio; López, Maria Otilia; Oria, Rosa; López-Buesa, Pascual

    2005-11-01

    IGF2, insulin-like growth factor 2, is implicated in myogenesis and lean meat content. A mutation in a single base (A for G substitution) of the gene for IGF2 (position 3072 in intron 3) has been recently described as the cause of a major QTL effect on muscle growth in pigs [Van Laere, A. S, Nguyen, M., Braunschweig, M., Nezer, C., Collete, C., & Moreau, L. et al. (2003). Nature, 425, 832-836]. We describe here a rapid assay based on real time PCR (RT-PCR) to detect this mutation. We have evaluated the incidence of the mutation in commercial pig crosses, in three populations of purebred Iberian or Iberian×Duroc crosses, and in cured meat products and wild boars. The incidence of the mutation varies among these groups. Penetrance of the A mutation is about 80% in the commercial population. Purebred Iberian pigs were all homozygous G/G whereas crosses of Iberian pigs were heterozygous (90%) or homozygous A/A (10%). The implications of this gene for the selection of Iberian pigs are discussed.

  20. Establishment of Real-Time TaqMan-Fluorescence Quantitative RT-PCR Assay for Detection and Quantification of Porcine Lipoprotein Lipase mRNA

    Institute of Scientific and Technical Information of China (English)

    LIAN Hong-xia; LU De-xun; GAO Min

    2009-01-01

    Porcine lipoprotein lipase (LPL) cDNA was cloned as the standard for real-time quantifying LPL mRNA and the TaqMan-fluorescence quantitative PCR assay for detection was established. The total RNA extracted from Longissimus dorsi of porcine was reverse-transcribed to cDNA. LPL cDNA was ligated with pGM-T vector and transformed into Escherichia coli TOP 10. Plasmid DNA extracted from positive clones was verified by PCR amplification and sequenced. LPL was amplified by real-time fluorescence quantitative PCR from the plasmid DNA. The concentration of DNA template purified was detected by analyzing absorbance in 260 nm and then the combined plasmid was diluted to series as standard for fluorescence quantitative PCR (FQ-PCR). The method of LPL mRNA real-time PCR was well established, which detected as low as 103 with the linear range 103 to 1010 copies. The standard curves showed high correlations (R2=0.9871). A series of standards for real-time PCR analysis have been constructed successfully, and real-time TaqMan-fluorescence quantitative RT-PCR is reliable to quantitatively evaluate FQ-PCR mRNA in L. dorsi of porcine.

  1. A novel pancoronavirus RT-PCR assay: frequent detection of human coronavirus NL63 in children hospitalized with respiratory tract infections in Belgium

    Directory of Open Access Journals (Sweden)

    Berkhout Ben

    2005-02-01

    Full Text Available Abstract Background Four human coronaviruses are currently known to infect the respiratory tract: human coronaviruses OC43 (HCoV-OC43 and 229E (HCoV-229E, SARS associated coronavirus (SARS-CoV and the recently identified human coronavirus NL63 (HCoV-NL63. In this study we explored the incidence of HCoV-NL63 infection in children diagnosed with respiratory tract infections in Belgium. Methods Samples from children hospitalized with respiratory diseases during the winter seasons of 2003 and 2004 were evaluated for the presence of HCoV-NL63 using a optimized pancoronavirus RT-PCR assay. Results Seven HCoV-NL63 positive samples were identified, six were collected during January/February 2003 and one at the end of February 2004. Conclusions Our results support the notation that HCoV-NL63 can cause serious respiratory symptoms in children. Sequence analysis of the S gene showed that our isolates could be classified into two subtypes corresponding to the two prototype HCoV-NL63 sequences isolated in The Netherlands in 1988 and 2003, indicating that these two subtypes may currently be cocirculating.

  2. Development and Characterization of A Multiplexed RT-PCR Species Specific Assay for Bovine and one for Porcine Foot-and-Mouth Disease Virus Rule-Out

    Energy Technology Data Exchange (ETDEWEB)

    Smith, S M; Danganan, L; Tammero, L; Vitalis, B; Lenhoff, R; Naraghi-arani, P; Hindson, B

    2007-08-06

    Lawrence Livermore National Laboratory (LLNL), in collaboration with the Department of Homeland Security (DHS) and the United States Department of Agriculture (USDA), Animal and Plant Health Inspection Services (APHIS) has developed candidate multiplexed assays that may potentially be used within the National Animal Health Laboratory Network (NAHLN), the National Veterinary Services Laboratory (Ames, Iowa) and the Plum Island Animal Disease Center (PIADC). This effort has the ability to improve our nation's capability to discriminate between foreign animal diseases and those that are endemic using a single assay, thereby increasing our ability to protect food and agricultural resources with a diagnostic test which could enhance the nation's capabilities for early detection of a foreign animal disease. In FY2005 with funding from the DHS, LLNL developed the first version (Version 1.0) of a multiplexed (MUX) nucleic-acid-based RT-PCR assay that included signatures for foot-and-mouth disease virus (FMDV) detection with rule-out tests for two other foreign animal diseases (FADs) of swine, Vesicular Exanthema of Swine (VESV) and Swine Vesicular Disease Virus (SVDV), and four other domestic viral diseases Bovine Viral Diarrhea Virus (BVDV), Bovine Herpes Virus 1 (BHV-1), Bluetongue virus (BTV) and Parapox virus complex (which includes Bovine Papular Stomatitis Virus [BPSV], Orf of sheep, and Pseudocowpox). In FY06, LLNL has developed Bovine and Porcine species-specific panel which included existing signatures from Version 1.0 panel as well as new signatures. The MUX RT-PCR porcine assay for detection of FMDV includes the FADs, VESV and SVD in addition to vesicular stomatitis virus (VSV) and porcine reproductive and respiratory syndrome (PRRS). LLNL has also developed a MUX RT-PCR bovine assay for detection of FMDV with rule out tests for the two bovine FADs malignant catarrhal fever (MCF), rinderpest virus (RPV) and the domestic diseases vesicular stomatitis

  3. Design and feasibility of a novel, rapid, and simple fluorescence 26-plex rt-PCR assay for simultaneous detection of 24 fusion transcripts in adult acute myeloid leukemia.

    Science.gov (United States)

    Laforêt, Marie-Pierre; Turlure, Pascal; Lippert, Eric; Cornillet-Lefebvre, Pascale; Pigneux, Arnaud; Pradeau, Rachel; Feuillard, Jean; Gachard, Nathalie

    2013-03-01

    Identification of chromosomal abnormalities is mandatory for classification of acute myeloid leukemia (AML), and the abnormalities have to be determined quickly, to allow patient enrollment in multicenter protocols and/or for selecting therapeutic strategies. Rapid AML molecular diagnosis is often difficult to achieve, however, because it is based on numerous different RT-PCR protocols. We developed a new RT-PCR method, one that does not require a nested step, to simultaneously detect all AML fusion transcripts from six major recurrent translocations found in adults: t(15;17)(q22;q12), inv(16)(p13.1q22) [t(16;16)(p13.1;q22)], t(8;21)(q22;q22), t(6;9)(p23;q34), t(9;22)(q34;q11), and t(10;11)(p13;q14). Specific primers for RT-PCR detection of the 24 fusion transcripts, along with two transcripts for controls, were designed for this 26-plex RT-PCR. Each PCR product had a different size and was separated by capillary electrophoresis. We also designed a multiplex positive control with 24 chimeric RNAs, corresponding to all chimeric RNAs tested. Compared with classical molecular biology protocols and cytogenetic analyses used as reference standards, results of the 26-plex RT-PCR method were concordant in all 204 (100%) cases of adult AML tested. Results were obtained in less than 24 hours. Because of the multiplex positive control, interpretation of the peaks was very easy, without any ambiguity. The tumor cell detection threshold was 1.5%.

  4. The development of a real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay using TaqMan technology for the pan detection of bluetongue virus (BTV).

    Science.gov (United States)

    Mulholland, Catherine; McMenamy, Michael J; Hoffmann, Bernd; Earley, Bernadette; Markey, Bryan; Cassidy, Joseph; Allan, Gordon; Welsh, Michael D; McKillen, John

    2017-07-01

    Bluetongue virus (BTV) is an infectious, non-contagious viral disease of domestic and wild ruminants that is transmitted by adult females of certain Culicoides species. Since 2006, several serotypes including BTV-1, 2, 4, 6, 8, 9 and 16, have spread from the Mediterranean basin into Northern Europe for the first time. BTV-8 in particular, caused a major epidemic in northern Europe. As a result, it is evident that most European countries are at risk of BTV infection. The objective of this study was to develop and validate a real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assay based on TaqMan technology for the detection of representative strains of all BTV serotypes. Primers and probes were based on genome segment 10 of the virus, the NS3 gene. The assay was tested for sensitivity, and specificity. The analytical sensitivity of the rRT-PCR assay was 200 copies of RNA per reaction. The assay did not amplify the closely related orbivirus epizootic hemorrhagic disease virus (EHDV) but successfully detected all BTV reference strains including clinical samples from animals experimentally infected with BTV-8. This real time RT-PCR assay offers a sensitive, specific and rapid alternative assay for the pan detection of BTV that could be used as part of a panel of diagnostic assays for the detection of all serotypes of BTV. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  5. Analytical approach for selecting normalizing genes from a cDNA microarray platform to be used in q-RT-PCR assays: a cnidarian case study.

    Science.gov (United States)

    Rodriguez-Lanetty, Mauricio; Phillips, Wendy S; Dove, Sophie; Hoegh-Guldberg, Ove; Weis, Virginia M

    2008-04-24

    Research in gene function using Quantitative Reverse Transcription PCR (q-RT-PCR) and microarray approaches are emerging and just about to explode in the field of coral and cnidarian biology. These approaches are showing the great potential to significantly advance our understanding of how corals respond to abiotic and biotic stresses, and how host cnidarians/dinoflagellates symbioses are maintained and regulated. With these genomic advances, however, new analytical challenges are also emerging, such as the normalization of gene expression data derived from q-RT-PCR. In this study, an effective analytical method is introduced to identify candidate housekeeping genes (HKG) from a sea anemone (Anthopleura elegantissima) cDNA microarray platform that can be used as internal control genes to normalize q-RT-PCR gene expression data. It is shown that the identified HKGs were stable among the experimental conditions tested in this study. The three most stables genes identified, in term of gene expression, were beta-actin, ribosomal protein L12, and a Poly(a) binding protein. The applications of these HKGs in other cnidarian systems are further discussed.

  6. Screening and detection of human enterovirus 71 infection by a real-time RT-PCR assay in Marseille, France, 2009-2011

    OpenAIRE

    Tan, C. Y. Q.; Gonfrier, G.; Ninove, L.; Zandotti, C.; Dubot Pérès, Audrey; De Lamballerie, Xavier; Charrel, R.N.

    2012-01-01

    Enterovirus-positive samples diagnosed in Marseille (January 2009 to September 2011) were screened for EV71 by real-time RT-PCR. EV71 was detected in three children below the age of 2 years with no history of overseas travel; two of these cases were associated with severe clinical presentation. Viruses demonstrated genetic similarity to other European genogroup C2 strains. Strain MRS/09/3663 complete sequencing revealed 97.6% identity across the entire genome with a 2008 Singapore isolate, wi...

  7. Assessment of Preparation of Samples Under the Field Conditions and a Portable Real-Time RT-PCR Assay for the Rapid On-Site Detection of Newcastle Disease Virus.

    Science.gov (United States)

    Liu, L; Benyeda, Z; Zohari, S; Yacoub, A; Isaksson, M; Leijon, M; LeBlanc, N; Benyeda, J; Belák, S

    2016-04-01

    Newcastle disease virus (NDV), also known as virulent forms of avian paramyxovirus serotype 1 (AMPV-1), is the causative agent of Newcastle disease affecting many species of birds and causing heavy losses to the poultry industry worldwide. Early, rapid and sensitive detection of the viruses or the viral nucleic acids is very important for disease diagnosis and control. This study aimed to evaluate sample preparation under field conditions and the application of a real-time RT-PCR method in the portable T-COR4 platform for the rapid, on-site detection of NDV on a farm. In the laboratory setting, the portable real-time RT-PCR assay had a similar performance compared with that obtained with a larger, stationary Rotor Gene real-time thermocycler. In the field conditions, viral nucleic acids were manually extracted just outside of animal units with minimal equipment and real-time RT-PCR detection was performed with the portable thermocycler T-COR4 placed in a nearby room. The portable assay at the farm detected viral RNA in 15 samples and reached an agreement of 83% (39/47) when the same RNA preparations were tested in the Rotor Gene thermocycler under the laboratory setting. The results demonstrated the feasibility of performing field detection but also the need to improve and further simplify sample preparation procedures.

  8. Development of tailored real-time RT-PCR assays for the detection and differentiation of serotype O, A and Asia-1 foot-and-mouth disease virus lineages circulating in the Middle East

    DEFF Research Database (Denmark)

    Reid, Scott M.; Mioulet, Valerie; Knowles, Nick J.

    2014-01-01

    transcription polymerase chain reaction (rRT-PCR) assays using primer/probe sets designed from the VP1 coding region of the virus genomes were developed for the specific detection of serotype O, A and Asia-1 FMD viruses (FMDVs) circulating in the Middle East. These assays were evaluated using representative...... three primer/probe sets detected the RNA from homotypic viruses and no cross-reactivity was observed with heterotypic viruses. Similar results were obtained using both single- and multiplex assay formats. Using plasmid standards, the minimum detection level of these tests was found to be lower than two...

  9. Development of a One-Step Multiplex PCR Assay for Differential Detection of Major Mycobacterium Species.

    Science.gov (United States)

    Chae, Hansong; Han, Seung Jung; Kim, Su-Young; Ki, Chang-Seok; Huh, Hee Jae; Yong, Dongeun; Koh, Won-Jung; Shin, Sung Jae

    2017-09-01

    The prevalence of tuberculosis continues to be high, and nontuberculous mycobacterial (NTM) infection has also emerged worldwide. Moreover, differential and accurate identification of mycobacteria to the species or subspecies level is an unmet clinical need. Here, we developed a one-step multiplex PCR assay using whole-genome analysis and bioinformatics to identify novel molecular targets. The aims of this assay were to (i) discriminate between the Mycobacterium tuberculosis complex (MTBC) and NTM using rv0577 or RD750, (ii) differentiate M. tuberculosis (M. tuberculosis) from MTBC using RD9, (iii) selectively identify the widespread M. tuberculosis Beijing genotype by targeting mtbk_20680, and (iv) simultaneously detect five clinically important NTM (M. avium, M. intracellulare, M. abscessus, M. massiliense, and M. kansasii) by targeting IS1311, DT1, mass_3210, and mkan_rs12360 An initial evaluation of the multiplex PCR assay using reference strains demonstrated 100% specificity for the targeted Mycobacterium species. Analytical sensitivity ranged from 1 to 10 pg for extracted DNA and was 10(3) and 10(4) CFU for pure cultures and nonhomogenized artificial sputum cultures, respectively, of the targeted species. The accuracy of the multiplex PCR assay was further evaluated using 55 reference strains and 94 mycobacterial clinical isolates. Spoligotyping, multilocus sequence analysis, and a commercial real-time PCR assay were employed as standard assays to evaluate the multiplex PCR assay with clinical M. tuberculosis and NTM isolates. The PCR assay displayed 100% identification agreement with the standard assays. Our multiplex PCR assay is a simple, convenient, and reliable technique for differential identification of MTBC, M. tuberculosis, M. tuberculosis Beijing genotype, and major NTM species. Copyright © 2017 American Society for Microbiology.

  10. Performance of a commercial assay for the diagnosis of influenza A (H1N1 infection in comparison to the Centers for Disease Control and Prevention protocol of real time RT-PCR

    Directory of Open Access Journals (Sweden)

    María G Barbás

    2012-03-01

    Full Text Available At the time of influenza A (H1N1 emergency, the WHO responded with remarkable speed by releasing guidelines and a protocol for a real-time RT-PCR assay (rRT-PCR. The aim of the present study was to evalúate the performance of the "Real Time Ready Influenza A/H1N1 Detection Set" (June 2009-Roche kit in comparison to the CDC reference rRT-PCR protocol. The overall sensitivity of the Roche assay for detection of the Inf A gene in the presence or absence of the H1 gene was 74.5 %. The sensitivity for detecting samples that were only positive for the Inf A gene (absence of the H1 gene was 53.3 % whereas the sensitivity for H1N1-positive samples (presence of the Inf A gene and any other swine gene was 76.4 %. The specificity of the assay was 97.1 %. A new version of the kit (November 2009 is now available, and a recent evaluation of its performance showed good sensitivity to detect pandemic H1N1 compared to other molecular assays.Durante la pandemia de influenza A (H1N1, la OMS recomendó algoritmos y protocolos de detección del virus mediante RT-PCR en tiempo real. El objetivo del presente estudio fue evaluar el desempeño del equipo que comercializa la empresa Roche, Real Time Ready Influenza A/H1N1 Detection Set (junio de 2009, en comparación con el protocolo de RT-PCR en tiempo real de los CDC. La sensibilidad global del ensayo de Roche para la detección del gen Inf A en presencia o ausencia del gen H1 fue 74,5 %. La sensibilidad para la detección de muestras positivas solo para el gen Inf A (ausencia del gen H1 fue 53,3 % y la sensibilidad para la detección de muestras positivas para H1N1 (presencia del gen Inf A y cualquier otro gen porcino fue 76,4 %. La especificidad fue 97,1 %. Existe una nueva versión del equipo (noviembre 2009 que, según se ha descrito, presenta buena sensibilidad en comparación con otros ensayos moleculares para detectar H1N1 pandémica.

  11. Establishment of a novel one-step reverse transcription loop-mediated isothermal amplification assay for rapid identification of RNA from the severe fever with thrombocytopenia syndrome virus.

    Science.gov (United States)

    Xu, Haihong; Zhang, Lei; Shen, Guangqiang; Feng, Cen; Wang, Xinying; Yan, Jie; Zhang, Yanjun

    2013-12-01

    As an emerging infectious disease, severe fever with thrombocytopenia syndrome virus (SFTSV) infection has been found in many areas of China. Suitable laboratory diagnostic method is urgently needed in clinical detections and epidemiological investigations. In this study, a modified, low-cost and rapid visualized one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of RNA from the SFTSV has been established. In order to avoid the risk of aerosol contamination and facilitate the naked eye to observe, a microcrystalline wax-dye capsule wrapping the highly sensitive DNA fluorescence dye SYBR Green I was added to the RT-LAMP reaction tube before the initiation of the assay. The detection limit of the established RT-LAMP assay was 10 fg template RNA per reaction mixture. The RT-LAMP assay was confirmed to be high specific to SFTSV, and no cross-reaction was found with the detection of the Chikungunya fever virus, Hemorrhagic Fever with Renal Syndrome virus (HFRSV), and Dengue fever virus. The assay was then applied for the detection of SFTSV RNA in 32 clinical serum samples and showed 94.4% consistence with the detection results of the real-time RT-PCR. The whole process, from sample preparation to result reporting, can be completed within 2h. This adapted, cost efficient and quick visualized RT-LAMP method is feasible for SFTSV field diagnosis in resource-limited field settings.

  12. Comparison of real-time SYBR green dengue assay with real-time taqman RT-PCR dengue assay and the conventional nested PCR for diagnosis of primary and secondary dengue infection

    Directory of Open Access Journals (Sweden)

    Damodar Paudel

    2011-01-01

    Full Text Available Background : Dengue fever and dengue hemorrhagic fever are caused by dengue virus. Dengue infection remains a burning problem of many countries. To diagnose acute dengue in the early phase we improve the low cost, rapid SYBR green real time assay and compared the sensitivity and specificity with real time Taqman® assay and conventional nested PCR assay. Aims: To develop low cost, rapid and reliable real time SYBR green diagnostic dengue assay and compare with Taqman real-time assay and conventional nested PCR (modified Lanciotti. Materials and Methods: Eight cultured virus strains were diluted in tenth dilution down to undetectable level by the PCR to optimize the primer, temperature (annealing, and extension and to detect the limit of detection of the assay. Hundred and ninety three ELISA and PCR proved dengue clinical samples were tested with real time SYBR® Green assay, real time Taqman® assay to compare the sensitivity and specificity. Results: Sensitivity and specificity of real time SYBR® green dengue assay (84% and 66%, respectively was almost comparable to those (81% and 74% of Taqman real time PCR dengue assay. Real time SYBR® green RT-PCR was equally sensitive in primary and secondary infection while real time Taqman was less sensitive in the secondary infection. Sensitivity of real time Taqman on DENV3 (87% was equal to SYBR green real time PCR dengue assay. Conclusion: We developed low cost rapid diagnostic SYBR green dengue assay. Further study is needed to make duplex primer assay for the serotyping of dengue virus.

  13. Rapid one-step assays for on-site monitoring of mouse and rat urinary allergens.

    Science.gov (United States)

    Koets, Marjo; Renström, Anne; Zahradnik, Eva; Bogdanovic, Jelena; Wouters, Inge M; van Amerongen, Aart

    2011-12-01

    Allergy to rodent proteins is common among laboratory animal workers. Sensitive methods to measure exposure to these allergens have been developed. These assays are, however, expensive, time-consuming, and require a laboratory facility and methodological expertise. A simple method to screen for allergen spread, or to test whether hygiene standards are maintained, would be useful. Lateral flow immunoassays (LFIAs) are especially suited for field settings; the tests are simple and results are visible within minutes. LFIAs were developed for detection of the rodent urinary allergens Mus m 1 and Rat n 1. Pilot studies were performed in animal facilities in three countries using both extracts from airborne dust samples and samples collected by wiping surfaces. For comparison and determination of sensitivity, the concentrations of rodent urinary allergens in the samples were also measured using enzyme immunoassays (EIAs). The LFIAs for rat and mouse urinary allergens had a detection limit of 31 pg allergen per mL in a buffer system with purified allergen standards. Results of environmental dust extracts tested in LFIAs correlated well with levels obtained using EIAs. Spread of rodent allergens, or non-adherence to hygiene around laboratory animal facilities, may aggravate rodent allergy. Using a simple, sensitive one-step assay, allergens can be detected to prevent allergen exposure. The results reveal that the rapid assays are suited for on-site demonstration of exposure to rodent allergens, and thus, useful in occupational hygiene practice.

  14. Development and validation of a real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay for investigation of wild poliovirus type 1-South Asian (SOAS) strain reintroduced into Israel, 2013 to 2014.

    Science.gov (United States)

    Hindiyeh, M Y; Moran-Gilad, J; Manor, Y; Ram, D; Shulman, L M; Sofer, D; Mendelson, E

    2014-02-20

    In February 2013, wild poliovirus type 1 (WPV1) was reintroduced into southern Israel and resulted in continuous silent circulation in the highly immune population. As a part of the public health emergency response, a novel real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed, to allow for the sensitive and specific detection of the circulatingWPV1-South Asian (SOAS) strain. Specific primers and probes derived from the VP-1 region were designed, based on sequenced sewage isolates, and used to simultaneously amplify this WPV1-SOAS sequence together with bacteriophage MS-2 as internal control. High titre WPV1-SOAS stock virus was used for assay optimisation and 50 processed sewage samples collected from southern Israel and tested by reference culture based methods were used for analytical validation of the assay’s performance. The limit of detection of the multiplex qRT-PCR (SOAS/MS-2) assay was 0.1 plaque-forming unit (pfu)/reaction (20 pfu/mL) for WPV1-SOAS RNA with 100% sensitivity, specificity, positive and negative predictive values when compared to the culture based method. The turnaround time was rapid, providing results for environmental samples within 24 to 48 hours from completion of sewage processing, instead of five to seven days by culture-based analysis. Direct sewage testing by qRT-PCR assay proved to be a useful tool for rapid detection and environmental surveillance of WPV1-SOAS circulating strain during emergency response. Application of the approach for detection of WPV1-SOAS in stool samples obtained during acute flaccid paralysis (AFP) surveillance or field surveys should be further evaluated.

  15. Real-time quantitative RT-PCR assay for detection of Maedi-visna virus%梅迪-维斯纳病毒实时荧光定量PCR检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    张太翔; 肖越强; 杨慧; 凌宗帅; 孙涛; 袁涛; 徐彪; 梁成珠; 刘文鹏; 孙军; 王洪兵

    2011-01-01

    为建立检测梅迪-维斯纳病毒(MVV)的TagMan实时荧光定量PCR方法,本研究根据MVV核苷酸保守序列设计引物和探针.以梯度稀释的含有MVV目的扩增片段的重组质粒作为标准品,进行定量PCR反应.结果显示:5.0×105~5.0×101,拷贝范围内定量PCR均有"S"型扩增曲线,检测灵敏度为50拷贝.对羊的其他病毒核酸均无扩增反应.本研究建立的实时定量PCR方法,灵敏度高,特异性好,在NM的快速检测中具有良好的应用前景.%A real-time quantitative RT-PCR assay was developed for detection of Maedi-visna virus (MVV). Primers and probes were designed based on the sequence of MW by Primer Express 2.0. The recombinant plasmid containing the target sequence was constructed to detect the sensitivity and prepare the standard curve. The real-time RT-PCR assay had a detection limit of 50 copies, with a dynamic range of detection between 5 × 105 copies to 5 × 101 copies. The primers and probe were specific for MVV and did not react with other virus, including Capripox virus, pseudorabies virus and Foot-mouth disease virus.The real-time RT-PCR assay described here with high sensitivity, specificity and accuracy is considered to be a powerful tool for the rapid detection and quantification of MVV.

  16. Development and evaluation of a real-time one step Reverse-Transcriptase PCR for quantitation of Chandipura Virus

    Directory of Open Access Journals (Sweden)

    Tandale Babasaheb V

    2008-12-01

    Full Text Available Abstract Background Chandipura virus (CHPV, a member of family Rhabdoviridae was attributed to an explosive outbreak of acute encephalitis in children in Andhra Pradesh, India in 2003 and a small outbreak among tribal children from Gujarat, Western India in 2004. The case-fatality rate ranged from 55–75%. Considering the rapid progression of the disease and high mortality, a highly sensitive method for quantifying CHPV RNA by real-time one step reverse transcriptase PCR (real-time one step RT-PCR using TaqMan technology was developed for rapid diagnosis. Methods Primers and probe for P gene were designed and used to standardize real-time one step RT-PCR assay for CHPV RNA quantitation. Standard RNA was prepared by PCR amplification, TA cloning and run off transcription. The optimized real-time one step RT-PCR assay was compared with the diagnostic nested RT-PCR and different virus isolation systems [in vivo (mice in ovo (eggs, in vitro (Vero E6, PS, RD and Sand fly cell line] for the detection of CHPV. Sensitivity and specificity of real-time one step RT-PCR assay was evaluated with diagnostic nested RT-PCR, which is considered as a gold standard. Results Real-time one step RT-PCR was optimized using in vitro transcribed (IVT RNA. Standard curve showed linear relationship for wide range of 102-1010 (r2 = 0.99 with maximum Coefficient of variation (CV = 5.91% for IVT RNA. The newly developed real-time RT-PCR was at par with nested RT-PCR in sensitivity and superior to cell lines and other living systems (embryonated eggs and infant mice used for the isolation of the virus. Detection limit of real-time one step RT-PCR and nested RT-PCR was found to be 1.2 × 100 PFU/ml. RD cells, sand fly cells, infant mice, and embryonated eggs showed almost equal sensitivity (1.2 × 102 PFU/ml. Vero and PS cell-lines (1.2 × 103 PFU/ml were least sensitive to CHPV infection. Specificity of the assay was found to be 100% when RNA from other viruses or healthy

  17. Reverse-transcription PCR (RT-PCR).

    Science.gov (United States)

    Bachman, Julia

    2013-01-01

    RT-PCR is commonly used to test for genetic diseases and to characterize gene expression in various tissue types, cell types, and over developmental time courses. This serves as a form of expression profiling, but typically as a candidate approach. RT-PCR is also commonly used to clone cDNAs for further use with other molecular biology techniques (e.g., see Oligo(dT)-primed RT-PCR isolation of polyadenylated RNA degradation intermediates and Circularized RT-PCR (cRT-PCR): analysis of RNA 5' ends, 3' ends, and poly(A) tails).

  18. Use of dual priming oligonucleotide system-based multiplex RT-PCR combined with high performance liquid chromatography assay for simultaneous detection of five enteric viruses associated with acute enteritis.

    Science.gov (United States)

    Fan, Wen-Lu; Wang, Zi-Wei; Qin, Yue; Sun, Chao; Liu, Zhong-Mei; Jiang, Yan-Ping; Qiao, Xin-Yuan; Tang, Li-Jie; Li, Yi-Jing; Xu, Yi-Gang

    2017-05-01

    In this study, a specific and sensitive method for simultaneous detection of human astrovirus, human rotavirus, norovirus, sapovirus and enteric adenovirus associated with acute enteritis was developed, based on the specific dual priming oligonucleotide (DPO) system and the sensitive high-performance liquid chromatography (HPLC) analysis. The DPO system-based multiplex reverse transcription-polymerase chain reaction (RT-PCR) combined with HPLC assay was more sensitive than agarose gel electrophoresis analysis and real-time SYBR Green PCR assay, and showed a specificity of 100% and sensitivity of 96%-100%. The high sensitivity and specificity of the assay indicates its great potential to be a useful tool for the accurate diagnosis of enteric virus infections.

  19. Real-time RT-PCR high-resolution melting curve analysis and multiplex RT-PCR to detect and differentiate grapevine leafroll-associated associated virus 3 variant groups I, II, III and VI

    Directory of Open Access Journals (Sweden)

    Bester Rachelle

    2012-09-01

    Full Text Available Abstract Background Grapevine leafroll-associated virus 3 (GLRaV-3 is the main contributing agent of leafroll disease worldwide. Four of the six GLRaV-3 variant groups known have been found in South Africa, but their individual contribution to leafroll disease is unknown. In order to study the pathogenesis of leafroll disease, a sensitive and accurate diagnostic assay is required that can detect different variant groups of GLRaV-3. Methods In this study, a one-step real-time RT-PCR, followed by high-resolution melting (HRM curve analysis for the simultaneous detection and identification of GLRaV-3 variants of groups I, II, III and VI, was developed. A melting point confidence interval for each variant group was calculated to include at least 90% of all melting points observed. A multiplex RT-PCR protocol was developed to these four variant groups in order to assess the efficacy of the real-time RT-PCR HRM assay. Results A universal primer set for GLRaV-3 targeting the heat shock protein 70 homologue (Hsp70h gene of GLRaV-3 was designed that is able to detect GLRaV-3 variant groups I, II, III and VI and differentiate between them with high-resolution melting curve analysis. The real-time RT-PCR HRM and the multiplex RT-PCR were optimized using 121 GLRaV-3 positive samples. Due to a considerable variation in melting profile observed within each GLRaV-3 group, a confidence interval of above 90% was calculated for each variant group, based on the range and distribution of melting points. The intervals of groups I and II could not be distinguished and a 95% joint confidence interval was calculated for simultaneous detection of group I and II variants. An additional primer pair targeting GLRaV-3 ORF1a was developed that can be used in a subsequent real-time RT-PCR HRM to differentiate between variants of groups I and II. Additionally, the multiplex RT-PCR successfully validated 94.64% of the infections detected with the real-time RT-PCR HRM

  20. Real-time Quantitative RT-PCR Assay for Specific Identification of Panthera tigris Materials%基于TaqMan探针的虎制品实时荧光定量PCR的特异性鉴定

    Institute of Scientific and Technical Information of China (English)

    张太翔; 凌宗帅; 赵立娜; 袁涛; 徐彪; 梁成珠; 刘文鹏; 孙军; 张艺兵

    2012-01-01

    根据虎线粒体细胞色素b核苷酸保守序列设计引物和探针,建立了一种快速鉴定虎制品的TaqMan实时荧光定量PCR方法.以梯度稀释的含有目的扩增片段的重组质粒作为标准品,进行定量PCR反应.结果显示,5.0×105~5.0×101拷贝/μL范围内定量PCR均有“S”型扩增曲线,检测灵敏度为50拷贝/μL.对豹、狮子、猫等7种非虎哺乳动物DNA样本的检测结果均为阴性.本研究建立的实时荧光定量PCR方法具有灵敏度高、特异性和重复性好、方便经济的特性,在虎制品的检测与鉴定中具有良好的应用前景.%Primers and probes were designed based on the conservative sequence of cytochondriome cytochrome b, a realtime quan quantitative RT-PCR assay was developed for indentification of Panthera tigris. The recombinant plasmid containing the target sequence was constructed to detect the sensitivity and prepare the standard curve. The real-time RT-PCR assay had a detection limit of 50 copies/μL, and with a dynamic range of detection between 5 × 105 to 5 × 101 copies /μL. The primers and probe were specific for Panthera tigris' DNA and did not react with other non- Panthera tigris ' DNA. This real-time RT-PCR assay with high sensitivity, specificity and accuracy is considered to be a powerful tool for the rapid indentification and quantification of Panthera tigris.

  1. Establishment and Application of a RT-PCR Assay for Detection of Bovine Viral Diarrhea Virus%牛病毒性腹泻病毒RT-PCR检测方法的建立及应用

    Institute of Scientific and Technical Information of China (English)

    张淑琴; 郭利; 张亭亭; 吴永旺; 武华

    2011-01-01

    根据GenBank中登录的牛病毒性腹泻病毒(BVDV)基因序列,设计合成了1对特异性引物,建立了检测BVDV的RT-PCR方法.通过对该方法的特异性、敏感性和重复性进行试验,结果显示,该方法可从BVDV标准毒株Oregon C24V中扩增出471 bp的特异性片段,而对猪瘟病毒、牛传染性鼻气管炎病毒、牛呼吸道合胞体病毒、牛副流感病毒、MDBK正常细胞的扩增结果均为阴性.经对标准毒株的细胞毒进行检测,其敏感度达10-1TCIDso/mL应用该方法对临床腹泻病牛各脏器样品进行检测,结果比病毒分离方法更为敏感,操作简便.表明建立的RT-PCR方法具有特异、灵敏、高效、快速的特点,可用于BVDV的临床检测及流行病学监测.%A reverse transcription PCR (RT-PCR) assay for detection of bovine viral diarrhea virus (BVDV)was established using a pair of specific primers of BVDV.This method could specifically amplify a 471 bp fragment from BVDV Oregon C24V strain, but not from classical swine fever virus (CSFV) ,infectious bovine rhinotracheitis virus (IBRV), bovine respiratory syncytial virus (BRSV) ,parainfluenza virus-3 (PI3) and normal MDBK cells.The organs were detected by RT-PCR and virus isolated that collected from a sick calf with bovine viral diarrhea (BVD) like syndrome.These results indicated that the established RT-PCR assay was specific, sensitive, efficient and rapid for detection, monitoring and epidemiological investigation of BVDV infection.

  2. Detection of Common Foodborne Pathogens in Emulsifiable Dust by Multiplex RT-PCR Assay%乳粉中常见致病菌的多重荧光PCR检测

    Institute of Scientific and Technical Information of China (English)

    秦丽; 周巍; 张明; 杨帆; 贾昭斌; 张岩

    2014-01-01

    利用多重荧光PCR技术快速检测乳粉中的沙门氏菌、金黄色葡萄球菌、志贺氏菌。以沙门氏菌的invA基因、志贺氏菌的ipaH基因、金黄色葡萄球菌的nuc基因为靶基因,选择特异性引物,以参照菌种为对象,验证引物的特异性,建立多重荧光PCR检测方法,人工添加沙门氏菌、金黄色葡萄球菌、志贺氏菌,确定方法的检出限。结果表明该方法特异性强、灵敏度高,乳粉中检测的灵敏度为1CFU/mL,可在8h内完成对乳品中沙门氏菌、金黄色葡萄球菌、志贺氏菌的检测。%A multiplex RT-PCR assay was designed for prompt detection of Staphylococcus aureus , Salmonella spp ,and Shigella spp in milk powder .In this study ,the target genes were the invA of Salmo-nella ,the ipaH of Shigella and the nuc of Staphylococcus ,and specific primers were designed accordingly . Staphylococcus aureus ,Salmonella spp ,and Shigella spp were artificially added to determine the detection limit of the method .The results indicated that detection of Staphylococcus aureus ,Salmonella spp ,and Shigella spp in milk powder by multiplex RT-PCR was of strong specificity and high sensitivity ,and the sensitivity of the multiplex RT-PCR was 1 CFU/mL .The assay was capable of detecting Staphylococcus aureus ,Salmonella spp ,and Shigella spp in milk powder within 8 hours .

  3. Evaluation of the suitability of six host genes as internal control in real-time RT-PCR assays in chicken embryo cell cultures infected with infectious bursal disease virus

    DEFF Research Database (Denmark)

    Li, Yiping; Bang, Dang Duong; Handberg, Kurt

    2005-01-01

    -time RT-PCR is needed to a suitable internal control. We thus investigated the expression pattern of six chicken genes, including P-actin, 28S rRNA, 18S rRNA, glyceral dehyde-3-phosphate dehydrogenase (GAPDH), TATA box-binding protein (TBP) and beta-2-microglobulin, in chicken embryo (CE) cell cultures...... and GAPDH had a lower expression level in CE cell cultures. Also, beta-actin showed no significant variation in both normalized and non-normalized assays and virus dose-independent of inoculation, while other genes did. beta-Actin was further successfully used as an internal control to quantitate Bursine-2...... virus-specific RNA load in CE cell cultures. Thus, beta-actin was suggested as a suitable internal control in studying gene expression as well as virus-specific RNA load in CE cell after IBDV infection....

  4. Validation of a norovirus multiplex real-time RT-PCR assay for the detection of norovirus GI and GII from faeces samples.

    LENUS (Irish Health Repository)

    Jones, S

    2011-01-01

    Norovirus is a leading cause of infectious non-bacterial gastroenteritis. The virus is highly contagious and has multiple modes of transmission, presenting a growing challenge to hospital-based healthcare. In this study, a total of 120 stool samples are tested for the presence of norovirus GI and GII by the Roche two-step Lightcycler 2.0 assay incorporating primers and probes produced by TIB Molbiol, and the results are compared with results from the National Virus Reference Laboratory. The Roche\\/TIB Molbiol assay produced 51 positive results and 69 negative results. Discrepancy analysis was performed for six conflicting results using a second real-time polymerase chain reaction (PCR) assay (Roche\\/TIB Molbiol) and this confirmed that four of the five discrepant positive results were true positives. A single discrepant negative result generated by the Roche assay remained negative using the second assay. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated to be 98%, 98.6%, 98.0% and 98.6%, respectively. Melting curve analysis was used to differentiate genogroups I and II and this showed that 92% of strains belonged to genogroup II.

  5. Development and evaluation of a SYBR Green real-time RT-PCR assay for evaluation of cytokine gene expression in horse.

    Science.gov (United States)

    Sánchez-Matamoros, A; Kukielka, D; De las Heras, A I; Sánchez-Vizcaíno, J M

    2013-01-01

    Cytokine secretion is one of the main mechanisms by which the immune system is regulated in response to pathogens. Therefore, the measurement of cytokine expression is fundamental to characterizing the immune response to infections. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) is widely used to measure cytokine mRNA levels, but assay conditions should be properly evaluated before analyzing important equine infections through relative quantification of gene expression. The aim of this study was to develop and evaluate a set of RT-qPCR assays for a panel of the most common cytokines in horses involved in innate and adaptive immune responses. Eight cytokines (interleukin (IL)-1β, IL-2, IL-4, IL-10, IL-12, TNFα, IFNβ and IFNγ) and a housekeeping gene (β-actin) were detected and amplified with the same annealing temperature in a SYBR Green RT-qPCR assay of samples of mitogen-stimulated peripheral blood mononuclear cells from a healthy horse and whole blood from a horse infected with African horse sickness virus. The method gave good efficiency for all genes tested, allowing quantification of relative expression levels. These SYBR Green RT-qPCR assays may be useful for examining cytokine gene expression in horses in response to exposure to economically important pathogens.

  6. Development of a real-time RT-PCR assay based on primer-probe energy transfer for the detection of all serotypes of bluetongue virus

    DEFF Research Database (Denmark)

    Leblanc, N; Rasmussen, Thomas Bruun; Fernandez, J

    2010-01-01

    that circulate currently in Europe was also determined. BTV 2, 4, 9 and 16 were tested on available cell culture samples and the detection limits were 109, 12, 13 and 24 copies, respectively. This assay provides an important tool for early and rapid detection of a wide range of BTV strains, including emerging...

  7. Quantification of Simian Immunodeficiency Virus by SYBR Green RT-PCR Technique

    Institute of Scientific and Technical Information of China (English)

    Jing LU; Li QIN; Guang-jie LIU; Si-ting ZHAO; Xiao-ping CHEN

    2008-01-01

    Plasma viral RNA load is widely accepted as the most relevant parameter to assess the status and progression of Simian immunodeficiency virus (SIV) infections. To accurately measure RNA levels of the virus, a one-step fluorescent quantitative assay was established based on the SYBR green Real-time reverse transcription-polymerase chain reaction (RT-PCR). The lower detection limit of the assay was 10 copies per reaction for the virus. This method was successfully applied to quantify SIVmac251 and SIVmac239 viruses produced in CEM×174 cells. Additionally, the performance of the SYBR green RT-PCR was assessed in a SIVmac251 infected rhesus macaque. The result demonstrated that the method could detect as little as 215 copies per milliliter of plasma and the dynamic pattern of viral load was highly consistent with previous results. With regard to convenience, sensitivity and accuracy our assay represents a realistic alternative to both branched-chain DNA (b-DNA) assays or real-time PCR assays based on TaqMan probes.

  8. One-step assay for the quantification of T4 DNA ligase.

    Science.gov (United States)

    Franke, Steffi; Kreisig, Thomas; Buettner, Karin; Zuchner, Thole

    2015-02-01

    As one of the most commonly used enzyme in molecular biology, the T4 DNA ligase presents an important tool for the manipulation of DNA. T4 DNA ligase activity measurements are based on the use of radioactivity or rather labor-intense procedures including gel-based analysis. We therefore established a homogeneous T4 DNA ligase assay utilizing a specifically designed fluorescein- and dark quencher-labeled DNA molecule. Upon ligation of both DNA molecules, a quenching occurs and the fluorescence intensity decreases with increasing ligase concentrations. The assay allows a sensitive and precise quantification (CV, 4.6-5.5 %) of T4 DNA ligase activities and showed a high specificity when tested against other ligases of related and different species. Most importantly, this T4 DNA ligase assay requires only one working and incubation step before measurement can take place at room temperature and may therefore offer an interesting alternative to existing, more laborious ligase assays.

  9. Development of a molecular-beacon-based multi-allelic real-time RT-PCR assay for the detection of human coronavirus causing severe acute respiratory syndrome (SARS-CoV): a general methodology for detecting rapidly mutating viruses.

    Science.gov (United States)

    Hadjinicolaou, Andreas V; Farcas, Gabriella A; Demetriou, Victoria L; Mazzulli, Tony; Poutanen, Susan M; Willey, Barbara M; Low, Donald E; Butany, Jagdish; Asa, Sylvia L; Kain, Kevin C; Kostrikis, Leondios G

    2011-04-01

    Emerging infectious diseases have caused a global effort for development of fast and accurate detection techniques. The rapidly mutating nature of viruses presents a major difficulty, highlighting the need for specific detection of genetically diverse strains. One such infectious agent is SARS-associated coronavirus (SARS-CoV), which emerged in 2003. This study aimed to develop a real-time RT-PCR detection assay specific for SARS-CoV, taking into account its intrinsic polymorphic nature due to genetic drift and recombination and the possibility of continuous and multiple introductions of genetically non-identical strains into the human population, by using mismatch-tolerant molecular beacons designed to specifically detect the SARS-CoV S, E, M and N genes. These were applied in simple, reproducible duplex and multiplex real-time PCR assays on 25 post-mortem samples and constructed RNA controls, and they demonstrated high target detection ability and specificity. This assay can readily be adapted for detection of other emerging and rapidly mutating pathogens.

  10. 扎伊尔型埃博拉病毒的实时荧光RT-PCR检测方法研究%Development of Real-time RT-PCR assay for detection of Zaire Ebolavirus

    Institute of Scientific and Technical Information of China (English)

    李阿茜; 李伟红; 李建东; 张硕; 曲靖; 李川; 张全福; 梁米芳; 李德新

    2014-01-01

    目的 建立扎伊尔型埃博拉病毒的核酸检测方法,以期用于埃博拉出血热临床标本的检测.方法 针对扎伊尔型埃博拉病毒核蛋白和糖蛋白基因设计引物和探针,建立单重和双重实时荧光RT-PCR检测方法,利用体外转录病毒RNA和埃博拉病毒系列参考品RNA评价其敏感性,利用马尔堡病毒、健康人、登革热患者和发热伴血小板减少综合征患者血清评价其特异性.结果 所建立的实时荧光RT-PCR检测方法扩增效率在95%~105%,可特异性地检测扎伊尔型埃博拉病毒核蛋白和糖蛋白基因,与马尔堡病毒、登革热和发热伴血小板减少综合征病毒均无交叉反应,体外转录的病毒RNA可检出10~100拷贝/μl.双重检测方法通过细胞培养的扎伊尔型埃博拉病毒RNA验证,可检出100 pfu/ml病毒.结论 本研究建立的检测扎伊尔型埃博拉病毒的实时荧光RT-PCR方法具有良好的特异性和敏感性,可用于埃博拉出血热临床标本的检测.%Objective To establish a method for Zaire Ebolavirus (ZEBOV) RNA detection and laboratory diagnosis of suspected ZEBOV infected cases.Methods Primers/probe sets for Nucleoprotein (NP) and Glycoprotein(GP) detection were designed and used to develop monoplex and duplex real-time RT-PCR assays.The sensitivity was evaluated by in vitro transcribed RNA and ZEBOV RNA reference,and the specificity was identified by Marburg virus,healthy human sera and sera from Dengue fever patients and Severe fever with thrombocytopenia syndrome (SFTS) patients.Results The developed real-time RT-PCR assay scould be used for ZEBOV NP and GP detection specifically,and the amplification efficiency was 95% -105%.There was no cross reaction with Marburg virus,Dengue fever virus and SFTS virus.The sensitivity was evaluated with serial dilutions of synthesized viral RNAs,and the results showed that in vitro transcribed RNA of 10-100 copies/μl could be detected.The developed duplex

  11. GeXP多重PCR技术同时检测12种常见呼吸道病毒%A GeXP based Multiplex RT-PCR Assay for Simultaneous Detection of Twelve Human Respiratory Viruses

    Institute of Scientific and Technical Information of China (English)

    李瑾; 毛乃颖; 秦萌; 胡秀梅; 杨梦婕; 王淼; 张晨; 许文波; 马学军

    2011-01-01

    本研究建立了一种基于GeXP多重基因表达遗传分析系统的多重RT-PCR检测方法,该方法可以同时检测12种呼吸道病毒,包括流感病毒A型和B型、季节性H1N1、副流感病毒1~3型、人鼻病毒、人偏肺病毒、腺病毒、呼吸道合胞病毒A型和B型、人博卡病毒.针对病原体保守区序列设计12种病毒的特异性引物,分别用已验证的阳性标本为模板检验多重体系的特异性.多重检测体系在103拷贝/μL水平可同时检测到12种病毒.另检测24份临床标本,以real-time RT-PCR为参考标准,进一步验证检测体系.结果表明,这种基于GeXP系统的新方法灵敏度高、特异性强,可以快速同时检测12种常见呼吸道病毒.%A GeXP based multiplex RT-PCR assay was developed to simultaneously detect twelve different respiratory viruses types/subtypes including influenza A virus, influenza B virus, influenza A virus sHlNl, parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3 , human rhinovi-rus, human metapneumovirus, adenovirus, respiratory syncytial virus A, respiratory syncytial virus B and human bocavirus. Twelve sets of specific primers were designed based on the conserved sequences of available respiratory-virus sequence database The specificity of the multiplex system was examined by positive specimens confirmed previously. The sensitivity to detect twelve respiratory viruses simultaneously was 103 copies/μL. Twenty four clinical specimens were further detected by this novel assay and the results were compared with that of the real-time RT-PCR. These results showed that this novel assay based on GeXP is a fast, sensitive, and high throughput test for the detection of respiratory virus infections.

  12. One-step thickness shear mode acoustic assay for plasminogen activators.

    Science.gov (United States)

    Ghazali, Mirnader; Hayward, Gordon L

    2008-07-01

    A new procedure is presented for the measurement of plasminogen activators using a thickness shear mode sensor and a modified version of the fibrin plate assay at the micro-scale. Separate, well-mixed solutions of the substrates fibrinogen and plasminogen, and enzymes thrombin and the plasminogen activator sample were mixed together and placed on the sensor surface. The temperature and evaporation were controlled during the assay. The clot dissolution time correlated well with the quantity of the plasminogen activator in the sample. The average relative standard deviation was 12.5%.

  13. Rapid one-step assays for on-site monitoring of mouse and rat urinary allergens

    NARCIS (Netherlands)

    Koets, M.; Renström, A.; Zahradnik, E.; Bogdanovic, J.; Wouters, I.M.; Amerongen, van A.

    2011-01-01

    llergy to rodent proteins is common among laboratory animal workers. Sensitive methods to measure exposure to these allergens have been developed. These assays are, however, expensive, time-consuming, and require a laboratory facility and methodological expertise. A simple method to screen for aller

  14. Establishment and Application of a RT-PCR Assay for Detection of Newly Emerged Porcine Deltacoronavirus%新现猪Delta冠状病毒RT-PCR检测方法的建立及其应用

    Institute of Scientific and Technical Information of China (English)

    张帆帆; 唐玉新; 宋德平; 周信荣; 黄冬艳; 李安琪; 彭棋; 陈燕君; 吴琼; 何后军

    2016-01-01

    及PEDV、TGEV的相应N基因片段序列构建进化树,结果表明26条PDCoV序列属于同一个分支,而PEDV和TGEV则分别属于不同的分支。试验获得的PDCoV序列与韩国PDCoV毒株KNU14-04亲缘关系最近,同源性高达99.1%;与两株香港毒株HKU 15-44和HKU 15-155亲缘关系相对较远;③本试验建立的RT-PCR方法特异性强,仅能扩增出PDCoV,而对PEDV、TGEV、PKoV、PAstV、PRRSV及CSFV核酸无交叉扩增现象;④所建立的RT-PCR方法灵敏度高,将含扩增片段的重组质粒以1.0×106拷贝/μL为起始浓度依次10倍稀释至1.0×101拷贝/μL作为模板验证方法的灵敏度,结果表明所建立的方法对 PDCoV 最低可检出量为1.0×103拷贝/μL;⑤应用建立的 RT-PCR 方法检测了249份2012—2015年采集自江西省各地区的腹泻母猪粪便及腹泻仔猪粪便/肠道样本,结果显示腹泻样本中 PDCoV 的检出率为31.33%(78/249),母猪粪便中PDCoV的检出率(27.78%)稍低于仔猪粪便及肠道样品PDCoV的检出率(31.92%),最早可从2012年的样品中检测出PDCoV。【结论】试验成功建立了检测新现PDCoV的RT-PCR方法;应用所建立的RT-PCR方法检测了249份2012—2015年江西地区猪群临床腹泻样品中PDCoV存在情况,结果表明PDCoV是一种普遍存在于腹泻猪群中的病毒;研究建立的RT-PCR方法对猪群PDCoV的临床诊断和流行病学调查等具有应用价值。%Objective]Porcine Deltacoronavirus (PDCoV) is a newly emerged coronavirus, which has caused diarrhea in pigs, especially in newborn piglets leading to high morbidity and mortality, such that it has become one of the most important causes of death in newborn piglets. In this study, we attempted to establish a reverse transcription-polymerase chain reaction (RT-PCR) assay for detection of newly emerged PDCoV in diarrheal pigs, and investigate the infection situation of PDCoV in diarrheal samples of pigs.[Method]Based on

  15. A novel DANP-coupled hairpin RT-PCR for rapid detection of Chikungunya virus.

    Science.gov (United States)

    Chen, Huixin; Takei, Fumie; Koay, Evelyn Siew-Chuan; Nakatani, Kazuhiko; Chu, Justin Jang Hann

    2013-03-01

    Chikungunya has re-emerged as an important arboviral infection of global health significance. Because of lack of a vaccine and effective treatment, rapid diagnosis plays an important role in early clinical management of patients. In this study, we have developed a novel molecular diagnostic platform that ensures a rapid and cost-effective one-step RT-PCR assay, with high sensitivity and specificity, for the early detection of the Chikungunya virus (CHIKV). It uses 2,7-diamino-1,8-naphthyridine derivative (DANP)-labeled cytosine-bulge hairpin primers to amplify the nsP2 region of the CHIKV genome, followed by measurement of the fluorescence emitted from DANP-primer complexes after PCRs. The detection limit of our assay was 0.01 plaque-forming units per reaction of CHIKV. Furthermore, the HP-nsP2 primers were highly specific in detecting CHIKV, without any cross-reactivity with the panel of RNA viruses validated in this study. The feasibility of the DANP-coupled hairpin RT-PCR for clinical diagnosis was evaluated using clinical serum samples from CHIKV-infected patients, and the specificity and sensitivity were 100% (95% CI, 80.0% to 100%) and 95.5% (95% CI, 75.1% to 99.8%), respectively. These findings confirmed its potential as a point-of-care clinical molecular diagnostic assay for CHIKV in acute-phase patient serum samples.

  16. Silver nanoparticle enhanced immunoassays: one step real time kinetic assay for insulin in serum.

    Science.gov (United States)

    Lochner, Nina; Lobmaier, Christina; Wirth, Michael; Leitner, Alfred; Pittner, Fritz; Gabor, Franz

    2003-11-01

    Silver nanoparticle enhanced fluorescence is introduced as an alternative method to surface plasmon resonance techniques for real time monitoring of biorecognitive interactions or immunoassays. This method relies on the phenomenon that an electromagnetic near field is generated upon illumination on the surface of silver nanoparticles. The interaction of this field with nearby fluorophores results in fluorescence enhancement. Thus, fluorophores in the bulk solution can be discriminated from surface bound fluorophores. Anti-insulin-antibodies were immobilized on the surface of silver colloids in the following order: A ready to use microplate was prepared by bottom up coating with layers of aminosilane, silver nanoparticles, Fc-recognizing F(ab)(2)-fragments and anti-insulin-antibodies. At equilibrium conditions fluorescein-labeled insulin could only be detected in the presence of the colloid; the detection limit was 250 nM, and a fourfold increase in fluorescence was observed upon real time monitoring. The competitive assay of labeled and unlabeled insulin revealed a working range of 10-200 nM insulin in serum. The rapid single step immunoassay is easy to perform even in microplate format, its sensitivity is comparable to ELISA techniques, and offers broad application for real time monitoring of molecular recognitive processes.

  17. One-step seeding of neural stem cells with vitronectin-supplemented medium for high throughput screening assays

    Science.gov (United States)

    Dai, Sheng; Li, Rong; Long, Yan; Titus, Steve; Zhao, Jinghua; Huang, Ruili; Xia, Menghang; Zheng, Wei

    2017-01-01

    Human neuronal cells differentiated from induced pluripotent cells have emerged as a new model system for the study of disease pathophysiology and evaluation of drug efficacy. Differentiated neuronal cells are more similar in genetics and biological content to the human brain cells than other animal disease models. However, culture of neuronal cells in assay plates requires a labor-intensive procedure of plate pre-coating, hampering its applications in high throughput screening (HTS). We developed a simplified method with one-step seeding of neural stem cells in assay plates by supplementing the medium with a recombinant human vitronectin (VTN), thus avoiding plate pre-coating. Robust results were obtained from cell viability, calcium response, and neurite outgrowth assays using this new method. Our data demonstrate that this approach greatly simplifies high throughput assays using neuronal cells differentiated from human stem cells for translational research. PMID:27647668

  18. A Multiplex RT-PCR Assay for Detection and Differentiation of Avian-Origin Canine H3N2, Equine-Origin H3N8, Human-Origin H3N2, and H1N1/2009 Canine Influenza Viruses

    Science.gov (United States)

    Sun, Honglei; Pu, Juan; Liu, Jinhua; Sun, Yipeng

    2017-01-01

    Virological and serological surveys have documented that H1N1/2009, avian-origin canine H3N2 (cH3N2), seasonal human-origin H3N2 (hH3N2), and equine-origin H3N8 influenza viruses are consistently circulating in dogs. In the present study, a multiplex reverse-transcriptase polymerase chain reaction (mRT-PCR) assay was developed for simultaneous detection and differentiation of these influenza viruses. Four primer sets were designed to target the hemagglutinin genes of H1N1/2009, cH3N2, hH3N2, and H3N8 canine influenza viruses (CIVs). This mRT-PCR assay demonstrated high specificity and sensitivity for the four CIV subtypes. Additionally, mRT-PCR results obtained from 420 clinical samples were consistent with those obtained by the conventional virus isolation method. Our mRT-PCR assay is reliable for clinical diagnosis and rapid identification of CIVs. PMID:28107507

  19. Development and application of a real-time TaqMan RT-PCR assay for detection of duck hepatitis virus type 1%I型鸭肝炎病毒 TaqMan 荧光定量 RT-PCR 方法的建立及初步应用

    Institute of Scientific and Technical Information of China (English)

    刘海燕; 赵丽丽; 牛银杰; 祝明皓; 刘胜旺; 陈洪岩

    2015-01-01

    Objective To develop a real-time RT-PCR assay ( rRT-PCR) for efficient detection of duck hepatitis virus type 1 ( DHV-I) .Method According to the different gene sequences of DHV-I from different provinces download from NCBI and to find the conserved sequences.One pair of the specific primers and one TaqMan probe were designed. Then reaction parameters were optimized to develop a real-time RT-PCR assay ( rRT-PCR) .Results This developed rRT-PCR assay could detect 20 template copies of RNA, and its sensitivity was higher than that of the conventional RT-PCR. This rRT-PCR assay was found to be specific and able to detect DHV-I, and no positive results were observed when nucleic acid from Muscovy duck parvovirus, goose parvovims, Newcastle disease and avian influenza virus, egg drop syndrome virus, reticuloendotheliosis virus, duck Tembusu virus, poultry intestinal arc virus were used as rRT-PCR templates.The results of this developed rRT-PCR assay used for 100 duck clinical samples showed a positive rate of 92%, indicating that DHV exists in duck group of Jiangsu province in China.Conclusion This rRT-PCR assay can be used as a rapid tool for detection of DHV-I.%目的:建立快速诊断I型鸭肝炎病毒的荧光定量RT-PCR方法。方法根据NCBI下载的20个来自我国不同省份的的I型鸭肝炎病毒的基因序列,找出其保守序列,设计合成一对引物和一条TaqMan探针,进行条件优化,检测其特异性,敏感性,稳定性。结果该方法敏感性达20拷贝,比常规PCR敏感性高。其特异性强,对番鸭细小病毒(MDPV),鹅细小病毒(GPV),新城疫病毒(NDV)和禽流感(AIV),鸭减蛋综合征病毒(EDSV),禽网状内皮组织增生症病毒(REV),鸭坦布苏病毒(DTMUV),禽呼肠弧病毒(ARV)8种病毒的检测均为阴性,I型鸭肝炎病毒检测结果为阳性。用建立的方法检测了江苏徐州采集100份样品,阳性率为92%。说明I型鸭

  20. Development and Characterization of a Multiplexed RT-PCR Species Specific Assay for Bovine and one for Porcine Foot-and-Mouth Disease Virus Rule-Out Supplemental Materials

    Energy Technology Data Exchange (ETDEWEB)

    Smith, S; Danganan, L; Tammero, L; Lenhoff, R; Naraghi-arani, P; Hindson, B

    2007-08-06

    viruses which are of two bovine types bovine papular stomatitis virus (BPSV) and psuedocowpox (PCP). This document provides details of signature generation, evaluation, and testing, as well as the specific methods and materials used. A condensed summary of the development, testing and performance of the multiplexed assay panel was presented in a 126 page separate document, entitled 'Development and Characterization of A Multiplexed RT-PCR Species Specific Assay for Bovine and one for Porcine Foot-and-Mouth Disease Virus Rule-Out'. This supplemental document provides additional details of large amount of data collected for signature generation, evaluation, and testing, as well as the specific methods and materials used for all steps in the assay development and utilization processes. In contrast to last years effort, the development of the bovine and porcine panels is pending additional work to complete analytical characterization of FMDV, VESV, VSV, SVD, RPV and MCF. The signature screening process and final panel composition impacts this effort. The unique challenge presented this year was having strict predecessor limitations in completing characterization, where efforts at LLNL must preceed efforts at PIADC, such challenges were alleviated in the 2006 reporting by having characterization data from the interlaboratory comparison and at Plum Island under AgDDAP project. We will present an addendum at a later date with additional data on the characterization of the porcine and bovine multiplex assays when that data is available.

  1. Rapid detection of sacbrood virus (SBV by one-step reverse transcription loop-mediated isothermal amplification assay

    Directory of Open Access Journals (Sweden)

    Jin-Long Yang

    2012-02-01

    Full Text Available Abstract Background Sacbrood virus (SBV primarily infects honeybee broods, and in order to deal with the problem cost effective detection methods are required. Findings A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP assay was developed for the rapid identification of SBV. The data demonstrated that, in a simple water bath, SBV RNA could be detected as early as 20 min at 65°C, and a positive amplification reaction was visible to the naked eye due to a color change brought on by the addition of nucleic acid stain SYBR Green. Conclusions The current study presents a method for the rapid and simple detection of SBV by RT-LAMP with high sensitivity and analytic specificity.

  2. Simultaneous detection of three lily viruses using Triplex IC-RT-PCR.

    Science.gov (United States)

    Zhang, Yubao; Wang, Yajun; Xie, Zhongkui; Yang, Guo; Guo, Zhihong; Wang, Le

    2017-08-25

    Viruses commonly infecting lily (Lilium spp.) include: Lily symptomless virus (LSV), Cucumber mosaic virus (CMV) and Lily mottle virus (LMoV). These viruses usually co-infect lilies causing severe economic losses in terms of quantity and quality of flower and bulb production around the world. Reliable and precise detection systems need to be developed for virus identification. We describe the development of a triplex immunocapture (IC) reverse transcription (RT) polymerase chain reaction (PCR) assay for the simultaneous detection of LSV, CMV and LMoV. The triplex IC-RT-PCR was compared with a quadruplex RT-PCR assay. Relative to the quadruplex RT-PCR, the specificity of the triplex IC-RT-PCR system for LSV, CMV and LMoV was 100% for field samples. The sensitivity of the triplex IC-RT-PCR system was 99.4%, 81.4% and 98.7% for LSV, CMV and LMoV, respectively. Agreement (κ) between the results obtained from the two tests was 0.968, 0.844 and 0.984 for LSV, CMV and LMoV, respectively. This is the first report of the simultaneous detection of LSV, CMV and LMoV in a triplex IC-RT-PCR assay. In particular we believe this convenient and reliable triplex IC-RT-PCR method could be used routinely for large-scale field surveys or crop health monitoring of lily. Copyright © 2017. Published by Elsevier B.V.

  3. Evaluation and application of a one-step duplex real-time reverse transcription polymerase chain reaction assay for the rapid detection of influenza A (H7N9) virus from poultry samples.

    Science.gov (United States)

    Bao, Hongmei; Ma, Yong; Shi, Jianzhong; Zeng, Xianying; Zhao, Yuhui; Wang, Xiurong; Chen, Hualan

    2015-10-01

    In China, a novel reassortant influenza A (H7N9) virus, which has caused 435 cases of human infection, has recently emerged. Most cases of human infections with the H7N9 virus are known to be associated with a poultry farm and live-poultry markets. In this study, a one-step duplex real-time reverse transcription polymerase chain reaction (RRT-PCR) assay was developed for the simultaneous detection of the hemagglutinin (HA) and neuraminidase (NA) genes of the H7N9 virus for effective surveillance and early diagnosis of cases from clinical samples collected from live-poultry markets or poultry farms. The detection limit of this assay was as low as 0.1 EID50 of H7N9 viruses, which is similar to the detection limit of the real-time RT-PCR assay released by the Word Health Organization. The coefficients of variation (CVs) of both inter-assay and intra-assay reproducibility were less than 1.55 %, showing good reproducibility. No cross-reactivity was observed with RNA of other subtypes of influenza virus or other avian respiratory viruses. The assay can effectively detect H7N9 influenza virus RNA from multiple sources, including chickens, pigeons, ducks, humans, and the environment. Furthermore, the RRT-PCR assay was evaluated with more than 700 clinical samples collected from live-poultry markets and 120 experimentally infected chicken samples. Together, these results indicate that the duplex RRT-PCR assay is a specific, sensitive, and efficient diagnostic method for the epidemiological surveillance and diagnosis of H7N9 virus from different sources, particularly poultry samples.

  4. Avaliação da urina e de leucócitos como amostras biológicas para a detecção ante mortem do vírus da cinomose canina por RT-PCR em cães naturalmente infectados Evaluation of the urine and leucocytes as biological samples for ante mortem detection of canine distemper virus by RT-PCR assay in naturally infected dogs

    Directory of Open Access Journals (Sweden)

    F.J. Negrão

    2007-02-01

    Full Text Available Urine and leucocytes were comparatively evaluated as clinical samples for ante mortem detection of the canine distemper virus (CDV by a reverse transcription-polymerase chain reaction (RT-PCR assay. One hundred and eighty eight dogs with clinical symptoms of distemper, were distributed in three groups. The group A was constituted of 93 dogs with systemic signs of distemper; the group B by 11 dogs with neurological signs, and the group C by 84 dogs that presented simultaneously systemic and neurological signs. In 66.5% (125/188 of the dogs was amplified an amplicon with 287 base pair of the CDV nucleoprotein gene. In 60.8% (76/125 of the animals the CDV was detected simultaneously in the urine and leucocytes, and in 39.2% (49/125 of the dogs just a type of clinical sample (urine: n=37; leucocytes: n=12 was positive. These results demonstrate that the different forms of clinical distemper disease can hinder the choice of only one type of clinical sample to carry out the ante mortem etiological diagnosis of CDV infection, and false-negative results can be generated.

  5. Comparison of quantitative RT-PCR with cell culture to detect viral hemorrhagic septicemia virus (VHSV) IVb infections in the Great Lakes.

    Science.gov (United States)

    Hope, Kristine M; Casey, Rufina N; Groocock, Geoffrey H; Getchell, Rodman G; Bowser, Paul R; Casey, James W

    2010-03-01

    Viral hemorrhagic septicemia virus (VHSV) is an important pathogen of cultured and wild fish in marine and freshwater environments. A new genotype, VHSV IVb, was isolated from a fish collected from the Great Lakes in 2003. Since the first isolation, VHSV IVb has been confirmed in 28 species, signaling the early invasion and continued spread of this Office International des Epizooties-reportable agent. For surveillance of this virus in both wild and experimental settings, we have developed a rapid and sensitive one-step quantitative real-time polymerase chain reaction (qRT-PCR) assay that amplifies a 100-base-pair conserved segment from both the genomic negative strand and the mRNA positive strand of the nucleoprotein (N) gene of VHSV IVb. This assay is linear over seven orders of magnitude, with an analytical capability of detecting a single copy of viral RNA and reproducibility at 100 copies. The assay is approximately linear with RNA input from 50 to 1000 ng per assay and works equally well with RNA prepared from a column-based or phenol-chloroform-based method. In wild-caught fish, 97% of the cases were found to be more than three orders of magnitude more sensitive using qRT-PCR than using cell culture. Of the 1,428 fish from the Great Lakes region tested in 2006 and 2007, 24% were positive by qRT-PCR whereas only 5% were positive by cell culture. All of the fish that were positive by cell culture were also positive by qRT-PCR. Importantly, qRT-PCR sensitivity is comparable to that of cell culture detection when comparing VHSV viral RNA levels with viral titer stocks, confirming that the high qRT-PCR signals obtained with diagnostic samples are due to the accumulation of N gene mRNA by transcriptional attenuation. The qRT-PCR assay is particularly valuable for rapid and high-throughput prescreening of fish before confirmatory testing by cell culture or sequencing tissue-derived amplicons and especially in detecting infection in fish that do not show clinical

  6. A One-Step PCR-Based Assay to Evaluate the Efficiency and Precision of Genomic DNA-Editing Tools.

    Science.gov (United States)

    Germini, Diego; Bou Saada, Yara; Tsfasman, Tatiana; Osina, Kristina; Robin, Chloé; Lomov, Nikolay; Rubtsov, Mikhail; Sjakste, Nikolajs; Lipinski, Mar; Vassetzky, Yegor

    2017-06-16

    Despite rapid progress, many problems and limitations persist and limit the applicability of gene-editing techniques. Making use of meganucleases, TALENs, or CRISPR/Cas9-based tools requires an initial step of pre-screening to determine the efficiency and specificity of the designed tools. This step remains time consuming and material consuming. Here we propose a simple, cheap, reliable, time-saving, and highly sensitive method to evaluate a given gene-editing tool based on its capacity to induce chromosomal translocations when combined with a reference engineered nuclease. In the proposed technique, designated engineered nuclease-induced translocations (ENIT), a plasmid coding for the DNA-editing tool to be tested is co-transfected into carefully chosen target cells along with that for an engineered nuclease of known specificity and efficiency. If the new enzyme efficiently cuts within the desired region, then specific chromosomal translocations will be generated between the two targeted genomic regions and be readily detectable by a one-step PCR or qPCR assay. The PCR product thus obtained can be directly sequenced, thereby determining the exact position of the double-strand breaks induced by the gene-editing tools. As a proof of concept, ENIT was successfully tested in different cell types and with different meganucleases, TALENs, and CRISPR/Cas9-based editing tools.

  7. Comparison of real-time SYBR green dengue assay with real-time taqman RT-PCR dengue assay and the conventional nested PCR for diagnosis of primary and secondary dengue infection

    OpenAIRE

    Damodar Paudel; Richard Jarman; Kriengsak Limkittikul; Chonticha Klungthong; Supat Chamnanchanunt; Ananda Nisalak; Robert Gibbons; Watcharee Chokejindachai

    2011-01-01

    Background : Dengue fever and dengue hemorrhagic fever are caused by dengue virus. Dengue infection remains a burning problem of many countries. To diagnose acute dengue in the early phase we improve the low cost, rapid SYBR green real time assay and compared the sensitivity and specificity with real time Taqman® assay and conventional nested PCR assay. Aims: To develop low cost, rapid and reliable real time SYBR green diagnostic dengue assay and compare with Taqman real-time assay and conven...

  8. EDTA-treated cotton-thread microfluidic device used for one-step whole blood plasma separation and assay.

    Science.gov (United States)

    Ulum, Mokhamad Fakhrul; Maylina, Leni; Noviana, Deni; Wicaksono, Dedy Hermawan Bagus

    2016-04-21

    This study aims to observe the wicking and separation characteristics of blood plasma in a cotton thread matrix functioning as a microfluidic thread-based analytical device (μTAD). We investigated several cotton thread treatment methods using ethylenediaminetetraacetic acid (EDTA) anticoagulant solution for wicking whole blood samples and separating its plasma. The blood of healthy Indonesian thin tailed sheep was used in this study to understand the properties of horizontal wicking and separation on the EDTA-treated μTAD. The wicking distance and blood cell separation from its plasma was observed for 120 s and documented using a digital phone camera. The results show that untreated cotton-threads stopped the blood wicking process on the μTAD. On the other hand, the deposition of EDTA anticoagulant followed by its drying on the thread at room temperature for 10 s provides the longest blood wicking with gradual blood plasma separation. Furthermore, the best results in terms of the longest wicking and the clearest on-thread separation boundary between blood cells and its plasma were obtained using the μTAD treated with EDTA deposition followed by 60 min drying at refrigerated temperature (2-8 °C). The separation length of blood plasma in the μTADs treated with dried-EDTA at both room and refrigerated temperatures was not statistically different (P > 0.05). This separation occurs through the synergy of three factors, cotton fiber, EDTA anticoagulant and blood platelets, which induce the formation of a fibrin-filter via a partial coagulation process in the EDTA-treated μTAD. An albumin assay was employed to demonstrate the efficiency of this plasma separation method during a one-step assay on the μTAD. Albumin in blood is an important biomarker for kidney and heart disease. The μTAD has a slightly better limit of detection (LOD) than conventional blood analysis, with an LOD of 114 mg L(-1) compared to 133 mg L(-1), respectively. However, the μTAD performed

  9. RT-PCR-ELISA as a tool for diagnosis of low-pathogenicity avian influenza

    DEFF Research Database (Denmark)

    Dybkaer, Karen; Munch, Mette; Handberg, Kurt Jensen;

    2003-01-01

    A one-tube reverse transcriptase/polymerase chain reaction coupled with an enzyme-linked immunosorbent assay (RT-PCR-ELISA) was developed for the rapid detection of avian influenza virus (AIV) in clinical specimens. A total of 419 swab pools were analyzed from chickens experimentally infected wit...... of the twenty-three VI-positive specimens were negative when tested by RT-PCR-ELISA. The diagnostic sensitivity and specificity of the RT-PCR-ELISA was 91% and 97%, respectively, using VI in SPF eggs as the gold reference standard.......A one-tube reverse transcriptase/polymerase chain reaction coupled with an enzyme-linked immunosorbent assay (RT-PCR-ELISA) was developed for the rapid detection of avian influenza virus (AIV) in clinical specimens. A total of 419 swab pools were analyzed from chickens experimentally infected...... with low-pathogenicity AIV, from wild aquatic birds, and from domestic ducks. The AIV was detected in 32 swab pools by RT-PCR-ELISA compared to 23 by virus isolation (VI) in embryonated specific pathogen free (SPF) chicken eggs. Thus, 39% more specimens were positive by RT-PCR-ELISA than by VI. Two...

  10. Comparison of a nucleoprotein gene based RT-PCR with real time RT-PCR for diagnosis of avian influenza in clinical specimens.

    Science.gov (United States)

    Nagarajan, S; Murugkar, H V; Tosh, C; Behera, P; Khandia, R; Jain, R; Katare, M; Syed, Z; Tripati, S; Dubey, S C

    2012-08-01

    A nucleoprotein (NP) gene based reverse transcription polymerase chain reaction (npRT-PCR) assay was developed in our laboratory which could detect 35.09% of the experimental samples negative for virus isolation in first passage but positive by third passage. Reducing the reaction volume to 12.5 μl did not alter the test sensitivity and the results did not vary when duplicate samples were run in a different thermal cycler. The positive and negative agreements of this test in clinical specimens were compared with a matrix gene based real time RT-PCR with virus isolation as standard. A total of 516 clinical specimens including tissues, swabs and feces submitted from various States of India as part of active surveillance for avian influenza were tested by npRT-PCR, RRT-PCR and virus isolation in 9-11 day old embryonated specific pathogen free chicken eggs. The positive and negative agreements of npRT-PCR with virus isolation were found to be 0.909±0.022 and 0.980±0.004 respectively and that of RRT-PCR with virus isolation were 0.902±0.023 and 0.977±0.005 respectively. Since the positive and negative agreements of both npRT-PCR and RRT-PCR tests were similar, we suggest that this test can be used by peripheral veterinary laboratories that do not have real time PCR facility for active surveillance of AIV.

  11. Cross-Platform Evaluation of Commercial Real-Time SYBR Green RT-PCR Kits for Sensitive and Rapid Detection of European Bat Lyssavirus Type 1

    Directory of Open Access Journals (Sweden)

    Evelyne Picard-Meyer

    2015-01-01

    Full Text Available This study evaluates the performance of five two-step SYBR Green RT-qPCR kits and five one-step SYBR Green qRT-PCR kits using real-time PCR assays. Two real-time thermocyclers showing different throughput capacities were used. The analysed performance evaluation criteria included the generation of standard curve, reaction efficiency, analytical sensitivity, intra- and interassay repeatability as well as the costs and the practicability of kits, and thermocycling times. We found that the optimised one-step PCR assays had a higher detection sensitivity than the optimised two-step assays regardless of the machine used, while no difference was detected in reaction efficiency, R2 values, and intra- and interreproducibility between the two methods. The limit of detection at the 95% confidence level varied between 15 to 981 copies/µL and 41 to 171 for one-step kits and two-step kits, respectively. Of the ten kits tested, the most efficient kit was the Quantitect SYBR Green qRT-PCR with a limit of detection at 95% of confidence of 20 and 22 copies/µL on the thermocyclers Rotor gene Q MDx and MX3005P, respectively. The study demonstrated the pivotal influence of the thermocycler on PCR performance for the detection of rabies RNA, as well as that of the master mixes.

  12. Application of Multiplex RT-PCR for Detection of Cucurbit-infecting Tobamovirus

    Directory of Open Access Journals (Sweden)

    Budi Setiadi Daryono

    2015-11-01

    Full Text Available Cucumber green mottle mosaic virus (CGMMV and Kyuri green mottle mosaic virus (KGMMV are seed borne viruses and they are also transmitted mechanically during agricultural practice and through water. Hence, these viruses have potential diseases widely distributed throughout the world. To detect different strains of CGMMV and KGMMV, several specific primers for each virus were designed for single and multiplex RT-PCR. The results of single and multiplex RT-PCR showed that CGMMV was detected in zucchini isolated in Bali-Indonesia, while KGMMV was detected both in zucchini isolated in Bali-Indonesia and Cucumis metuliferus isolated in Thailand. Furthermore, artificial co-infection of these two viruses was prepared and carried out using two different ways of viral RNAs extraction. Based on the results, it could be reported that viral RNAs for cDNA amplification by multiplex RT-PCR could be extracted from a mixture of infected leaves or separate extraction of each viruses infected leaves. In addition, results presented in this study demonstrated the application of multiplex RT-PCR to simultaneously detect CGMMV and KGMMV from cucurbit leaves using a mixture of four primers and its feasibility as a sensitive and rapid laboratory assay. Since, no multiplex RT-PCR technique has been described for the detection of CGMMV and KGMMV, this technique can be a good option for sensitive and reliable tool for detection of two major cucurbit infecting Tobamoviruses.Keywords : Cucurbit infecting Tobamovirus, multiplex RT-PCR, seed borne viruses

  13. One-Step Multiplex PCR Assay for Differentiating Proposed New Species "Clostridium neonatale" from Closely Related Species.

    Science.gov (United States)

    Ferraris, Laurent; Schönherr, Sophia; Bouvet, Philippe; Dauphin, Brunhilde; Popoff, Michel; Butel, Marie Jose; Aires, Julio

    2015-11-01

    "Clostridium neonatale" sp. nov., previously involved in an outbreak of neonatal necrotizing enterocolitis, was recently proposed as a new species of the Clostridium genus sensu stricto. We developed a one-step multiplex colony PCR for C. neonatale identification and investigated C. neonatale intestinal colonization frequency in healthy preterm neonates. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  14. GeXP多重基因表达遗传分析系统在手足口病病原分型检测中的应用%Development of a GeXP based Multiplex RT-PCR Assay for Simultaneous Differentiation of Nine Human Hand Food Mouth Disease Pathogens

    Institute of Scientific and Technical Information of China (English)

    胡秀梅; 许文波; 马学军; 张勇; 徐邦牢; 杨梦; 王淼; 张晨; 李瑾; 白如银; 周小棉

    2011-01-01

    利用GeXP多重基因表达遗传分析系统,建立一种多重逆转录-聚合酶链反应(RT-PCR)方法,同时检测引起手足口病的9种常见的人肠道病毒一人肠道病毒71型(HEV71)、柯萨奇病毒A组(CVA)16、4、5、9、10型和柯萨奇病毒B组(CVB)1、3、5型.优化多重反应体系中针对5'UTR区的肠道病毒通用引物和11对针对9种血清型人肠道病毒VP1区的特异性引物的浓度比例,分别以病毒细胞培养物和阳性粪便标本来验证多重反应体系的特异性,以TCID50定量的细胞培养物和克隆质粒体外转录的RNA梯度稀释液来检测多重检测体系的灵敏度.结果表明,优化后的多重检测体系,可扩增出人肠道病毒共有的保守片段的和型特异性片段,HEV71和CVA16细胞培养物的检测下限为100.5TCID50/μL,并可在103copies/μL水平同时、特异地检测出9种病毒RNA.该方法灵敏度高、特异性强,可快速对大量临床样本进行高通量检测,用于手足口病的分子流行病学调查.%A multiplex RT-PCR assay based on GeXP system was developed in order to detect simultaneously human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) and other coxsackieviruses (CVA4, 5,9 and 10, CVB1, 3 and 5). Enterovirus detection was performed with a mixture of 12 pairs of oligonu-cleotide primers including one pair of published primers for amplifying all known pan-enterovirus genomes and eleven primer pairs specific for detection of the VP1 genes of EV71, C A16, CVA4, CVA5, CVA9,CVA10, CVB1, CVB3 and CVB5, respectively. The specificity of multiplex RT-PCR system was examined using enterovirus cell cultures and positive strains identified previously from hand-foot-and-mouth disease (HFMD) patients. Serial dilution of titrated EV71 and C A16 cell cultures and in vitro transcripted RNA of enterovirus VP1 regions were used to detect the sensitivity of the multiplex RT-PCR system. The limit of detection for this multiplex RT-PCR system was 100

  15. Prognostic Value of RT-PCR Tyrosinase Detection in Peripheral Blood of Melanoma Patients

    Directory of Open Access Journals (Sweden)

    Esmeralda Carrillo

    2006-01-01

    Full Text Available Malignant melanoma (MM prognosis has been related to tumour thickness and clinical stage and metastasis risk has been associated with presence of tumour cells in peripheral blood. The aim of this study was to determine the relationship between presence of tyrosinase in peripheral blood of MM patients and their clinical prognosis. Blood samples from 58 MM patients (stage I–IV were analysed, using RT-PCR assay to detect tyrosinase mRNA. The results showed that positive RT-PCR assay for tyrosinase were significantly associated with clinical status and tumour thickness. After a median follow-up of 24 months, RT-PCR results were found to be significant correlated with recurrence (p < 0.05 and clinical stage III (p < 0.05. Separate analysis of stage III tumours to determine the prognostic value of tyrosinase presence in peripheral blood showed an overall 24-month survival rate of 70% in the RT-PCR negative group versus 10% in the positive group (p < 0.02. These results suggest that detection of circulating melanoma cells may be especially relevant in stage III patients, in whom RT-PCR positivity defines a subpopulation at high risk of recurrence.

  16. 鸭黄病毒荧光定量RT-PCR检测方法的建立及应用%Development and application of real-time RT-PCR assay for duck flavivirus

    Institute of Scientific and Technical Information of China (English)

    高凤; 于可响; 马秀丽; 李玉峰; 王友令; 李建亮; 崔言顺

    2013-01-01

    A real-time RT-PCR method was developed for the detection of duck flavivirus (DFV). The specific primers and TaqMan probe were designed and synthesized according to the E gene sequence of duck flavivirus. The standard curve (Y=-3. 39 X + 43. 18,r = 0. 973) was plotted based on the linear relationship between the amount of plasmid DNA and cycle threshold (Ct). This method was specific for duck flavivirus(DFV) but not for duck hepatitis virus,avian influenza virus,Newcastle disease virus,adenovirus,duck plague virus and porcine Japanese encephalitis virus. The detection limit reached 1. 9 TCID50 of the virus under the optimized conditions. Tissues in five ducks after 86 h artificial infected with DFV were tested positive by this method, without parts of cecal tonsils. And the positive detection rate of the method was consistent with routine virus isolation method. These results indicated that the real-time RT-PCR approach provides a powerful diagnostic tool with high sensitivity and specificity for the identification and quantitation of DFV and the whole process of the test could be completed within 3 hours.%根据鸭黄病毒E蛋白基因序列设计了1对特异性引物和TaqMan探针,建立了实时荧光定量RT-PCR检测鸭黄病毒的方法.根据舍鸭黄病毒目的扩增片段的质粒拷贝数与定量反应Ct值的关系,绘制了标准曲线(y=-3.39 X+43.18,r=0.973).该方法具有特异性,对鸭病毒性肝炎病毒、禽流感病毒、新城疫病毒、腺病毒、鸭瘟病毒、猪乙脑病毒的核酸都没有扩增反应.敏感性试验显示,建立的此方法最低可检出1.9个TCID50病毒核酸.该方法对5只健康雏鸭人工感染86 h后的组织器官样品(盲肠扁桃体除外)的检测结果均为阳性,与病毒分离鉴定的阳性符合率为100%.结果表明,该方法真实可靠,而且从核酸提取到报告检测结果耗时不超过3h,为鸭黄病毒病提供了一种敏感特异的定量检测方法.

  17. Comparison of electron microscopy, ELISA, real time RT-PCR and insulated isothermal RT-PCR for the detection of Rotavirus group A (RVA) in feces of different animal species.

    Science.gov (United States)

    Soltan, Mohamed A; Tsai, Yun-Long; Lee, Pei-Yu A; Tsai, Chuan-Fu; Chang, Hsiao-Fen G; Wang, Hwa-Tang T; Wilkes, Rebecca P

    2016-09-01

    There is no gold standard for detection of Rotavirus Group A (RVA), one of the main causes of diarrhea in neonatal animals. Sensitive and specific real-time RT-PCR (rtRT-PCR) assays are available for RVA but require submission of the clinical samples to diagnostic laboratories. Patient-side immunoassays for RVA protein detection have shown variable results, particularly with samples from unintended species. A sensitive and specific test for detection of RVA on the farm would facilitate rapid management decisions. The insulated isothermal RT-PCR (RT-iiPCR) assay works in a portable machine to allow sensitive and specific on-site testing. The aim of this investigation was to evaluate a commercially available RT-iiPCR assay for RVA detection in feces from different animal species. This assay was compared to an in-house rtRT-PCR assay and a commercially available rtRT-PCR kit, as well as an ELISA and EM for RVA detection. All three PCR assays targeted the well-conserved NSP5 gene. Clinical fecal samples from 108 diarrheic animals (mainly cattle and horses) were tested. The percentage of positive samples by ELISA, EM, in-house rtRT-PCR, commercial rtRT-PCR, and RT-iiPCR was 29.4%, 31%, 36.7%, 51.4%, 56.9%, respectively. The agreement between different assays was high (81.3-100%) in samples containing high viral loads. The sensitivity of the RT-iiPCR assay appeared to be higher than the commercially available rtRT-PCR assay, with a limit of detection (95% confidence index) of 3-4 copies of in vitro transcribed dsRNA. In conclusion, the user-friendly, field-deployable RT-iiPCR system holds substantial promise for on-site detection of RVA.

  18. Duplex real-time qRT-PCR for the detection of hepatitis A virus in water and raspberries using the MS2 bacteriophage as a process control.

    Science.gov (United States)

    Blaise-Boisseau, Sandra; Hennechart-Collette, Catherine; Guillier, Laurent; Perelle, Sylvie

    2010-06-01

    Hepatitis A virus (HAV) infection is the leading worldwide cause of acute viral hepatitis. An important aspect of viral control is rapid diagnosis. Epidemiological studies have linked hepatitis A outbreaks to the consumption of drinking water or soft fruits exposed to faecal contamination. Real-time reverse transcriptase PCR (qRT-PCR) is now widely used for detecting RNA viruses in food samples. Efficiency of viral concentration, nucleic acid extraction and the presence of potential inhibitors of the RT-PCR reaction must be monitored to prevent false negative results. In this study, the MS2 bacteriophage used as a process control was detected simultaneously with HAV in a one-step duplex real-time qRT-PCR. The assay was developed for testing water and raspberries. Adding MS2 showed no loss of sensitivity for HAV detection in water and raspberry samples. The limit of detection of HAV with this new approach was 10PFU for 1.5L of bottled water, 100PFU for 1.5L of tap water, 50PFU for 25g of fresh raspberries and 100PFU for 25g of frozen raspberries. The data show that the MS2 offers a very reliable and simple way to monitor false-negative results, making it a valuable tool in the routine diagnostics laboratory.

  19. 鸭Ⅰ型肝炎病毒和番鸭细小病毒二重荧光定量RT-PCR方法的建立%Development of duplex real-time RT-PCR assay for detection of duck hepatitis virus type Ⅰ and muscovy duck parvovirus

    Institute of Scientific and Technical Information of China (English)

    谢丽基; 谢芝勋; 邓显文; 谢志勤; 庞耀珊; 范晴; 刘加波

    2013-01-01

    A duplex real-time reverse polymerase chain reaction (drRT-PCR) assay was developed and optimized to simultaneously detect duck hepatitis virus type Ⅰ and muscovy duck parvovirus in one reaction.Two sets of specific oligonucleotide primers for duck hepatitis virus type Ⅰ and muscovy duck parvovirus,along with two TaqMan probes specific for each virus were used in the assay.This drRT-PCR assay was found to be specific and to be able to detect and differentiate duck hepatitis virus type Ⅰ and muscovy duck parvovirus,and no positive results were observed when nucleic acid from duck circovirus,gosling plague virus,duck parvovirus,duck plague virus and avian influenza virus were used as drRT-PCR templates.The sensitivity of this drRT-PCR assay was 200 template copies for duck hepatitis virus type Ⅰ and muscovy duck parvovirus.This drRT-PCR assay is a quick,sensitive,and specific test for detection of duck hepatitis virus type Ⅰ and muscovy duck parvovirus,and will be useful for the control of these viruses in ducks.%根据基因库中鸭Ⅰ型肝炎病毒和番鸭细小病毒的基因序列,设计2对特异性引物和2条用不同荧光基团标记的TaqMan探针.对反应条件和试剂浓度进行优化,建立能够同时检测鸭Ⅰ型肝炎病毒和番鸭细小病毒的二重荧光定量RT-PCR方法.该方法敏感性好,对鸭Ⅰ型肝炎病毒和番鸭细小病毒的检测敏感性均达到200个模板拷贝数;该方法特异性强,对鸭圆环病毒、鹅细小病毒、鸭副黏病毒、鸭瘟病毒和禽流感病毒等病原体的检测全为阴性.本研究建立的鸭Ⅰ型肝炎病毒和番鸭细小病毒的二重荧光定量RT-PCR方法,具有快速、敏感、特异、定量和重复性好等优点,可用于临床上鸭Ⅰ型肝炎病毒和番鸭细小病毒感染的检测.

  20. Development of a novel recombinant encapsidated RNA particle: evaluation as an internal control for diagnostic RT-PCR

    DEFF Research Database (Denmark)

    King, Donald P.; Montague, Nick; Ebert, Katja

    2007-01-01

    This report describes the generation of novel encapsidated RNA particles and their evaluation as in-tube internal controls in diagnostic real-time reverse-transcription PCR (rRT-PCR) assays for the detection of RNA viruses. A cassette containing sequences of 2 diagnostic primer sets for foot...... unguiculata). RNA contained in these particles was amplified in diagnostic rRT-PCR assays used for detection of FMDV and SVDV. Amplification of these internal controls was used to confirm that rRT-PCR inhibitors were absent from clinical samples, thereby verifying negative assay results. The recombinant CPMVs...... did not reduce the analytical sensitivity of the rRT-PCRs when amplification of the insert was performed in the same tube as the diagnostic target. This system provides an attractive solution to the production of internal controls for rRT-PCR assays since CPMV grows to high yields in plants...

  1. Development of a novel recombinant encapsidated RNA particle: evaluation as an internal control for diagnostic RT-PCR

    DEFF Research Database (Denmark)

    King, Donald P.; Montague, Nick; Ebert, Katja

    2007-01-01

    did not reduce the analytical sensitivity of the rRT-PCRs when amplification of the insert was performed in the same tube as the diagnostic target. This system provides an attractive solution to the production of internal controls for rRT-PCR assays since CPMV grows to high yields in plants......This report describes the generation of novel encapsidated RNA particles and their evaluation as in-tube internal controls in diagnostic real-time reverse-transcription PCR (rRT-PCR) assays for the detection of RNA viruses. A cassette containing sequences of 2 diagnostic primer sets for foot...... unguiculata). RNA contained in these particles was amplified in diagnostic rRT-PCR assays used for detection of FMDV and SVDV. Amplification of these internal controls was used to confirm that rRT-PCR inhibitors were absent from clinical samples, thereby verifying negative assay results. The recombinant CPMVs...

  2. Development of a One-Step Probe Based Molecular Assay for Rapid Immunodiagnosis of Infection with M. tuberculosis Using Dried Blood Spots

    DEFF Research Database (Denmark)

    Blauenfeldt, Thomas; Heyckendorf, Jan; Graff Jensen, Sidse

    2014-01-01

    %, p = ns.). CONCLUSION: We developed a rapid, robust and accurate molecular immunodiagnostic test for M. tuberculosis infection. By combining DBS based sample acquisition, mail or currier based sample transport with centralized molecular detection, this immunodiagnostic test concept can reduce...... detection. AIM: To develop a robust IP-10 based molecular assay for the diagnosis of infection with M. tubercuolsis from whole blood and DBS. METHOD: We developed a one-step probe based multiplex RT-qPCR assay for detecting IP-10 and IFN-γ mRNA expression from whole blood and DBS samples. The assay...

  3. Detection and identification of infectious bronchitis virus by RT-PCR in Iran.

    Science.gov (United States)

    Homayounimehr, Alireza; Pakbin, Ahmad; Momayyez, Reza; Fatemi, Seyyedeh Mahsa Rastegar

    2016-06-01

    Infectious bronchitis virus (IBV) causes severe diseases in poultry with significant economic consequences to the poultry industry in Iran. The aim of this study was the detection and identification of IBV by reverse transcription(RT)-PCR in Iran. Ten IB virus strains were detected by testing trachea, cecal tonsil, and kidney tissues collected from broiler and layer farms in Iran. In order to detect infectious bronchitis virus, an optimized RT-PCR was used. Primers targeting the conserved region of known IBV serotypes were used in the RT-PCR assay. Primers selectively detecting Massachusetts and 793/B type IB viruses were designed to amplify the S1 gene of the virus and used in the nested PCR test. Our findings indicate the circulation of at least three genotypes of IB viruses (Massachusetts, 793/B, and variant 2) among poultry flocks.

  4. An isothermal, label-free, and rapid one-step RNA amplification/detection assay for diagnosis of respiratory viral infections.

    Science.gov (United States)

    Koo, Bonhan; Jin, Choong Eun; Lee, Tae Yoon; Lee, Jeong Hoon; Park, Mi Kyoung; Sung, Heungsup; Park, Se Yoon; Lee, Hyun Jung; Kim, Sun Mi; Kim, Ji Yeun; Kim, Sung-Han; Shin, Yong

    2017-04-15

    Recently, RNA viral infections caused by respiratory viruses, such as influenza, parainfluenza, respiratory syncytial virus, coronavirus, and Middle East respiratory syndrome-coronavirus (MERS-CoV), and Zika virus, are a major public health threats in the world. Although myriads of diagnostic methods based on RNA amplification have been developed in the last decades, they continue to lack speed, sensitivity, and specificity for clinical use. A rapid and accurate diagnostic method is needed for appropriate control, including isolation and treatment of the patients. Here, we report an isothermal, label-free, one-step RNA amplification and detection system, termed as iROAD, for the diagnosis of respiratory diseases. It couples a one-step isothermal RNA amplification method and a bio-optical sensor for simultaneous viral RNA amplification/detection in a label-free and real-time manner. The iROAD assay offers a one-step viral RNA amplification/detection example to rapid analysis (<20min). The detection limit of iROAD assay was found to be 10-times more sensitive than that of real-time reverse transcription-PCR method. We confirmed the clinical utility of the iROAD assay by detecting viral RNAs obtained from 63 human respiratory samples. We envision that the iROAD assay will be useful and potentially adaptable for better diagnosis of emerging infectious diseases including respiratory diseases.

  5. RT-PCR-ELISA as a tool for diagnosis of low-pathogenicity avian influenza

    DEFF Research Database (Denmark)

    Dybkær, Karen; Munch, Mette; Handberg, Kurt

    2003-01-01

    A one-tube reverse transcriptase/polymerase chain reaction coupled with an enzyme-linked immunosorbent assay (RT-PCR-ELISA) was developed for the rapid detection of avian influenza virus (AIV) in clinical specimens. A total of 419 swab pools were analyzed from chickens experimentally infected...

  6. Molecular detection of Papaya meleira virus in the latex of Carica papaya by RT-PCR

    NARCIS (Netherlands)

    Araujo, de M.M.M.; Tavares, E.T.; Silva, da F.R.; Marinho, V.L.D.; Souza, M.T.

    2007-01-01

    A RT-PCR assay was developed for early and accurate detection of Papaya meleira virus (PMeV) in the latex from infected papayas. The meleira disease is characterized by an excessive exudation of more fluidic latex from fruits, leaves and stems. This latex oxidises and gives the fruit a ¿sticky¿ text

  7. Predicting Gene Structures from Multiple RT-PCR Tests

    Science.gov (United States)

    Kováč, Jakub; Vinař, Tomáš; Brejová, Broňa

    It has been demonstrated that the use of additional information such as ESTs and protein homology can significantly improve accuracy of gene prediction. However, many sources of external information are still being omitted from consideration. Here, we investigate the use of product lengths from RT-PCR experiments in gene finding. We present hardness results and practical algorithms for several variants of the problem and apply our methods to a real RT-PCR data set in the Drosophila genome. We conclude that the use of RT-PCR data can improve the sensitivity of gene prediction and locate novel splicing variants.

  8. 双重胶体金层析法快速检测番茄环斑病毒和烟草环斑病毒RT-PCR扩增子%Duplex Detection of Tomato Ringspot Virus and Tobacco Ringspot Virus RT-PCR Amplicons by Dipstick Assay

    Institute of Scientific and Technical Information of China (English)

    曹成; 魏梅生; 张永江; 李桂芬; 吴兴泉

    2011-01-01

    Both tomato ringspot virus(ToRSV)and tobacco ringspot virus(TRSV) are quarantine pests for China. We have developed dipstick assay for detection of RT-PCR amplicons of the two viruses within 15 minutes. The single dipstick assay is performed by labeling primers of the virus with biotin and fluorescein (or digoxigenin). Monoclonal antibody against fluorescein (or digoxigenin) is spotted on nitrocellulose membrane as T dot. The dual-labeled(biotin-fluorescein or biotin-digoxigenin) RT-PCR amplicons can be detected on the test dot(T dot). The duplex dipstick assay is performed by labeling primers of ToRSV with biotin and fluorescein (or digoxigenin) and primers of TRSV with biotin and digoxigenin(or fluorescein). Monoclonal antibody against fluorescein and digoxigenin is spotted on nitrocellulose membrane respectively as T1 and T2 dot . The dual-labeled(biotin-fluorescein and biotin-digoxigenin) RT-PCR amplicons of the two viruses can be detected on the Tl and T2 dot simultaneously.%番茄环斑病毒和烟草环斑病毒是中国进境检疫性有害生物,此研究建立了单个和双重胶体金层析快速检测方法,在15 min即可获得对双标记PCR产物的检测结果.单个胶体金层析是在一张层析膜上点上荧光素(或地高辛)的单克隆抗体作为T检测点,经生物素-荧光素(或生物素-地高辛)双标记的RT-PCR扩增产物可在T点上被检测到.双重胶体金层析是在一张层析膜上分别点上荧光素和地高辛的单克隆抗体作为T1和T2检测点,经生物素-荧光素和生物素-地高辛双标记的两种病毒的RT-PCR扩增产物可分别在T1和T2点上被检测到.

  9. Infectious bronchitis virus: detection and vaccine Strain differentiation by semi-nested RT-PCR

    Directory of Open Access Journals (Sweden)

    CH Okino

    2005-03-01

    Full Text Available A semi-nested reverse transcription-polymerase chain reaction (Semi-N-RT-PCR was developed and used to detect the S glycoprotein gene of infectious bronchitis virus (IBV strains and to discriminate H120 vaccine strain from other strains. Viral RNA was extracted from the allantoic fluid of chicken embryos and from tissues of chickens experimentally infected with different strains of IBV. Amplification and identification of the viral RNA was performed using two sets of primers complementary to a region of the S glycoprotein gene in the Semi-N-RT-PCR assay. The pair of primers used in the first PCR consisted of universal oligonucleotides flanking a more variable region of S1-S2 gene. The second primer pair was used in the Semi-N-RT-PCR and was comprised of one of the primers from the first universal pair together with either another universal internal oligolucleotide or a oligonucleotide sequence specific for the H120 strain of IBV. The universal primers detected all reference IBV strains and field isolates tested herein. The Semi-N-RT-PCR had high sensitivity and specificity, and was able to differentiate the H120 vaccine strain from other reference IBV strains; including M41 strain. All tissue samples collected from chickens experimentally infected with H120 or M41 strains were positive in the semi-nested RT-PCR using universal primers, while only the H120-infected tissue samples were amplified by the set of primers containing the H120-oligonucleotide. In conclusion, the ability of Semi-N-RT-PCR to detect distinct IBV strains and preliminarily discriminate the vaccine strain (H120 closes a diagnostic gap and offers the opportunity to use comprehensive PCR procedures for the IBV diagnosis.

  10. RT-PCR and real-time RT-PCR methods for the detection of potato virus Y in potato leaves and tubers.

    Science.gov (United States)

    MacKenzie, Tyler D B; Nie, Xianzhou; Singh, Mathuresh

    2015-01-01

    Potato virus Y (PVY) is a major threat to potato crops around the world. It is an RNA virus of the family Potyviridae, exhibiting many different strains that cause a range of symptoms in potato. ELISA detection of viral proteins has traditionally been used to quantify virus incidence in a crop or seed lot. ELISA, however, cannot reliably detect the virus directly in dormant tubers, requiring several weeks of sprouting tubers to produce detectable levels of virus. Nor can ELISA fully discriminate between the wide range of strains of the virus. Several techniques for directly detecting the viral RNA have been developed which allow rapid detection of PVY in leaf or tuber tissue, and that can be used to easily distinguish between different strains of the virus. Described in this chapter are several protocols for the extraction of RNA from leaf and tuber tissues, and three detection methods based upon reverse-transcription-PCR (RT-PCR). First described is a traditional two-step protocol with separate reverse transcription of viral RNA into cDNA, then PCR to amplify the viral cDNA fragment. Second described is a one-step RT-PCR protocol combining the cDNA production and PCR in one tube and one step, which greatly reduces material and labor costs for PVY detection. The third protocol is a real-time RT-PCR procedure which not only saves on labor but also allows for more precise quantification of PVY titre. The three protocols are described in detail, and accompanied with a discussion of their relative advantages, costs, and possibilities for cost-saving modifications. While these techniques have primarily been developed for large-scale screening of many samples for determining viral incidence in commercial fields or seed lots, they are also amenable to use in smaller-scale research applications.

  11. Detection and quantitation of two cucurbit criniviruses in mixed infection by real-time RT-PCR.

    Science.gov (United States)

    Abrahamian, Peter E; Seblani, Rewa; Sobh, Hana; Abou-Jawdah, Yusuf

    2013-11-01

    Cucurbit chlorotic yellows virus (CCYV) and Cucurbit yellow stunting disorder virus (CYSDV) are whitefly-transmitted criniviruses infecting cucurbit crops inducing similar symptoms. Single and multiplex RT-PCR protocols were developed and evaluated on cucurbit samples collected from commercial greenhouses. Primers and probes were designed from the highly conserved heat shock protein 70 homolog (Hsp70h) gene. Conventional RT-PCR and multiplex RT-PCR assays showed high specificity and suitability for routine screening. TaqMan-based quantitative real-time RT-PCR (RT-qPCR) protocols were also developed for the detection and quantitation of both viruses occurring in single or mixed infection. The assays proved to be highly specific with no cross amplification. RT-qPCR assays showed a 100-1000 times improved sensitivity over conventional RT-PCR. Virus titers in mixed infections were compared to singly infected plants by RT-qPCR. CYSDV and CCYV titers decreased in double infected plants. This paper reports highly specific conventional RT-PCR and quantitative real-time PCR assays for detection, quantitation and differentiation between two closely related cucurbit-infecting criniviruses.

  12. Development and Application of Real-time RT-PCR Assay for Chicken Infectious Bronchitis Virus%鸡传染性支气管炎病毒Real-time RT-PCR检测方法的建立及应用

    Institute of Scientific and Technical Information of China (English)

    王鹏; 高峰; 王园; 杨莹; 路红; 周双海; 刘凤华

    2013-01-01

    The objective of this study was to develop a Real-time RT-PCR assay of chicken Infectious Bronchitis Virus (IBV) and therefore to detect the load IBV quantitatively. A fragment of N gene in IBV was amplified with RT-PCR method, and was cloned into the vector pEASY-T3. Then, the recombinant plasmid containing the N gene fragment was constructed. The standard curve and corresponding linear regression equation of IBV nucleic acid level were developed by Real-time PCR based on SYBR Green I with the recombinant plasmid. This method showed a high specificity and had a detection limit of 5.58X102 copies/p,L, and its coefficient of variations was less than 3.2% in the reproducible assays. The virus nucleic acid in tissue samples from chickens inoculated experimentally with IBV M41 strain was quantitatively determined with the established Real-time RT-PCR. The detection results showed that the load of IBV in the kidney was more than that in bronchus and lung after inoculation, and the virus load in the bronchus and lung on 3 days post-inoculation (DPI) were higher than those on 7 DPI and 10 DPI. Moreover, the correlation between the clinical manifestations and viral load was confirmed. The results indicated that this Real-time RT-PCR assay was of high specificity, sensitivity and reproducibility, and could be used for the quantitative detection of IBV.%为定量检测鸡传染性支气管炎病毒(IBV)载量,建立IBV的Real-time RT-PCR方法.用RT-PCR方法扩增出IBV的N基因片段,并克隆到pEASY-T3载体中,构建成含有N基因片段的重组质粒.应用该重组质粒进行SYBR Green I Real-time PCR,建立了定量检测IBV核酸的标准曲线与直线回归方程,该方法显示:特异性强,检测下限至少达到5.58× 102拷贝/μL,其重复性试验的变异系数小于3.2%;用建立的方法对实验接种IBV M41株的雏鸡组织中的病毒核酸进行了定量检测,检测结果显示:攻毒后肾脏中IBV含量高于支气管和肺脏,支气管

  13. Development and evaluation of a real-time RT-PCR assay for the detection of Ebola virus (Zaire) during an Ebola outbreak in Guinea in 2014-2015.

    Science.gov (United States)

    Dedkov, V G; Magassouba, N' F; Safonova, M V; Deviatkin, A A; Dolgova, A S; Pyankov, O V; Sergeev, A A; Utkin, D V; Odinokov, G N; Safronov, V A; Agafonov, A P; Maleev, V V; Shipulin, G A

    2016-02-01

    In early February 2014, an outbreak of the Ebola virus disease caused by Zaire ebolavirus (EBOV) occurred in Guinea; cases were also recorded in other West African countries with a combined population of approximately 25 million. A rapid, sensitive and inexpensive method for detecting EBOV is needed to effectively control such outbreak. Here, we report a real-time reverse-transcription PCR assay for Z. ebolavirus detection used by the Specialized Anti-epidemic Team of the Russian Federation during the Ebola virus disease prevention mission in the Republic of Guinea. The analytical sensitivity of the assay is 5 × 10(2) viral particles per ml, and high specificity is demonstrated using representative sampling of viral, bacterial and human nucleic acids. This assay can be applied successfully for detecting the West African strains of Z. ebolavirus as well as on strains isolated in the Democratic Republic of the Congo in 2014.

  14. Development and Characterization of Probe-Based Real Time Quantitative RT-PCR Assays for Detection and Serotyping of Foot-And-Mouth Disease Viruses Circulating in West Eurasia

    DEFF Research Database (Denmark)

    Jamal, Syed M.; Belsham, Graham

    2015-01-01

    Asia,A-Iran05 and Asia-1 (Group-II and Group-VII (Sindh-08)). In addition, field samples from Iran and Bulgaria, containing FMDVs belonging to the O-PanAsiaANT-10 subline-agewere also tested. Each of the three primer/probe sets was designed to be specific for just one of the serotypes O, A and Asia-1 of FMDV....... Comparisons of the sequences of the primers and probes used in these assays and the corresponding regions of the circulating viruses provided explanations for the poor recognition of some of the viruses by the earlier assays. These new assays should help in the early detection and typing of serotype O...

  15. 猪繁殖与呼吸综合征病毒和牛病毒性腹泻病毒双重RT-PCR检测方法的建立%Establishment of duplex RT-PCR assay for detection of bovine viral diarrhea virus and porcine reproductive and respiratory syndrome virus

    Institute of Scientific and Technical Information of China (English)

    王克伟; 李玉峰; 王先炜; 马涛; 张国龙; 姜平

    2011-01-01

    根据牛病毒性腹泻病毒(BVDV)5'端非编码区基因序列,设计合成了1对特异性引物,参考本实验室针对猪繁殖与呼吸综合征病毒(PRRSV)N蛋白设计的引物,经过PCR反应条件的优化,建立了BVDV和PRRSV双重RT-PCR的检测方法.对于PRRSV和BVDV的cDNA最低检测量分别为3.8×10-4 ng和7×1014 ng,对于猪瘟病毒(CFSV)、脑心肌炎病毒(EMCV)和猪圆环病毒2型(PCV-2)的PCR扩增结果均为阴性;用该方法对江苏省不同地区采集的75份仔猪的肺脏、脾脏和淋巴结等病料进行了检测,结果PRRSV有55份阳性,BVDV有14份阳性,PRRSV和BVDV混合感染的有12份,与PRRSV和BVDV单一RT-PCR的检测结果符合率分别为89.3%和92%.证明建立的双重RT-PCR检测方法可用于临床样品中BVDV和PRRSV的检测.%According to the sequence of 5' untranslated region (5'UTR) of the bovine viral diarrhea virus ( BVDV) genome, a pair of primers was designed and synthesized. Combined with the established primers of the N protein of porcine reproductive and respiratory syndrome vims ( PRRSV) , a duplex RT-PCR was established for detection of the two viruses simultaneously based on the optimization of the reaction condition. The specific PCR products were 290 bp for BVDV and 374 bp for PRRSV in length. The detection limit of cDNA template in the RT-PCR was 3. 8×1O-4 ng for PRRSV and 7×10-4 ng for BVDV. The high specificity of the method was demonstrated by detecting classical swine fever virus ( CSFV) , encephalomyocarditis virus ( FMCV) and porcine circovirus 2 ( PCV-2). Among 75 clinical samples,there were 55 samples with PRRSV positive, 14 samples with BVDV positive, and 12 samples with coinfection of BVDV and PRRSV. The coincidence rates were 89. 3% and 92% with the single RT-PCR for PRRSV and BVDV, respectively. It suggested that the duplex RT-PCR assay in this study could be applied for the detection of BVDV and PRRSV in clinical samples.

  16. DETECTION OF CLASSICAL SWINE FEVER VIRUS BY RT-PCR IN WEST BENGAL, INDIA

    Directory of Open Access Journals (Sweden)

    Sumit Chowdhury

    2016-12-01

    Full Text Available Classical swine fever is a deadly disease of swine, caused by a RNA virus. The present study has identified presence of the classical swine fever virus (CSFV in pigs of West Bengal by one step reverse transcriptase PCR (RT-PCR performed using 5’ NTR specific primers. Internal organs from clinically affected pigs were examined from three districts of West Bengal. RT-PCT has identified presence of CSFV in all the tissues examined confirming presence of CSFV in different parts of the state.

  17. Quantification of transcript levels with quantitative RT-PCR.

    Science.gov (United States)

    Carleton, Karen L

    2011-01-01

    Differential gene expression is a key factor driving phenotypic divergence. Determining when and where gene expression has diverged between organisms requires a quantitative method. While large-scale approaches such as microarrays or high-throughput mRNA sequencing can identify candidates, quantitative RT-PCR is the definitive method for confirming gene expression differences. Here, we describe the steps for performing qRT-PCR including extracting total RNA, reverse-transcribing it to make a pool of cDNA, and then quantifying relative expression of a few candidate genes using real-time or quantitative PCR.

  18. RT-PCR detection of HIV in Republic of Macedonia.

    Science.gov (United States)

    Bosevska, Golubinka; Panovski, Nikola; Dokić, Eleni; Grunevska, Violeta

    2008-11-01

    The aim of the study was to detect HIV RNA in seropositive patients using RT-PCR method and thus, to establish PCR methodology in the routine laboratory works. The total of 33 examined persons were divided in two groups: 1) 13 persons seropositive for HIV; and 2) 20 healthy persons - randomly selected blood donors that made the case control group. The subjects age was between 25 and 52 years (average 38,5). ELFA test for combined detection of HIV p24 antigen and anti HIV-1+2 IgG and ELISA test for detection of antibodies against HIV-1 and HIV-2, were performed for each examined person. RNA from the whole blood was extracted using a commercial kit based on salt precipitation. Detection of HIV RNA was performed using RT-PCR kit. Following nested PCR, the product was separated by electrophoresis in 1,5 % agarose gel. The result was scored positive if the band of 210bp was visible regardless of intensity. Measures of precaution were taken during all the steps of the work and HIV infected materials were disposed of accordingly. In the group of blood donors ELFA, ELISA and RT-PCR were negative. Assuming that prevalence of HIV infection is zero, the clinical specificity of RT-PCR is 100 %. The analytical specificity of RT-PCR method was tested against Hepatitis C and B, Human Papiloma Virus, Cytomegalovirus, Herpes Simplex Virus, Rubella Virus, Mycobacterium tuberculosis, Chlamydia trachomatis. None of these templates yielded amplicon. In the group of 13 seropositive persons, 33 samples were analyzed. HIV RNA was detected in 15 samples. ELISA and ELFA test were positive in all samples. Different aliquots of the samples were tested independently and showed the same results. After different periods of storing the RNA samples at -70 masculineC, RT-PCR reaction was identical to the one performed initially. The obtained amplicons were maintained frozen at -20 masculineC for a week and the subsequently performed electrophoresis was identical to the previous one. The reaction is

  19. Rapid identification viruses from nasal pharyngeal aspirates in acute viral respiratory infections by RT-PCR and electrospray ionization mass spectrometry.

    Science.gov (United States)

    Chen, Kuan-Fu; Rothman, Richard E; Ramachandran, Padmini; Blyn, Lawrence; Sampath, Rangarajan; Ecker, David J; Valsamakis, Alexandra; Gaydos, Charlotte A

    2011-04-01

    Diagnosis of the etiologic agent of respiratory viral infection relies traditionally on culture or antigen detection. This pilot evaluation compared performance characteristics of the RT-PCR and electrospray ionization mass spectrometry (RT-PCR/ESI-MS) platform to conventional virologic methods for identifying multiple clinically relevant respiratory viruses in nasopharyngeal aspirates. The RT-PCR/ESI-MS respiratory virus surveillance kit was designed to detect respiratory syncytial virus, influenza A and B, parainfluenza types 1-4, adenoviridae types A-F, coronaviridae, human bocavirus, and human metapneumovirus. Patients (N=192) attending an emergency department during the 2007-2008 respiratory season consented, and "excess" frozen archived nasopharyngeal aspirates were analysed; 46 were positive by conventional virology and 69 by RT-PCR/ESI-MS, among which there were six samples with multiple viral pathogens detected. The sensitivity and specificity of the assay were 89.1% and 80.3%, respectively. Additional viruses that were not identified by conventional virology assays were detected (4 human bocaviruses and 7 coronaviruses). Samples in which the RT-PCR/ESI-MS results disagreed with conventional virology were sent for analysis by a third method using a commercial RT-PCR-based assay, which can identify viruses not detectable by conventional virologic procedures. Time to first result of RT-PCR/ESI-MS was 8h. RT-PCR/ESI-MS demonstrated capacity to detect respiratory viruses identifiable and unidentifiable by conventional methods rapidly.

  20. A new quantitative RT-PCR method for sensitive detection of dengue virus in serum samples.

    Science.gov (United States)

    Sadon, Nadine; Delers, Anne; Jarman, Richard G; Klungthong, Chonticha; Nisalak, Ananda; Gibbons, Robert V; Vassilev, Ventzislav

    2008-10-01

    In order to detect and identify dengue serotypes in serum samples, we developed a single-step quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) assay (referred to as Q-PCR). Sets of primers were selected from the capsid region of the viral genome. Dengue serotypes 1/3 and 2/4 were detected in two separate duplex amplification reactions using specific primers and fluorogenic TaqMan probes. Results obtained with this Q-PCR and the classical nested RT-PCR (N-PCR) assays were compared using a panel of 97 representative human sera collected from patients in Bangkok, Thailand. It is shown that the Q-PCR is a rapid, sensitive and reproducible tool for the detection and quantitation of the four dengue serotypes in clinical samples, and therefore of great interest for diagnostic use or for large cohort studies.

  1. One-Step Multiplex RT-qPCR Assay for the Detection of Peste des petits ruminants virus, Capripoxvirus, Pasteurella multocida and Mycoplasma capricolum subspecies (ssp.) capripneumoniae

    Science.gov (United States)

    Lamien, Charles Euloge; Spergser, Joachim; Lelenta, Mamadou; Wade, Abel; Gelaye, Esayas; Loitsch, Angelika; Minoungou, Germaine; Thiaucourt, Francois; Diallo, Adama

    2016-01-01

    Respiratory infections, although showing common clinical symptoms like pneumonia, are caused by bacterial, viral or parasitic agents. These are often reported in sheep and goats populations and cause huge economic losses to the animal owners in developing countries. Detection of these diseases is routinely done using ELISA or microbiological methods which are being reinforced or replaced by molecular based detection methods including multiplex assays, where detection of different pathogens is carried out in a single reaction. In the present study, a one-step multiplex RT-qPCR assay was developed for simultaneous detection of Capripoxvirus (CaPV), Peste de petits ruminants virus (PPRV), Pasteurella multocida (PM) and Mycoplasma capricolum ssp. capripneumonia (Mccp) in pathological samples collected from small ruminants with respiratory disease symptoms. The test performed efficiently without any cross-amplification. The multiplex PCR efficiency was 98.31%, 95.48%, 102.77% and 91.46% whereas the singleplex efficiency was 93.43%, 98.82%, 102.55% and 92.0% for CaPV, PPRV, PM and Mccp, respectively. The correlation coefficient was greater than 0.99 for all the targets in both multiplex and singleplex. Based on cycle threshold values, intra and inter assay variability, ranged between the limits of 2%–4%, except for lower concentrations of Mccp. The detection limits at 95% confidence interval (CI) were 12, 163, 13 and 23 copies/reaction for CaPV, PPRV, PM and Mccp, respectively. The multiplex assay was able to detect CaPVs from all genotypes, PPRV from the four lineages, PM and Mccp without amplifying the other subspecies of mycoplasmas. The discriminating power of the assay was proven by accurate detection of the targeted pathogen (s) by screening 58 viral and bacterial isolates representing all four targeted pathogens. Furthermore, by screening 81 pathological samples collected from small ruminants showing respiratory disease symptoms, CaPV was detected in 17 samples

  2. One-Step Multiplex RT-qPCR Assay for the Detection of Peste des petits ruminants virus, Capripoxvirus, Pasteurella multocida and Mycoplasma capricolum subspecies (ssp. capripneumoniae.

    Directory of Open Access Journals (Sweden)

    Tirumala Bharani Kumar Settypalli

    Full Text Available Respiratory infections, although showing common clinical symptoms like pneumonia, are caused by bacterial, viral or parasitic agents. These are often reported in sheep and goats populations and cause huge economic losses to the animal owners in developing countries. Detection of these diseases is routinely done using ELISA or microbiological methods which are being reinforced or replaced by molecular based detection methods including multiplex assays, where detection of different pathogens is carried out in a single reaction. In the present study, a one-step multiplex RT-qPCR assay was developed for simultaneous detection of Capripoxvirus (CaPV, Peste de petits ruminants virus (PPRV, Pasteurella multocida (PM and Mycoplasma capricolum ssp. capripneumonia (Mccp in pathological samples collected from small ruminants with respiratory disease symptoms. The test performed efficiently without any cross-amplification. The multiplex PCR efficiency was 98.31%, 95.48%, 102.77% and 91.46% whereas the singleplex efficiency was 93.43%, 98.82%, 102.55% and 92.0% for CaPV, PPRV, PM and Mccp, respectively. The correlation coefficient was greater than 0.99 for all the targets in both multiplex and singleplex. Based on cycle threshold values, intra and inter assay variability, ranged between the limits of 2%-4%, except for lower concentrations of Mccp. The detection limits at 95% confidence interval (CI were 12, 163, 13 and 23 copies/reaction for CaPV, PPRV, PM and Mccp, respectively. The multiplex assay was able to detect CaPVs from all genotypes, PPRV from the four lineages, PM and Mccp without amplifying the other subspecies of mycoplasmas. The discriminating power of the assay was proven by accurate detection of the targeted pathogen (s by screening 58 viral and bacterial isolates representing all four targeted pathogens. Furthermore, by screening 81 pathological samples collected from small ruminants showing respiratory disease symptoms, CaPV was detected in

  3. RT-PCR Protocols - Methods in Molecular Biology

    Directory of Open Access Journals (Sweden)

    Manuela Monti

    2011-03-01

    Full Text Available “The first record I have of it, is when I made a computer file which I usually did whenever I had an idea, that would have been on the Monday when I got back, and I called it Chain Reaction.POL, meaning polymerase. That was the identifier for it and later I called the thing the Polymerase Chain Reaction, which a lot of people thought was a dumb name for it, but it stuck, and it became PCR”. With these words the Nobel prize winner, Kary Mullis, explains how he named the PCR: one of the most important techniques ever invented and currently used in molecular biology. This book “RT-PCR Protocols” covers a wide range of aspects important for the setting of a PCR experiment for both beginners and advanced users. In my opinion the book is very well structured in three different sections. The first one describes the different technologies now available, like competitive RT-PCR, nested RT-PCR or RT-PCR for cloning. An important part regards the usage of PCR in single cell mouse embryos, stressing how important...........

  4. Development of a thermostabilized, one-step, nested, tetraplex PCR assay for simultaneous identification and differentiation of Entamoeba species, Entamoeba histolytica and Entamoeba dispar from stool samples.

    Science.gov (United States)

    Foo, Phiaw Chong; Chan, Yean Yean; See Too, Wei Cun; Tan, Zi Ning; Wong, Weng Kin; Lalitha, Pattabhiraman; Lim, Boon Huat

    2012-09-01

    Entamoeba histolytica is the only Entamoeba species that causes amoebiasis in humans. Approximately 50 million people are infected, with 100, 000 deaths annually in endemic countries. Molecular diagnosis of Entamoeba histolytica is important to differentiate it from the morphologically identical Entamoeba dispar to avoid unnecessary medication. Conventional molecular diagnostic tests require trained personnel, cold-chain transportation and/or are storage-dependent, which make them user-unfriendly. The aim of this study was to develop a thermostabilized, one-step, nested, tetraplex PCR assay for the detection of Entamoeba histolytica, Entamoeba dispar and Entamoeba species in cold-chain-free and ready-to-use form. The PCR test was designed based on the Entamoeba small subunit rRNA (SSU-rRNA) gene, which detects the presence of any Entamoeba species, and simultaneously can be used to differentiate Entamoeba histolytica from Entamoeba dispar. In addition, a pair of primers was designed to serve as an internal amplification control to help identify inhibitors in the samples. All PCR reagents together with the designed primers were thermostabilized by lyophilization and were stable at 24 °C for at least 6 months. The limit of detection of the tetraplex PCR was found to be 39 pg DNA or 1000 cells for Entamoeba histolytica and 78 pg DNA or 1000 cells for Entamoeba dispar, and the specificity was 100 %. In conclusion, this cold-chain-free, thermostabilized, one-step, nested, multiplex PCR assay was found to be efficacious in differentiating Entamoeba histolytica from other non-pathogenic Entamoeba species.

  5. 应用含内参的多重实时荧光RT-PCR方法快速检测登革病毒和基孔肯雅病毒%Rapid detection of Dengue virus and Chikungunya virus by multiplex real-time RT-PCR assay with an internal control

    Institute of Scientific and Technical Information of China (English)

    郑夔; 丁国允; 周惠琼; 谢雪妹; 李小波; 师永霞; 苏锦坤; 黄吉城

    2013-01-01

    目的 建立一种登革病毒、基孔肯雅病毒并含人类基因内参检测的多重实时荧光RT-PCR方法,能在同一反应管内同时检测目前发现的所有来源的登革病毒或基孔肯雅病毒.方法 针对登革病毒3′端非编码区和基孔肯雅病毒结构蛋白E2-6K-E1区以及人体各类组织细胞中均能稳定表达的RNAse P基因,设计了3套特异性引物和探针,建立了1套能同时检测登革病毒、基孔肯雅病毒及含有人类基因检测内参的多重实时荧光RT-PCR方法,对其灵敏性和特异性进行了验证,并对临床发热病人标本进行了应用评估.结果 该方法对检测体外转录合成的登革病毒和基孔肯雅病毒RNA的灵敏性可达最低每个反应10~100拷贝,对检测登革1型病毒和基孔肯雅病毒的灵敏性分别可达最低每个反应0.1 TCID50/mL和1 TCID50/mL.用20株登革病毒、4株基孔肯雅病毒和日本脑炎病毒、西尼罗病毒、黄热病毒、盖塔病毒、辛德毕斯病毒各1株进行检测,方法的特异性均为100%.方法应用于189份发热病人血清标本检测,可准确地鉴定出其中登革病毒或基孔肯雅病毒核酸阳性的标本,且所有血清标本均能被内参引物和探针有效地扩增和杂交.结论 本研究建立了一种高灵敏性、高特异性且含人类基因内参检测的登革病毒和基孔肯雅病毒多重实时荧光RT-PCR检测方法,可作为登革热或基孔肯雅热病人早期快速鉴别诊断的有效工具,也可用于蚊媒携带登革病毒或基孔肯雅病毒的高通量快速筛查.%The purpose was to establish a multiplex real-time RT-PCR assay for detection of Dengue virus and Chikun-gunya virus in one tube, which could detect all Dengue virus or Chikungunya virus strains from different origins . Based on sequences of 3 -UTR of Dengue virus , E2-6K-E1 region of Chikungunya virus's structural protein and RNAse P gene which stably expressed in all human organs , 3 pairs of

  6. Monitoring gene expression: quantitative real-time rt-PCR.

    Science.gov (United States)

    Wagner, Elke M

    2013-01-01

    Two-step quantitative real-time RT-PCR (RT-qPCR), also known as real-time RT-PCR, kinetic RT-PCR, or quantitative fluorescent RT-PCR, has become the method of choice for gene expression analysis during the last few years. It is a fast and convenient PCR method that combines traditional RT-PCR with the phenomenon of fluorescence resonance energy transfer (FRET) using fluorogenic primers. The detection of changes in fluorescence intensity during the reaction enables the user to follow the PCR reaction in real time.RT-qPCR comprises several steps: (1) RNA is isolated from target tissue/cells; (2) mRNA is reverse-transcribed to cDNA; (3) modified gene-specific PCR primers are used to amplify a segment of the cDNA of interest, following the reaction in real time; and (4) the initial concentration of the selected transcript in a specific tissue or cell type is calculated from the exponential phase of the reaction. Relative quantification or absolute quantification compared to standards that are run in parallel can be performed.This chapter describes the entire procedure from isolation of total RNA from liver and fatty tissues/cells to the use of RT-qPCR to study gene expression in these tissues. We perform relative quantification of transcripts to calculate the fold-difference of a certain mRNA level between different samples. In addition, tips for choosing primers and performing analyses are provided to help the beginner in understanding the technique.

  7. Combination of immunohistochemistry, FISH and RT-PCR shows high incidence of Xp11 translocation RCC: comparison of three different diagnostic methods.

    Science.gov (United States)

    Lee, Hyun Jung; Shin, Dong Hoon; Noh, Gyu You; Kim, Young Keum; Kim, Ahrong; Shin, Nari; Lee, Jung Hee; Choi, Kyung Un; Kim, Jee Yeon; Lee, Chang Hun; Sol, Mee Young; Rha, Seo Hee; Park, Sung Woo

    2017-05-09

    We evaluated the frequency of translocation renal cell carcinoma (RCC) by reverse transcription polymerase chain reaction (RT-PCR) and how well the TFE3 immunoreactivity is concordant with TFE3 gene translocation status proved by fluorescence in situ hybridization (FISH) assay and RT-PCR. TFE3 and Cathepsin K expression was analyzed by immunohistochemistry in 185 RCC cases, and 48 cases either of more than weak expression of TFE3 or of positivity for Cathepsin K were done for FISH analysis and RT-PCR. All the RT-PCR positive cases were confirmed by cloning and sequencing. Of the 14 cases with strong nuclear TFE3 expression, 12 showed a break-apart signal by FISH. ASPL- and PRCC-TFE3 translocations were detected in 13 and one case, respectively, by RT-PCR. Of 21 cases with weak TFE3 expression, five were translocation-positive by FISH. ASPL-, PRCC-, and PSF-TFE3 translocations were detected by RT-PCR (n=3, 3, and 1, respectively). All 13 TFE3-negative/cathepsin K-positive cases were negative by FISH and two each harbored ASPL- and PRCC-TFE3 translocations that were detected by RT-PCR. A high rate of TFE3 immunoreactivity (8.6%) was confirmed by RT-PCR (13.5%) and FISH (9.7%). Higher translocation rate of RT-PCR means RT-PCR detected translocation in TFE3 weak expression group and only cathepsin K positive group more specifically than FISH. Thus, RT-PCR would complement FISH analysis for detecting translocation RCC with fusion partners.

  8. Multiplex real-time RT-PCR for detecting chikungunya virus and dengue virus

    Institute of Scientific and Technical Information of China (English)

    Piyathida Pongsiri; Kesmanee Praianantathavorn; Apiradee Theamboonlers; Sunchai Payungporn; Yong Poovorawan

    2012-01-01

    ABSTRACT Objective:To develop diagnostic test for detection chikungunya virus (CHIKV and Dengue virus (DENV)infection.Methods:We have performed a rapid, accurate laboratory confirmative method to simultaneously detect, quantify and differentiateCHIKV and DENV infection by single-step multiplex real-timeRT-PCR.Results: The assay’s sensitivity was97.65%, specificity was 92.59% and accuracy was95.82% when compared to conventional RT-PCR. Additionally, there was no cross-reaction betweenCHIKV, DENV, Japanese encephalitis virus, hepatitis C, hepatitis A or hepatitis E virus.Conclusions:This rapid and reliable assay provides a means for simultaneous early diagnosis ofCHIKV andDENV in a single-step reaction.

  9. Role of real-time PCR (RT-PCR) in rapid diagnosis of tuberculous mycobacteria in different clinical samples.

    Science.gov (United States)

    2014-02-01

    The study was aimed for molecular detection of mycobacterial DNA in different clinical samples using real-time polymerase chain reaction (RT-PCR) system and rapid diagnosis of tuberculosis. A total of 508 clinical specimens (blood 343, menstrual fluid 53, endometrial tissue 43, body fluid 36, pus from lymph nodes 18, sputum 8, urine 5 and semen 2) were included in this study. We extracted DNA using QIAamp DNA Mini Kit (QIAGEN, Germany) and performed real-time assay using Rotor-Gene Q machine from Corbett Research, Australia for specific amplification of IS6110 sequence of mycobacterial genome. The RT-PCR result was also compared with bacterial culture and acid-fast bacillus staining. RT-PCR assay showed positivity in 52 cases and negative in 456 cases. Corresponding positive results in culture and acid-fast bacillus staining methods were 49 cases and 24 cases respectively. The sensitivity and specificity of detecting Mycobacterium tuberculosis by RT-PCR were 93.87% and 98.69% respectively taking positive culture results as reference standards. The overall positive and negative predictive values were 88.46% and 99.34% respectively. RT-PCR is a useful diagnostic tool for rapid and sensitive detection of mycobacteria in different clinical samples. The easy processing, fast reporting and relative lack of contamination issues make it worthy as a possible replacement to time consuming culture techniques. Moreover, it has added advantage of quantification of mycobacterial DNA, hence bacterial load.

  10. Nested RT-PCR method for the detection of European avian-like H1 swine inlfuenza A virus

    Institute of Scientific and Technical Information of China (English)

    WEI Yan-di; PEI Xing-yao; ZHANG Yuan; YU Chen-fang; SUN Hong-lei; LIU Jin-hua; PU Juan

    2016-01-01

    Swine inlfuenza A virus (swine IAV) circulates worldwide in pigs and poses a serious public health threat, as evidenced by the 2009 H1N1 inlfuenza pandemic. Among multiple subtypes/lineages of swine inlfuenza A viruses, European avian-like (EA) H1N1 swine IAV has been dominant since 2005 in China and caused infections in humans in 2010. Highly sensitive and speciifc methods of detection are required to differentiate EA H1N1 swine IAVs from viruses belonging to other lineages and subtypes. In this study, a nested reverse transcription (RT)-PCR assay was developed to detect EA H1 swine IAVs. Two primer sets (outer and inner) were designed speciifcaly to target the viral hemagglutinin genes. Speciifc PCR products were obtained from al tested EA H1N1 swine IAV isolates, but not from other lineages of H1 swine IAVs, other subtypes of swine IAVs, or other infectious swine viruses. The sensitivity of the nested RT-PCR was improved to 1 plaque forming unit (PFU) mL–1which was over 104 PFU mL–1 for a previously established multiplex RT-PCR method. The nested RT-PCR results obtained from screening 365 clinical samples were consistent with those obtained using conventional virus isolation methods combined with sequencing. Thus, the nested RT-PCR assay reported herein is more sensitive and suitable for the diagnosis of clinical infections and surveilance of EA H1 swine IAVs in pigs and humans.

  11. A novel enterovirus and parechovirus multiplex one-step real-time PCR-validation and clinical experience

    DEFF Research Database (Denmark)

    Nielsen, A. C. Y.; Bottiger, B.; Midgley, S. E.

    2013-01-01

    and human parechovirus type 3 had a similar seasonal pattern with a peak during the summer and autumn. Human parechovirus type 3 was almost invariably found in children less than 4 months of age. In conclusion, a multiplex assay was developed allowing simultaneous detection of 2 viruses, which can cause......As the number of new enteroviruses and human parechoviruses seems ever growing, the necessity for updated diagnostics is relevant. We have updated an enterovirus assay and combined it with a previously published assay for human parechovirus resulting in a multiplex one-step RT-PCR assay....... The multiplex assay was validated by analysing the sensitivity and specificity of the assay compared to the respective monoplex assays, and a good concordance was found. Furthermore, the enterovirus assay was able to detect 42 reference strains from all 4 species, and an additional 9 genotypes during panel...

  12. Development of a one-step PCR assay with nine primer pairs for the detection of five diarrheagenic Escherichia coli types.

    Science.gov (United States)

    Oh, Kyung-Hwan; Kim, Soo-Bok; Park, Mi-Sun; Cho, Seung-Hak

    2014-06-28

    Certain Escherichia coli (E. coli) strains have the ability to cause diarrheal disease. Five types of diarrheagenic E. coli have been identified, including EHEC, ETEC, EPEC, EAEC, and EIEC. To detect these five diarrheagenic types rapidly, we developed a one-step multiplex PCR (MPPCR) assay using nine primer pairs to amplify nine virulence genes specific to the different virotypes, with each group being represented (i.e., stx1 and stx2 for EHEC, lt, sth, and stp for ETEC, eaeA and bfpA for EPEC, aggR for EAEC, and ipaH for EIEC). The PCR primers were constructed using MultAlin. The sensitivity and specificity of the constructed multiplex PCR primers were measured using DNA isolated from diarrheagenic E. coli strains representing each group. The limits of detection were as follows: 5 × 10(1) CFU/ml for EHEC, 5 × 10(3) CFU/ml for ETEC expressing lt and sth, 5 × 10(4) CFU/ml for ETEC expressing stp, 5 × 102 CFU/ml for EPEC, 5 × 10(4) CFU/ml for EAEC, and 5 × 10(2) CFU/ml for EIEC. To confirm the specificity, C. jejuni, C. perfringens, S. Typhimurium, V. parahaemolyticus, L. monocytogenes, Y. enterocolitica, B. cereus, and S. aureus were used as negative controls, and no amplification was obtained for these. Moreover, this kit was validated using 100 fecal samples from patients with diarrhea and 150 diarrheagenic E. coli strains isolated in Korea. In conclusion, the multiplex PCR assay developed in this study is very useful for the rapid and specific detection of five diarrheagenic E. coli types. This single-step assay will be useful as a rapid and economical method, as it reduces the cost and time required for the identification of diarrheagenic E. coli.

  13. Rapid assay for H7N9 avian influenza virus genes by real-time fluorescence quantitative RT-PCR%实时荧光定量RT-PCR快速检测H7N9禽流感病毒

    Institute of Scientific and Technical Information of China (English)

    张舟听; 夏文英; 王忠发; 刘妙; 翁瑛瑛

    2014-01-01

    amplification curves,the detection time was 50 minutes,the detection limit of H7 gene and N9 gene was 28 copies/μL and 41 copies/μL,respectively.Repeatability assay showed that coefficients of variation of Ct value were 0.17%-0.89%and 0.33%-0.59%,respectively,amplification efficiencies were 0.9491 and 0.9713.No significant cross-reaction was found between H7N9 avian influenza virus and other common avian influenza viruses or enteroviruses.The positive rate was 5.43%(10/184) in 184 field suspicious samples,which was consistent with Kit Ⅱ.Conclusions The real-time fluorescence quantitative RT-PCR reaction system develops for rapid assay of H7N9 avian influnenza virus gene,good sensitivity,specificity,repeatability and reliable results,and can be used for emergency detection and early diagnosis of clinical suspected patients with H7N9 avian influenza virus infection,and for surveillance of the virus in external environments like chicken farms and live poultry markets.

  14. Application and evaluation of RT-PCR-ELISA for the nucleoprotein and RT-PCR for detection of low-pathogenic H5 and H7 subtypes of avian influenza virus

    DEFF Research Database (Denmark)

    Dybkær, Karen; Munch, Mette; Handberg, Kurt J.

    2004-01-01

    Three 1-tube Reverse Transcriptase Polymerase Chain Reactions (RT-PCR) directed against the genes encoding the nucleoprotein (NP) and the H5 and H7 hemagglutinin (HA) gene, respectively, were used for detection of avian influenza virus (AIV) in various specimens. A total of 1,040 samples...... originating from chickens experimentally infected with 2 different low pathogenic avian influenza viruses, from domestic ducks and from wild aquatic birds were examined. The outcome of 1) the universal AIV RT-PCR including a PCR-enzyme-linked immunosorbent assay (ELISA) procedure directed against NP (NP RT...

  15. Application and evaluation of RT-PCR-ELISA for the nucleoprotein and RT-PCR for detection of low-pathogenic H5 and H7 subtypes of avian influenza virus

    DEFF Research Database (Denmark)

    Dybkær, Karen; Munch, Mette; Handberg, Kurt J.

    2004-01-01

    Three 1-tube Reverse Transcriptase Polymerase Chain Reactions (RT-PCR) directed against the genes encoding the nucleoprotein (NP) and the H5 and H7 hemagglutinin (HA) gene, respectively, were used for detection of avian influenza virus (AIV) in various specimens. A total of 1,040 samples originat......Three 1-tube Reverse Transcriptase Polymerase Chain Reactions (RT-PCR) directed against the genes encoding the nucleoprotein (NP) and the H5 and H7 hemagglutinin (HA) gene, respectively, were used for detection of avian influenza virus (AIV) in various specimens. A total of 1,040 samples...... originating from chickens experimentally infected with 2 different low pathogenic avian influenza viruses, from domestic ducks and from wild aquatic birds were examined. The outcome of 1) the universal AIV RT-PCR including a PCR-enzyme-linked immunosorbent assay (ELISA) procedure directed against NP (NP RT...

  16. Development of real-time RT-PCR for the detection of low concentrations of Rift Valley fever virus.

    Science.gov (United States)

    Maquart, Marianne; Temmam, Sarah; Héraud, Jean-Michel; Leparc-Goffart, Isabelle; Cêtre-Sossah, Catherine; Dellagi, Koussay; Cardinale, Eric; Pascalis, Hervé

    2014-01-01

    In recent years, Madagascar and the Comoros archipelago have been affected by epidemics of Rift Valley fever (RVF), however detection of Rift Valley fever virus (RVFV) in zebu, sheep and goats during the post epidemic periods was frequently unsuccessful. Thus, a highly sensitive real-time RT-PCR assay was developed for the detection of RVFV at low viral loads. A new RVF SYBR Green RT-PCR targeting the M segment was tested on serum from different RVF seronegative ruminant species collected from May 2010 to August 2011 in Madagascar and the Comoros archipelago and compared with a RVF specific quantitative real time RT-PCR technique, which is considered as the reference technique. The specificity was tested on a wide range of arboviruses or other viruses giving RVF similar clinical signs. A total of 38 out of 2756 serum samples tested positive with the new RT-PCR, whereas the reference technique only detected 5 out of the 2756. The described RT-PCR is an efficient diagnostic tool for the investigation of enzootic circulation of the RVF virus. It allows the detection of low viral RNA loads adapted for the investigations of reservoirs or specific epidemiological situations such as inter-epizootic periods.

  17. Detection of enteroviruses and hepatitis a virus in water by consensus primer multiplex RT-PCR

    Institute of Scientific and Technical Information of China (English)

    Jun-Wen Li; Xin-Wei Wang; Chang-Qing Yuan; Jin-Lai Zheng; Min Jin; Nong Song; Xiu-Quan Shi; Fu-Huan Chao

    2002-01-01

    AIM: To develop a rapid detection method ofenteroviruses and Hepatitis A virus (HAV).METHODS: A one-step, single-tube consensus primersmultiplex RT-PCR was developed to simultaneouslydetect Poliovirus, Coxsackie virus, Echovirus and HAV.A general upstream primer and a HAV primer and fourdifferent sets of primers (5 primers) specific forPoliovirus, Coxsacki evirus, Echovirus and HAV cDNAwere mixed in the PCR mixture to reverse transcriptand amplify the target DNA.Four distinct amplified DNAsegments representing Poliovirus, Coxsackie virus,Echovirus and HAV were identified by gelelectrophoresis as 589-,671-, 1084-, and 1128bpsequences, respectively. Semi-nested PCR was used toconfirm the amplified products for each enterovirus andHAV.RESULTS: All four kinds of viral genome RNA weredetected, and producing four bands which could bedifferentiated by the band size on the gel.To confirmthe specificity of the multiplex PCR products, semi-nested PCR was performed. For all the four strainstested gave positive results .The detection sensitivityof multiplex PCR was similar to that of monoplex RT-PCR which was 24 PFU for Poliovrus, 21 PFU forCoxsackie virus,60 PFU for Echovirus and 105 TCID50for HAV. The minimum amount of enteric viral RNAdetected by semi-nested PCR was equivalent to 2.4 PFUfor Poliovrus, 2.1 PFU for Coxsackie virus, 6.0 PFU forEchovirus and 10.5 TCID50 for HAV.CONCLUSION: The consensus primers multiplex RT-PCRhas more advantages over monoplex RT-PCR for entericviruses detection, namely, the rapid turnaround timeand cost effectiveness.

  18. Sensitive detection and typing of porcine reproductive and respiratory syndrome virus by RT-PCR amplification of whole viral genes

    DEFF Research Database (Denmark)

    Oleksiewicz, M.B.; Bøtner, Anette; Madsen, K.G.;

    1998-01-01

    Following the recent use of a live vaccine against porcine reproductive and respiratory syndrome virus (PRRSV) in Denmark, both American (vaccine) and European-type PRRSV now coexist in Danish herds. This situation highlighted a requirement for supplementary tests for precise virus-typing. As a r...... and oligonucleotide hybridization) for confirmation of the specificity of ORF 7 RT-PCR reactions. As such, the RT PCR assay provides a new, powerful diagnostic tool to study the population dynamics between present and emerging PRRSV-types....

  19. Misidentification of Bordetella bronchiseptica as Bordetella pertussis using a Newly Described RT-PCR Targeting the Pertactin Gene

    Science.gov (United States)

    Recently a real-time PCR (RT-PCT) assay based on sequence from the gene for pertactin was proposed for identification of Bordetella pertussis. Here we report that the B. pertussis pertactin gene sequence for the region encompassing the RT-PCR probe and primers is nearly identical to that of many B....

  20. Development of a Duplex RT-PCR Assay for Detection of Duck Hepatitis Virus Type I and Muscovy Duck Parvovirus%鸭I型肝炎病毒和番鸭细小病毒二重RT-PCR方法的建立

    Institute of Scientific and Technical Information of China (English)

    谢丽基; 谢芝勋; 刘加波; 庞耀珊; 邓显文; 谢志勤; 范晴

    2012-01-01

      根据基因库中鸭I型肝炎病毒和番鸭细小病毒的基因序列,分别设计了两对特异性引物,通过对二重RT-PCR扩增条件的优化,研究建立了可同时鉴别检测鸭I型肝炎病毒和番鸭细小病毒的二重RT-PCR方法.该方法对同一样品中的鸭I型肝炎病毒和番鸭细小病毒模板进行扩增,结果均同时得到2条大小与实验设计相符的202 bp(鸭I型肝炎病毒)和474 bp(番鸭细小病毒)的特异性扩增条带,对鸭圆环病毒、鹅细小病毒、鸭副黏病毒、鸭瘟病毒和禽流感病毒等病原体的检测全为阴性.敏感性试验结果表明,该技术最低能检测到100 fg的鸭I型肝炎病毒RNA和番鸭细小病毒DNA.研究建立的鸭I型肝炎病毒和番鸭细小病毒的二重RT-PCR方法,具有快速、敏感、特异、定量和重复性好等优点,可用于临床上鸭I型肝炎病毒和番鸭细小病毒感染的检测.%  A duplex reverse polymerase chain reaction assay(duplex RT-PCR)was developed and optimized to simultaneously detect duck hepatitis virus Type I and Muscovy duck parvovirus in one reaction.Two pairs of specific primers were designed according to the conserved regions on the sequences of duck hepatitis virus Type I and Muscovy duck parvovirus in GenBank.It was shown that samples containg duck hepatitis virus Type I and Muscovy duck parvovirus could be amplified into the specific bands,202 bp for duck hepatitis virus Type I and 474 bp for Muscovy duck parvovirus by this duplex PCR,but no specific bands of the same sizes were amplified from duck circovirus, gosling plague virus,duck paramyxovirus,duck plague virus and avian influenza virus.As little as 100 fg of duck hepatitis virus Type I RNA and Muscovy duck parvovirus DNA could be detected.This duplex RT-PCR assay was a quick,sensitive,and specific test for detection of duck hepatitis virus Type I and Muscovy duck parvovirus,and could be useful for the control of these viruses in

  1. Strategy for the maximization of clinically relevant information from hepatitis C virus, RT-PCR quantification.

    LENUS (Irish Health Repository)

    Levis, J

    2012-02-03

    BACKGROUND: The increasing clinical application of viral load assays for monitoring viral infections has been an incentive for the development of standardized tests for the hepatitis C virus. OBJECTIVE: To develop a simple model for the prediction of baseline viral load in individuals infected with the hepatitis C virus. METHODOLOGY: Viral load quantification of each patient\\'s first sample was assessed by RT-PCR-ELISA using the Roche MONITOR assay in triplicate. Genotype of the infecting virus was identified by reverse line probe hybridization, using amplicons resulting from the qualitative HCV Roche AMPLICOR assay. RESULTS: Retrospective evaluation of first quantitative values suggested that 82.4% (n=168\\/204) of individuals had a viral load between 4.3 and 6.7 log(10) viral copies per ml. A few patients (3.4%; n=7\\/204) have a serum viremia less than the lower limit of the linear range of the RT-PCR assay. Subsequent, prospective evaluation of hepatitis C viral load of all new patients using a model based on the dynamic range of viral load in the retrospective group correctly predicted the dynamic range in 75.9% (n=33\\/54). CONCLUSION: The dynamic range of hepatitis C viremia extends beyond the linear range of the Roche MONITOR assay. Accurate determination of serum viremia is substantially improved by dilution of specimens prior to quantification.

  2. One-step microlithography

    Science.gov (United States)

    Kahlen, Franz-Josef; Sankaranarayanan, Srikanth; Kar, Aravinda

    1997-09-01

    Subject of this investigation is a one-step rapid machining process to create miniaturized 3D parts, using the original sample material. An experimental setup where metal powder is fed to the laser beam-material interaction region has been built. The powder is melted and forms planar, 2D geometries as the substrate is moved under the laser beam in XY- direction. After completing the geometry in the plane, the substrate is displaced in Z-direction, and a new layer of material is placed on top of the just completed deposit. By continuous repetition of this process, 3D parts wee created. In particular, the impact of the focal spot size of the high power laser beam on the smallest achievable structures was investigated. At a translation speed of 51 mm/s a minimum material thickness of 590 micrometers was achieved. Also, it was shown that a small Z-displacement has a negligible influence on the continuity of the material deposition over this power range. A high power CO2 laser was used as energy source, the material powder under investigation was stainless steel SS304L. Helium was used as shield gas at a flow rate of 15 1/min. The incident CO2 laser beam power was varied between 300 W and 400 W, with the laser beam intensity distribute in a donut mode. The laser beam was focused to a focal diameter of 600 (Mu) m.

  3. Enhancing the sensitivity of Dengue virus serotype detection by RT-PCR among infected children in India.

    Science.gov (United States)

    Ahamed, Syed Fazil; Vivek, Rosario; Kotabagi, Shalini; Nayak, Kaustuv; Chandele, Anmol; Kaja, Murali-Krishna; Shet, Anita

    2017-06-01

    Dengue surveillance relies on reverse transcription-polymerase chain reaction (RT-PCR), for confirmation of dengue virus (DENV) serotypes. We compared efficacies of published and modified primer sets targeting envelope (Env) and capsid-premembrane (C-prM) genes for detection of circulating DENV serotypes in southern India. Acute samples from children with clinically-diagnosed dengue were used for RT-PCR testing. All samples were also subjected to dengue serology (NS1 antigen and anti-dengue-IgM/IgG rapid immunochromatographic assay). Nested RT-PCR was performed on viral RNA using three methods targeting 654bp C-prM, 511bp C-prM and 641bp Env regions, respectively. RT-PCR-positive samples were validated by population sequencing. Among 171 children with suspected dengue, 121 were dengue serology-positive and 50 were dengue serology-negative. Among 121 serology-positives, RT-PCR detected 91 (75.2%) by CprM654, 72 (59.5%) by CprM511, and 74 (61.1%) by Env641. Among 50 serology-negatives, 10 (20.0%) were detected by CprM654, 12 (24.0%) by CprM511, and 11 (22.0%) by Env641. Overall detection rate using three methods sequentially was 82.6% (100/121) among serology-positive and 40.0% (20/50) among serology-negative samples; 6.6% (8/120) had co-infection with multiple DENV serotypes. We conclude that detection of acute dengue was enhanced by a modified RT-PCR method targeting the 654bp C-prM region, and further improved by using all three methods sequentially. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Quantitation of Rabbit Cytokine mRNA by Real-Time RT-PCR

    OpenAIRE

    Godornes, Charmie; Leader, Brandon Troy; Molini, Barbara J.; Centurion-Lara, Arturo; Lukehart, Sheila A.

    2007-01-01

    Fundamental understanding of rabbit immunology and the use of the rabbit as a disease model have long been hindered by the lack of immunological assays specific to this species. In the present study, we sought to develop a method to quantitate cytokine expression in rabbit cells and tissues. We report the development of a quantitative real-time RT-PCR method for measuring the relative levels of rabbit IFN-γ, IL-2, IL-4, IL-10 and TNF-α mRNA. Quantitation was accomplished by comparison to a st...

  5. Development of a simplified RT-PCR without RNA isolation for rapid detection of RNA viruses in a single small brown planthopper (Laodelphax striatellus Fallén).

    Science.gov (United States)

    Xu, Qiufang; Liu, Haoqiu; Yuan, Pingping; Zhang, Xiaoxia; Chen, Qingqing; Jiang, Xuanli; Zhou, Yijun

    2017-05-03

    The small brown planthopper (SBPH) is an important pest of cereal crops and acts as a transmission vector for multiple RNA viruses. Rapid diagnosis of virus in the vector is crucial for efficient forecast and control of viral disease. Reverse transcription polymerase chain reaction (RT-PCR) is a rapid, sensitive and reliable method for virus detection. The traditional RT-PCR contains a RNA isolation step and is widely used for virus detection in insect. However, using the traditional RT-PCR for detecting RNA virus in individual SBPHs becomes challenging because of the expensive reagents and laborious procedure associated with RNA isolation when processing a large number of samples. We established a simplified RT-PCR method without RNA isolation for RNA virus detection in a single SBPH. This method is achieved by grinding a single SBPH in sterile water and using the crude extract directly as the template for RT-PCR. The crude extract containing the virus RNA can be prepared in approximately two minutes. Rice stripe virus (RSV), rice black streaked dwarf virus (RBSDV) and Himetobi P virus (HiPV) were successfully detected using this simplified method. The detection results were validated by sequencing and dot immunobinding assay, indicating that this simplified method is reliable for detecting different viruses in insects. The evaluation of the sensitivity of this method showed that both RSV and HiPV can be detected when the cDNA from the crude extract was diluted up to 10(3) fold. Compared to the traditional RT-PCR with RNA isolation, the simplified RT-PCR method greatly reduces the sample processing time, decreases the detection cost, and improves the efficiency by avoiding RNA isolation. A simplified RT-PCR method is developed for rapid detection of RNA virus in a single SBPH without the laborious RNA isolation step. It offers a convenient alternative to the traditional RT-PCR method.

  6. Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs

    Directory of Open Access Journals (Sweden)

    Walton Eric F

    2007-10-01

    Full Text Available Abstract MicroRNAs (miRNAs are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 μl of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression.

  7. Development of a duplex real-time RT-PCR for the simultaneous detection and differentiation of Theiler's murine encephalomyelitis virus and rat theilovirus.

    Science.gov (United States)

    Yuan, Wen; Wang, Jing; Xu, Fengjiao; Huang, Bihong; Lian, Yuexiao; Rao, Dan; Yin, Xueqin; Wu, Miaoli; Zhu, Yujun; Zhang, Yu; Huang, Ren; Guo, Pengju

    2016-10-01

    Theiler's murine encephalomyelitis virus (TMEV) and rat theilovirus (RTV), the member of the genus Cardiovirus, are widespread in laboratory mice and rats, and are potential contaminants of biological materials. Cardioviruses infection may cause serious complications in biomedical research. To improve the efficiency of routine screening for Cardioviruses infection, a duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed for simultaneous detection and differentiation of TMEV and RTV. The duplex assay was specific for reference strains of TMEV and RTV, and no cross-reaction was found with seven other rodent viruses. The limits of detection of both TMEV and RTV were 4×10(1) copies RNA/reaction. Reproducibility was estimated using standard dilutions, with coefficients of variation PCR and conventional RT-PCR. For 439 clinical samples,95 samples were positive for TMEV and 72 samples were positive for RTV using duplex real-time RT-PCR approach, whereas only 77 samples were positive for TMEV and 66 samples were positive for RTV when conventional RT-PCR was applied. Mixed infections were found in 20 samples when analyzed by conventional RT-PCR whereas 30 samples were found to be mixed infection when duplex real-time RT-PCR was applied. This duplex assay provides a useful tool for routine health monitoring and screening of contaminated biological materials of these two viruses.

  8. Real-time onestep RT-PCR for the detection and differentiation of European and North American types of PRRSV in boar semen

    DEFF Research Database (Denmark)

    Hjulsager, Charlotte Kristiane; Larsen, Lars Erik

    ) and the virus can be transmitted by this route, creating a need for diagnostic tests to ensure a PRRSV-free semen supply. PCR is an obvious method for such testing, and especially nested and TwoStep RT-PCR methods have been extensively used for this purpose. However, OneStep RT-PCR offers a more convenient......Porcine Reproductive and respiratory syndrome virus (PRRRSV) is a single-stranded RNA virus and a worldwide cause of significant respiratory disease and reproductive failure in swine. Two different types of PRRSV, the European (EU) and North American (US) type exist. Boar semen can harbor PRRSV (1...

  9. Real-time onestep RT-PCR for the detection and differentiation of European and North American types of PRRSV in boar semen

    DEFF Research Database (Denmark)

    Hjulsager, Charlotte Kristiane; Larsen, Lars Erik

    Porcine Reproductive and respiratory syndrome virus (PRRRSV) is a single-stranded RNA virus and a worldwide cause of significant respiratory disease and reproductive failure in swine. Two different types of PRRSV, the European (EU) and North American (US) type exist. Boar semen can harbor PRRSV (1......) and the virus can be transmitted by this route, creating a need for diagnostic tests to ensure a PRRSV-free semen supply. PCR is an obvious method for such testing, and especially nested and TwoStep RT-PCR methods have been extensively used for this purpose. However, OneStep RT-PCR offers a more convenient...

  10. Identification of Euroasiatic and South Africa Serotype Foot-and-Mouth Disease Virus by One-Step Multiplex Real-Time Quantitative RT-PCR Assay%亚欧型及南非型口蹄疫一步法多重荧光RT-PCR检测方法建立

    Institute of Scientific and Technical Information of China (English)

    吴绍强; 查成刚; 邓俊花; 林祥梅; 刘建; 梅琳

    2010-01-01

    [目的]口蹄疫是一种严重的"政治经济病",世界各国面临的口岸疫情防控形势均十分严峻.针对南非型(SAT Ⅰ,SATⅡ,SATⅢ型)潜在入侵中国的严峻态势,为了防止疫情跨境传播,迫切需要建立有效甄别亚欧型(A、O、C及亚洲Ⅰ型)与南非型口蹄疫的检测方法,以进一步提高防控工作的准确性.[方法]选择口蹄疫病毒(FMDV)基因组保守的5'非翻译区(5'UTR)基因为靶序列,分别设计亚欧型及南非型FMDV特异的荧光PCR引物及Taqman探针,通过对反应体系和反应条件的优化,以插入扩增目的片段的阳性克隆质粒为模板,建立多重荧光PCR检测体系;进一步以体外转录cRNA作为阳性质控品,建立亚欧型及南非型FMDV一步法多重荧光RT-PCR鉴别方法,并进行敏感性、特异性确定及田间样本检测.[结果]本研究建立的FMDV多重荧光PCR检测方法,对亚欧型及南非型FMDV阳性质粒的扩增效率均在90%以上;以倍比稀释的体外转录cRNA作为模板,进行敏感性测试表明,本方法最低可检测至7.6×10-9ng南非型cRNA和6.3×108ng亚欧型cRNA;特异性试验结果显示本方法可以有效甄别亚欧型与南非型FMDV;田间样本检测结果与样品实际感染情况一致.[结论]本研究建立的FMDV一步法多重荧光RT-PCR检测方法敏感、特异而且快速,为亚欧型与南非型口蹄疫的鉴别诊断及流行病学调查提供了新的方法.

  11. Rapid detection of lineage IV peste des petits ruminants virus by real-time RT-PCR.

    Science.gov (United States)

    Li, Lin; Wu, Xiaodong; Liu, Fuxiao; Wang, Zhiliang; Liu, Chunju; Wang, Qinghua; Bao, Jingyue

    2016-09-01

    Peste des petits ruminants virus (PPRV) is the cause agent of peste des petitis ruminants (PPR). A novel lineage IV PPRV has reemerged in China in 2013 and 2014. Mass vaccination was implemented in most provinces in China. In order to detect lineage IV PPRV in clinical samples and to distinguish rapidly it from the other lineages PPRVs, a real-time RT-PCR assay was developed. This assay showed high sensitivity, specificity and efficiency in differentiating the lineage IV PPRV from others. The performance of this assay was evaluated by positive clinical samples of lineage IV viruses. This new real-time RT-PCR assay will facilitate epidemiological investigations and rapid differentiatial diagnosis in areas where lineage IV viruses are circulating. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Canine distemper virus detection by different methods of One-Step RT-qPCR

    Directory of Open Access Journals (Sweden)

    Claudia de Camargo Tozato

    2016-01-01

    Full Text Available ABSTRACT: Three commercial kits of One-Step RT-qPCR were evaluated for the molecular diagnosis of Canine Distemper Virus. Using the kit that showed better performance, two systems of Real-time RT-PCR (RT-qPCR assays were tested and compared for analytical sensitivity to Canine Distemper Virus RNA detection: a One-Step RT-qPCR (system A and a One-Step RT-qPCR combined with NESTED-qPCR (system B. Limits of detection for both systems were determined using a serial dilution of Canine Distemper Virus synthetic RNA or a positive urine sample. In addition, the same urine sample was tested using samples with prior centrifugation or ultracentrifugation. Commercial kits of One-Step RT-qPCR assays detected canine distemper virus RNA in 10 (100% urine samples from symptomatic animals tested. The One-Step RT-qPCR kit that showed better results was used to evaluate the analytical sensitivity of the A and B systems. Limit of detection using synthetic RNA for the system A was 11 RNA copies µL-1 and 110 RNA copies µl-1 for first round System B. The second round of the NESTED-qPCR for System B had a limit of detection of 11 copies µl-1. Relationship between Ct values and RNA concentration was linear. The RNA extracted from the urine dilutions was detected in dilutions of 10-3 and10-2 by System A and B respectively. Urine centrifugation increased the analytical sensitivity of the test and proved to be useful for routine diagnostics. The One-Step RT-qPCR is a fast, sensitive and specific method for canine distemper routine diagnosis and research projects that require sensitive and quantitative methodology.

  13. 非洲马瘟病毒VP7和NS2双重荧光RT-PCR检测技术的建立与应用%Development and Application of Duplex Real-time RT-PCR Assay for the Detection of VP7 and NS2 of African Horse Sickness Virus

    Institute of Scientific and Technical Information of China (English)

    高志强; 张鹤晓; 乔彩霞; 蒲静; 张伟; 谷强; 刘环; 张利峰; 马贵平

    2013-01-01

      Nucleic acid sequences of representative strains of different genotypes of African horse sickness viruses(AHSV)were aligned with the DNAMAN software. The two highly conservative NS2 and VP7 regions were then subjected to design primers and probes. The artificially synthesized nucleic acid fragments including amplification regions were used to prepare double strand RNA (dsRNA)by in vitro transcription in two directions(T7 and SP6). A duplex real-time TaqMan RT-PCR assay was developed to detect and quantify AHSV by optimization of reaction conditions using prepared dsRNA. The developed assay was used to detect a set of extracted pathogen RNA/DNA including those of AHSV,Eastern and Western equine encephalomyelitis virus(EEEV and WEEV), equine arteritis virus(EAV),equine influenza virus subtype H3N8(EIV H3N8),Salmonella abortus equi,Streptococcus equi subsp. zooepidimicus resulting in positive only for AHSV RNA,but negative for other virus nucleic acids,suggesting that the developed assay was specific and reliable. The detection limit of the real-time RT-PCR was 1.0×102copies per reaction,10 times more sensitive than that of the conventional RT–PCR based on gel electrophoresis. Also the double gene design could ensure the reliability, and efficiently reduce false negative results. By testing 248 clinical samples,it is confirmed that this assay was rapid,sensitive and repeatable,meeting the requirements for rapid diagnosis of AHSV.%  利用DNAMAN软件对非洲马瘟病毒不同基因型代表株的序列进行分析,选择其高度保守的VP7和NS2基因设计合成引物和探针。人工分别合成包含有扩增区域的VP7和NS2核苷酸片段进行双向(T7和SP6)体外转录制备双链RNA(dsRNA)。使用制备的dsRNA在对荧光定量RT-PCR的反应条件优化的基础上,建立了适用于非洲马瘟病毒检测的双重通用荧光定量RT-PCR检测技术。应用建立的方法对非洲马瘟病毒核酸,马流感病毒核酸

  14. Comparative evaluation of conventional RT-PCR and real-time RT-PCR (RRT-PCR for detection of avian metapneumovirus subtype A Comparação entre as técnicas de RT-PCR convencional e RT-PCR em tempo real para a detecção do metapneumovírus aviários subtipo A

    Directory of Open Access Journals (Sweden)

    Helena Lage Ferreira

    2009-08-01

    Full Text Available Avian metapneumovirus (AMPV belongs to Metapneumovirus genus of Paramyxoviridae family. Virus isolation, serology, and detection of genomic RNA are used as diagnostic methods for AMPV. The aim of the present study was to compare the detection of six subgroup A AMPV isolates (AMPV/A viral RNA by using different conventional and real time RT-PCR methods. Two new RT-PCR tests and two real time RT-PCR tests, both detecting fusion (F gene and nucleocapsid (N gene were compared with an established test for the attachment (G gene. All the RT-PCR tested assays were able to detect the AMPV/A. The lower detection limits were observed using the N-, F- based RRT-PCR and F-based conventional RT-PCR (10(0.3 to 10¹ TCID50 mL-1. The present study suggests that the conventional F-based RT-PCR presented similar detection limit when compared to N- and F-based RRT-PCR and they can be successfully used for AMPV/A detection.O metapneumovírus aviário (AMPV pertence ao gênero Metapneumovirus, família Paramyxoviridae. Isolamento viral, sorologia e detecção do RNA genômico são atualmente as técnicas utilizadas para o diagnóstico desse agente. O objetivo do presente estudo foi comparar a detecção de RNA viral de seis isolados de AMPV, subtipo A (AMPV/A, utilizando diferentes métodos de RT-PCR convencional e real time RT-PCR (RRT-PCR. Duas novas técnicas de RT-PCR convencional e duas técnicas de RRT-PCR, ambas para a detecção dos genes da nucleoproteína (N e da proteína de fusão (F, foram comparadas com um RT-PCR previamente estabelecido para a detecção do AMPV (gene da glicoproteína -G. Todos esses métodos foram capazes de detectar os isolados AMPV/A. As técnicas RRT-PCR (genes F e N mostraram os menores limites de detecção (10(0.3 to 10¹ TCID50 mL-1. Os resultados sugerem que as técnicas RT-PCR convencional (gene F e as técnicas de RRT-PCR (gene F e N desenvolvidas no presente estudo podem ser utilizadas com sucesso para a detecção do

  15. 江苏省葫芦科作物6种病毒的多重 RT-PCR方法及应用%Establishment and application of multiplex RT-PCR assays for simultane-ous detection of six viruses on Cucurbit crops in Jiangsu province

    Institute of Scientific and Technical Information of China (English)

    任春梅; 程兆榜; 杨柳; 缪倩; 周益军

    2015-01-01

    江苏省葫芦科作物上的病毒主要有小西葫芦黄花叶病毒( ZYMV)、西瓜花叶病毒( WMV)、黄瓜花叶病毒( CMV)、黄瓜绿斑驳花叶病毒( CGMMV)、烟草花叶病毒( TMV)和番木瓜环斑病毒( PRSV),通常发生复合侵染。基于GenBank中这6种病毒的核苷酸序列保守区设计特异性引物,在单重RT-PCR技术的基础上,通过对影响2种多重RT-PCR扩增的DNA聚合酶浓度、dNTPs及镁离子浓度、退火温度、延伸时间、循环次数等因素进行优化,对检测灵敏度进行测定,建立了2种PCR反应同时检测葫芦科作物上6种病毒的多重RT-PCR方法。结果显示:2种多重RT-PCR体系均能同时扩增出各自特异性片段,测序分析结果表明序列同源性在97%以上;优化的2种多重RT-PC体系检测灵敏度为总RNA稀释102~103倍,应用于江苏省210份样品的检测结果与单重RT-PCR一致,体现了检测的可靠性;检测结果表明近年来江苏省葫芦科作物以种子传播的ZYMV和CGMMV侵染为主,病毒的复合侵染率达75.24%。%Cucurbit crops are frequently co-infected by zucchini yellow mosaic virus ( ZYMV) , watermelon mosaic virus (WMV), cucumber mosaic virus (CMV), cucumber green mottle mosaic virus (CGMMV), tobacco mosaic virus ( TMV) and papaya ringspot virus ( PRSV) in Jiangsu province. Based on the RT-PCR for the detection of single virus, the two multiplex RT-PCR ( mRT-PCR) systems were established for simultaneous detection of the six viruses by using six sets of specific primers designed on the basis of conserved sequences of the viruses in GenBank. The main factors suches con-centrations of DNA polymerase, Mg2+ and dNTPs, PCR conditions of annealing temperature, extension time and amplifica-tion cycles, sensitivity of the detection were examined and optimized. The results showed that expected fragments of the six viruses were amplified in the two mRT-PCR systems and the sequences shared above 97 percent

  16. A diagnostic one-step real-time reverse transcription polymerase chain reaction method for accurate detection of influenza virus type A

    Science.gov (United States)

    Behzadi, Mohammad Amin; Alborzi, Abdolvahab

    2016-01-01

    Introduction Influenza A is known as a public health concern worldwide. In this study, a novel one-step real-time reverse transcription polymerase chain reaction (rtRT-PCR) assay was designed and optimized for the detection of influenza A viruses. Material and methods The primers and probe were designed based on the analysis of 90 matrix nucleotide sequence data of influenza type A subtypes from the GenBank database of the National Center for Biotechnology Information (NCBI). The influenza virus A/Tehran/5652/2010 (H1N1 pdm09) was used as a reference. The rtRT-PCR assay was optimized, compared with that of the World Health Organization (WHO), and its analytical sensitivity, specificity and reproducibility were evaluated. In total, 64 nasopharyngeal swabs from patients with influenza-like illness (ILI) and 41 samples without ILI symptoms were tested for the virus, using conventional cell culture, direct immunofluorescence antibody (DFA) methods, and one-step rtRT-PCR with the designed primer set and probe and the WHO’s. Results The optimized assay results were similar to the WHO’s. The optimized assay results were similar to WHO’s, with non-significant differences for 10–103 copies of viral RNA/reaction (p > 0.05). It detected 10 copies of viral RNA/reaction with high reproducibility and no cross reactivity with other respiratory viruses. A specific cytopathic effect was observed in 6/64 (9.37%) of the ILI group using conventional culture and DFA staining methods; however, it was not seen in non-ILI. Also, the results of our assay and the WHO’s were similar to those of viral isolation and DFA staining. Conclusions Given the high specificity, sensitivity and reproducibility of this novel assay, it can serve as a reliable diagnostic tool for the detection of influenza A viruses in clinical specimens and lab experiments. PMID:27904520

  17. A new StaRT-PCR approach to detect and quantify fish Viral Hemorrhagic Septicemia virus (VHSv): enhanced quality control with internal standards.

    Science.gov (United States)

    Pierce, Lindsey R; Willey, James C; Crawford, Erin L; Palsule, Vrushalee V; Leaman, Douglas W; Faisal, Mohamed; Kim, Robert K; Shepherd, Brian S; Stanoszek, Lauren M; Stepien, Carol A

    2013-04-01

    Viral Hemorrhagic Septicemia virus (VHSv) causes one of the world's most important finfish diseases, killing >80 species across Eurasia and North America. A new and especially virulent strain (IVb) emerged in the North American Great Lakes in 2003, threatening fisheries, baitfish, and aquaculture industries. Weeks-long and costly cell culture is the OIE and USDA-APHIS approved diagnostic. A new Standardized Reverse Transcriptase Polymerase Chain Reaction (StaRT-PCR) assay that uniquely incorporates internal standards to improve accuracy and prevent false negatives was developed and evaluated for its ability to detect and quantify VHSv. Results from StaRT-PCR, SYBR(®) green real time qRT-PCR, and cell culture were compared, as well as the effects of potential PCR inhibitors (EDTA and high RNA). Findings show that StaRT-PCR is sensitive, detecting a single molecule, with 100% accuracy at six molecules, and had no false negatives. In comparison, false negatives ranged from 14 to 47% in SYBR(®) green real time qRT-PCR tests, and 47-70% with cell culture. StaRT-PCR uniquely controlled for EDTA and RNA interference. Range of VHSv quantitation by StaRT-PCR was 1.0×10(0)-1.2×10(5) VHSv/10(6)actb1 molecules in wild caught fishes and 1.0×10(0)-8.4×10(5) molecules in laboratory challenged specimens. In the latter experiments, muskellunge with skin lesions had significantly more viral molecules (mean=1.9×10(4)) than those without (1.1×10(3)) (p<0.04). VHSv infection was detected earlier in injection than in immersion challenged yellow perch (two versus three days), with molecule numbers in both being comparable and relatively consistent over the remaining course of the experiment. Our results show that the StaRT-PCR test accurately and reliably detects and quantifies VHSv.

  18. Recent sequence variation in probe binding site affected detection of respiratory syncytial virus group B by real-time RT-PCR.

    Science.gov (United States)

    Kamau, Everlyn; Agoti, Charles N; Lewa, Clement S; Oketch, John; Owor, Betty E; Otieno, Grieven P; Bett, Anne; Cane, Patricia A; Nokes, D James

    2017-03-01

    Direct immuno-fluorescence test (IFAT) and multiplex real-time RT-PCR have been central to RSV diagnosis in Kilifi, Kenya. Recently, these two methods showed discrepancies with an increasing number of PCR undetectable RSV-B viruses. Establish if mismatches in the primer and probe binding sites could have reduced real-time RT-PCR sensitivity. Nucleoprotein (N) and glycoprotein (G) genes were sequenced for real-time RT-PCR positive and negative samples. Primer and probe binding regions in N gene were checked for mismatches and phylogenetic analyses done to determine molecular epidemiology of these viruses. New primers and probe were designed and tested on the previously real-time RT-PCR negative samples. N gene sequences revealed 3 different mismatches in the probe target site of PCR negative, IFAT positive viruses. The primers target sites had no mismatches. Phylogenetic analysis of N and G genes showed that real-time RT-PCR positive and negative samples fell into distinct clades. Newly designed primers-probe pair improved detection and recovered previous PCR undetectable viruses. An emerging RSV-B variant is undetectable by a quite widely used real-time RT-PCR assay due to polymorphisms that influence probe hybridization affecting PCR accuracy. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  19. Picoinjection Enables Digital Detection of RNA with Droplet RT-PCR

    Science.gov (United States)

    Abate, Adam R.

    2013-01-01

    The ability to add reagents to drops in a sequential fashion is necessary for numerous applications of microfluidics in biology. An important method for accomplishing this is picoinjection, a technique in which reagents are injected into aqueous drops using an electric field. While picoinjection has been shown to allow the precise addition of reagents to drops, its compatibility with biological reactions is yet to be thoroughly demonstrated. Here, we investigate the compatibility of picoinjection with digital RT-PCR Taqman assays, reactions that incorporate nucleic acids, enzymes, and other common biological reagents. We find that picoinjection is compatible with this assay and enables the detection of RNA transcripts at rates comparable to workflows not incorporating picoinjection. We also find that picoinjection results in negligible transfer of material between drops and that the drops faithfully retain their compartmentalization. PMID:23658657

  20. Picoinjection enables digital detection of RNA with droplet rt-PCR.

    Directory of Open Access Journals (Sweden)

    Dennis J Eastburn

    Full Text Available The ability to add reagents to drops in a sequential fashion is necessary for numerous applications of microfluidics in biology. An important method for accomplishing this is picoinjection, a technique in which reagents are injected into aqueous drops using an electric field. While picoinjection has been shown to allow the precise addition of reagents to drops, its compatibility with biological reactions is yet to be thoroughly demonstrated. Here, we investigate the compatibility of picoinjection with digital RT-PCR Taqman assays, reactions that incorporate nucleic acids, enzymes, and other common biological reagents. We find that picoinjection is compatible with this assay and enables the detection of RNA transcripts at rates comparable to workflows not incorporating picoinjection. We also find that picoinjection results in negligible transfer of material between drops and that the drops faithfully retain their compartmentalization.

  1. Picoinjection enables digital detection of RNA with droplet rt-PCR.

    Science.gov (United States)

    Eastburn, Dennis J; Sciambi, Adam; Abate, Adam R

    2013-01-01

    The ability to add reagents to drops in a sequential fashion is necessary for numerous applications of microfluidics in biology. An important method for accomplishing this is picoinjection, a technique in which reagents are injected into aqueous drops using an electric field. While picoinjection has been shown to allow the precise addition of reagents to drops, its compatibility with biological reactions is yet to be thoroughly demonstrated. Here, we investigate the compatibility of picoinjection with digital RT-PCR Taqman assays, reactions that incorporate nucleic acids, enzymes, and other common biological reagents. We find that picoinjection is compatible with this assay and enables the detection of RNA transcripts at rates comparable to workflows not incorporating picoinjection. We also find that picoinjection results in negligible transfer of material between drops and that the drops faithfully retain their compartmentalization.

  2. Specific detection of avian pneumovirus (APV) US isolates by RT-PCR.

    Science.gov (United States)

    Shin, H J; Rajashekara, G; Jirjis, F F; Shaw, D P; Goyal, S M; Halvorson, D A; Nagaraja, K V

    2000-01-01

    This report details the development of an RT-PCR assay for the specific detection of US isolates of avian pneumovirus (APV). Of the several primer pairs tested, two sets of primers derived from the matrix gene of APV were able to specifically detect the viral RNA of APV. The nucleotide sequence comparison of the PCR products of APV isolates from Minnesota suggested that these viruses were closely related to the Colorado strain of APV, but were distinct from subtypes A and B European isolates of turkey APV (turkey rhinotracheitis: TRT). This M gene-based PCR was found to be very specific and sensitive. APV as low as 8 x 10(-5) TCID50 (0.0323 microg/ml) could be detected using this assay. In addition, the two primers were able to differentiate isolates from turkeys in Minnesota.

  3. Genotyping of Rotavirus by Using RT-PCR Methods

    Directory of Open Access Journals (Sweden)

    Hera Nirwati

    2015-11-01

    Full Text Available There is a great diversity of rotavirus genotypes circulating worldwide, with dominant genotypes changing from year to year. Rotavirus genotyping was performed by using reverse transcription PCR with type-specifi c-primers. Since rotavirus is a RNA virus that has high mutation rate, there was a possibility of technical diffi culty in genotyping due to mutation in the primer binding sites. During Indonesian rotavirus surveillance study 2006-2009, it was reported that 17% of samples subjected for G type and 21% of samplessubjected for P type were untypeable. The objective of this study was to identify genotypes of the samples that were untypeable previously using RT-PCR based on the method described by Das et al. (1994 and Gentsch et al. (1992. There were 30 samples subjected to G type and 61 samples subjected to P type to be re-typed using method described by Gouvea et al. (1990 and Simmond et al. (2008 for G and P typing, respectively. By using another set of primer, the genotype of all samples was identifi ed. This study highlights the importance of a constant reconsideration of primer sequences employed for the molecular typing of rotaviruses.Key words: rotavirus, G typing, P typing

  4. Selective control of primer usage in multiplex one-step reverse transcription PCR

    Directory of Open Access Journals (Sweden)

    Paul Natasha

    2009-12-01

    Full Text Available Abstract Background Multiplex RT-PCR is a valuable technique used for pathogen identification, disease detection and relative quantification of gene expression. The simplification of this protocol into a one-step procedure saves time and reagents. However, intensive PCR optimization is often required to overcome competing undesired PCR primer extension during the RT step. Results Herein, we report multiplex one-step RT-PCR experiments in which the PCR primers contain thermolabile phosphotriester modification groups. The presence of these groups minimizes PCR primer extension during the RT step and allows for control of PCR primer extension until the more stringent, elevated temperatures of PCR are reached. Results reveal that the use of primers whose extension can be controlled in a temperature-mediated way provides improved one-step RT-PCR specificity in both singleplex and multiplex reaction formats. Conclusions The need for an accurate and sensitive technique to quantify mRNA expression levels makes the described modified primer technology a promising tool for use in multiplex one-step RT-PCR. A more accurate representation of the abundances in initial template sample is feasible with modified primers, as artifacts of biased PCR are reduced because of greater improvements in reaction specificity.

  5. Establishment of a Real-time Fluorescent Quantitative RT-PCR Assay for Detection of Porcine IFN-β and IRF-3 genes%猪IFN-β和IRF-3基因实时荧光定量RT-PCR检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    王蕊; 张改平; 乔松林; 刘永晖; 蒋志政; 万博; 鲍登克; 王爱萍

    2012-01-01

    为建立猪IFN-β及IRF-3基因实时荧光定量PCR检测方法,依据GenBank中IFN-β和IRF-3基因的保守序列,设计并合成各自特异性引物,并以β-actin为内参基因,采用SYBRGreen-Ⅰ为染料,建立实时荧光定量检测方法.提取猪肺泡巨噬细胞总RNA,经反转录得cDNA,用特异性引物经PCR扩增得到IFN-β和IRF-3基因,将其克隆至pMD-19T载体,经测序鉴定后得重组质粒,依次10倍稀释做为标准品,建立标准曲线及溶解曲线.结果表明,β3-actin基因、IFN-β基因和IRF-3基因标准曲线线性关系较好,R2≥0.997;溶解曲线为特异性单峰,无非特异性扩增,检测下限可达100个拷贝/μL.建立的猪IFN-β和IRF-3基因实时荧光定量RT-PCR检测方法,特异性强、重复性好,为从分子水平上研究猪的免疫应答奠定了基础.%To establish real-time fluorescent quantitative RT-PCR assays for detecting porcine IFN-β and IRF-3, sevral specific primer pairs were designed according to the porcine's IFN-β and IRF-3 gene sequences available in GenBank,and the porcine β-actin gene was used as an internal gene control. The total RNA was extracted from porcine alveolar macrophages. The reverse transcriptase PCR was used to obtain the first strand cDNA. Fragments for IFN-β and IRF-3 were amplified by PCR from the synthesized cDNA using the designed specific primers. The PCR products were purified and cloned into pMD-19T vector. The positive recombined plasmids were serially diluted and used as a standard. The standard and melting curve was analyzed. The results showed that the Ct value of β actin ,IFN-β and IRF-3 genes had a good linear relationship (R2≥ 0. 997) and the melting curve showed a single peak. The established real-time PCR methods can detect 100 copies of IFN-β and IRF-3 mRNA. The developed real-time PCR using SYBR Green I dye had high sensitivity,sepcifity and reproductivity.and could used as an effective tool for detection and quantification of IFN

  6. Total tumor load assessed by one-step nucleic acid amplification assay as an intraoperative predictor for non-sentinel lymph node metastasis in breast cancer.

    Science.gov (United States)

    Nabais, Celso; Figueiredo, Joana; Lopes, Paulina; Martins, Manuela; Araújo, António

    2017-04-01

    This study aimed to determine the relationship between CK19 mRNA copy number in sentinel lymph nodes (SLN) assessed by one-step nucleic acid amplification (OSNA) technique, and non-sentinel lymph nodes (NSLN) metastization in invasive breast cancer. A model using total tumor load (TTL) obtained by OSNA technique was also constructed to evaluate its predictability. We conducted an observational retrospective study including 598 patients with clinically T1-T3 and node negative invasive breast cancer. Of the 88 patients with positive SLN, 58 patients fulfill the inclusion criteria. In the analyzed group 25.86% had at least one positive NSLN in axillary lymph node dissection. Univariate analysis showed that tumor size, TTL and number of SLN macrometastases were predictive factors for NSLN metastases. In multivariate analysis just the TTL was predictive for positive NSLN (OR 2.67; 95% CI 1.06-6.70; P = 0.036). The ROC curve for the model using TTL alone was obtained and an AUC of 0.805 (95% CI 0.69-0.92) was achieved. For TTL >1.9 × 10(5) copies/μL we got 73.3% sensitivity, 74.4% specificity and 88.9% negative predictive value to predict NSLN metastases. When using OSNA technique to evaluate SLN, NSLN metastases can be predicted intraoperatively. This prediction tool could help in decision for axillary lymph node dissection. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. RT-PCR-ELISA as a tool for diagnosis of low-pathogenicity avian influenza

    DEFF Research Database (Denmark)

    Dybkaer, Karen; Munch, Mette; Handberg, Kurt Jensen

    2003-01-01

    with low-pathogenicity AIV, from wild aquatic birds, and from domestic ducks. The AIV was detected in 32 swab pools by RT-PCR-ELISA compared to 23 by virus isolation (VI) in embryonated specific pathogen free (SPF) chicken eggs. Thus, 39% more specimens were positive by RT-PCR-ELISA than by VI. Two...... of the twenty-three VI-positive specimens were negative when tested by RT-PCR-ELISA. The diagnostic sensitivity and specificity of the RT-PCR-ELISA was 91% and 97%, respectively, using VI in SPF eggs as the gold reference standard....

  8. MicroRNA markers for forensic body fluid identification obtained from microarray screening and quantitative RT-PCR confirmation.

    Science.gov (United States)

    Zubakov, Dmitry; Boersma, Anton W M; Choi, Ying; van Kuijk, Patricia F; Wiemer, Erik A C; Kayser, Manfred

    2010-05-01

    MicroRNAs (miRNAs) are non-protein coding molecules with important regulatory functions; many have tissue-specific expression patterns. Their very small size in principle makes them less prone to degradation processes, unlike messenger RNAs (mRNAs), which were previously proposed as molecular tools for forensic body fluid identification. To identify suitable miRNA markers for forensic body fluid identification, we first screened total RNA samples derived from saliva, semen, vaginal secretion, and venous and menstrual blood for the expression of 718 human miRNAs using a microarray platform. All body fluids could be easily distinguished from each other on the basis of complete array-based miRNA expression profiles. Results from quantitative reverse transcription PCR (RT-PCR; TaqMan) assays for microarray candidate markers confirmed strong over-expression in the targeting body fluid of several miRNAs for venous blood and several others for semen. However, no candidate markers from array experiments for other body fluids such as saliva, vaginal secretion, or menstrual blood could be confirmed by RT-PCR. Time-wise degradation of venous blood and semen stains for at least 1 year under lab conditions did not significantly affect the detection sensitivity of the identified miRNA markers. The detection limit of the TaqMan assays tested for selected venous blood and semen miRNA markers required only subpicogram amounts of total RNA per single RT-PCR test, which is considerably less than usually needed for reliable mRNA RT-PCR detection. We therefore propose the application of several stable miRNA markers for the forensic identification of blood stains and several others for semen stain identification, using commercially available TaqMan assays. Additional work remains necessary in search for suitable miRNA markers for other forensically relevant body fluids.

  9. Evaluation of viral extraction methods on a broad range of Ready-To-Eat foods with conventional and real-time RT-PCR for Norovirus GII detection.

    Science.gov (United States)

    Baert, Leen; Uyttendaele, Mieke; Debevere, Johan

    2008-03-31

    Noroviruses (NoV) are a common cause of foodborne outbreaks. In spite of that, no standard viral detection method is available for food products. Therefore, three viral elution-concentration methods and one direct RNA isolation method were evaluated on a broad range of Ready-To-Eat (RTE) food products (mixed lettuce, fruit salad, raspberries and two RTE dishes) artificially seeded with a diluted stool sample contaminated with NoV genogroup II. These seeding experiments revealed two categories of RTE products, fruits and vegetables grouped together and RTE dishes (penne and tagliatelle salads) which are rich in proteins and fat formed another category. The RNA extracts were amplified and detected with two conventional RT-PCR systems (Booster and Semi-nested GII) and one real-time RT-PCR (Real-time GII) assay. A fast direct RNA isolation method detected 10(2) RT-PCRU on 10 g penne and tagliatelle salads with the conventional RT-PCR assays. However real-time RT-PCR was less sensitive for penne salad. A viral elution-concentration method, including a buffer solution for the elution step and one polyethylene glycol (PEG) precipitation step, was able to detect 10(2) RT-PCRU on 50 g frozen raspberries with conventional and real-time RT-PCR assays. Moreover the latter extraction method used no environmental hazardous chemical reagents and was easy to perform.

  10. Two-step versus one-step FT/sub 4/ assays in heparin treated patients and non-thyroidal illness

    Energy Technology Data Exchange (ETDEWEB)

    Reiners, C.; Bieler, G.; Ertl, G.; Gloss, H.; Boerner, W.

    1985-11-01

    The primary intention of this study is to inform the clinician about the direction and the order of magnitude of possible disturbances of different FT/sub 4/ parameters under the condition mentioned last, which is not uncommon in daily routine. No single FT/sub 4/-RIA proved be valid in severe NTI. There is the risk to misinterprete hyperthyroidism as euthyroidism with 1-step assays and the possibility to classify euthyroid patients falsely as hyperthyroid with 2-step assays. In relation to this problem, the sometimes lowered FT/sub 4/ values by 1step methods are clinically not so important. It has to be established, wether TSH assays of high sensitivity are able to overcome some of the difficulties with determinations of peripheral thyroid hormones in NTI.

  11. Rapid detection and quantification of Ebola Zaire virus by one-step real-time quantitative reverse transcription-polymerase chain reaction.

    Science.gov (United States)

    Ro, Young-Tae; Ticer, Anysha; Carrion, Ricardo; Patterson, Jean L

    2017-04-01

    Given that Ebola virus causes severe hemorrhagic fever in humans with mortality rates as high as 90%, rapid and accurate detection of this virus is essential both for controlling infection and preventing further transmission. Here, a one-step qRT-PCR assay for rapid and quantitative detection of an Ebola Zaire strain using GP, VP24 or VP40 genes as a target is introduced. Routine assay conditions for hydrolysis probe detection were established from the manufacturer's protocol used in the assays. The analytical specificity and sensitivity of each assay was evaluated using in vitro synthesized viral RNA transcripts. The assays were highly specific for the RNA transcripts, no cross-reactivity being observed among them. The limits of detection of the assays ranged from 10(2) to 10(3) copies per reaction. The assays were also evaluated using viral RNAs extracted from cell culture-propagated viruses (Ebola Zaire, Sudan and Reston strains), confirming that they are gene- and strain-specific. The RT-PCR assays detected viral RNAs in blood samples from virus-infected animal, suggesting that they can be also a useful method for identifying Ebola virus in clinical samples. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

  12. Comparison of real time RT-PCR and flow cytometry methods for evaluation of biological activity of recombinant human erythropoietin

    Directory of Open Access Journals (Sweden)

    Sepehrizadeh Z

    2008-05-01

    Full Text Available Background: Evaluation of bioactivity of recombinant erythropoietin is essential for pharmaceutical industry, quality control authorities and researchers. The purpose of this study was to compare real time RT-PCR and flow cytometry for the assay of biological activity of recombinant erythropoietin. Methods: Three concentrations of recombinant erythropoietin BRP (80, 40 and 20 IU/ml were injected subcutaneously to mice. After 4 days the blood was collected and used for reticulocyte counts by flow cytometry and also for the RNA extraction. Real time RT-PCR amplification was carried out for β-globin. Results and conclusion: There was a significant correlation between the total RNA amounts (R2= 0.9995, relative quantity of β-globin mRNA (R2= 0.984 and reticulocyte counts (R2= 0.9742 with rhEpo concentrations. Total RNA and quantitative RT-PCR showed significant dose dependent results as well the reticulocyte counts by flow cytometry for the biological activity assay of rhEpo and so these methods could be considered as alternatives for flow cytometry.

  13. Selection of reference genes for quantitative RT-PCR studies in striped dolphin (Stenella coeruleoalba skin biopsies

    Directory of Open Access Journals (Sweden)

    Casini Silvia

    2006-09-01

    Full Text Available Abstract Background Odontocete cetaceans occupy the top position of the marine food-web and are particularly sensitive to the bioaccumulation of lipophilic contaminants. The effects of environmental pollution on these species are highly debated and various ecotoxicological studies have addressed the impact of xenobiotic compounds on marine mammals, raising conservational concerns. Despite its sensitivity, quantitative real-time PCR (qRT-PCR has never been used to quantify gene induction caused by exposure of cetaceans to contaminants. A limitation for the application of qRT-PCR is the need for appropriate reference genes which allow the correct quantification of gene expression. A systematic evaluation of potential reference genes in cetacean skin biopsies is presented, in order to validate future qRT-PCR studies aiming at using the expression of selected genes as non-lethal biomarkers. Results Ten commonly used housekeeping genes (HKGs were partially sequenced in the striped dolphin (Stenella coeruleoalba and, for each gene, PCR primer pairs were specifically designed and tested in qRT-PCR assays. The expression of these potential control genes was examined in 30 striped dolphin skin biopsy samples, obtained from specimens sampled in the north-western Mediterranean Sea. The stability of selected control genes was determined using three different specific VBA applets (geNorm, NormFinder and BestKeeper which produce highly comparable results. Glyceraldehyde-3P-dehydrogenase (GAPDH and tyrosine 3-monooxygenase (YWHAZ always rank as the two most stably expressed HKGs according to the analysis with geNorm and Normfinder, and are defined as optimal control genes by BestKepeer. Ribosomal protein L4 (RPL4 and S18 (RPS18 also exhibit a remarkable stability of their expression levels. On the other hand, transferrin receptor (TFRC, phosphoglycerate kinase 1 (PGK1, hypoxanthine ribosyltransferase (HPRT1 and β-2-microglobin (B2M show variable expression

  14. High-throughput screening for monoamine oxidase-A and monoamine oxidase-B inhibitors using one-step fluorescence assay

    Institute of Scientific and Technical Information of China (English)

    Hong-mei GUANG; Guan-hua DU

    2006-01-01

    Aim: To develop high-throughput screening (HTS) assays for monoamine oxidase (MAO)-A and MAO-B inhibitors. Methods: A fluorescence probe based method measuring MAO-A and MAO-B activity was established and optimized, with its sensitivity, stability and specificity evaluated. Reaction conditions including enzyme sources, substrate concentrations, incubation volume and reaction time in 384-well format were optimized to achieve sensitive and low consumptive goal. Results: In optimized conditions, dynamic parameters of MAO-A and MAO-B were obtained. The Km value of serotonin to MAO-A was 1.66 μmol/L, while that of benzylamine to MAO-B was 0.80 umol/L. The IC50 value of clorgyline to MAO-A was 2.99 nmol/L, and that of deprenyl to MAO-B was 7.04 nmol/L, matching those obtained from traditional spectrometric assays. Among tested samples, one compound exerted an inhibitory effect on MAO-A activity with IC50 as 0.36 μmol/L, and three compounds had an inhibitory effect on MAO-B activity with IC50 as 0.13,0.19, and 0.13 μmol/L. The Z' factor was 0.71±0.03 and 0.75±0.03 in MAO-A-inhibitor and MAO-B-inhibitor HTS system, respectively. Conclusion: The established assays can be well applied to MAO-A and MAO-B inhibitor screening with high quality, precision and reproducibility.

  15. Production of monoclonal antibody for okadaic acid and its utilization in an ultrasensitive enzyme-linked immunosorbent assay and one-step immunochromatographic strip.

    Science.gov (United States)

    Liu, Biing-Hui; Hung, Chun-Tse; Lu, Chuan-Chen; Chou, Hong-Non; Yu, Feng-Yih

    2014-02-12

    Okadaic acid (OA) is a common marine biotoxin that accumulates in bivalves and causes diarrhetic shellfish poisoning (DSP). This study generated a monoclonal antibody (mAb) specific to OA from a hybridoma cell line, 6B1A3, which was obtained by fusion of myeloma cells (P3/NS1/1-AG4-1) with spleen cells isolated from a BALB/c mouse immunized with OA-γ-globulin. The 6B1A3 mAb belongs to the immunoglobulin G1 (κ chain) isotype. Both competitive direct and indirect enzyme-linked immunosorbent assays (ELISAs) were established for characterization of the antibody. The concentrations causing 50% inhibition of binding of OA-horseradish peroxidase to the antibody by OA were calculated to be 0.077 ng/mL in the cdELISA. A rapid and sensitive mAb-based gold nanoparticle immunochromatographic strip was also established. This proposed strip has a detection limit of 5 ng/mL for OA and can be finished in 10 min. Extensive analyses of 20 seafood samples with ELISA revealed that 10 were slightly contaminated with OA, with a mean concentration of 0.892 ng/g. Analysis of OA in shellfish samples showed that data acquired by the immunochromatographic strip agreed well with those acquired by the ELISA. The mAb-based ELISA and immunochromatographic strip assay developed in this study have adequate sensitivity and accuracy for rapid screening of OA in shellfish samples.

  16. Quantitative real-time RT-PCR and chromogenic in situ hybridization

    DEFF Research Database (Denmark)

    Rosa, Fabíola E; Silveira, Sara M; Silveira, Cássia G T;

    2009-01-01

    . METHODS: To elucidate the molecular profile of HER-2 status, mRNA and protein expression in 75 invasive breast carcinomas were analyzed by real time quantitative RT-PCR (qRT-PCR) and IHC, respectively. Amplifications were evaluated in 43 of these cases by CISH and in 11 by FISH. RESULTS: The concordance...

  17. Diagnostic evaluation of RT-PCR-ELISA for the detection of rabies virus.

    Science.gov (United States)

    Aravindhbabu, R P; Manoharan, S; Ramadass, P

    2014-01-01

    Rabies is primarily a disease of terrestrial and airborne mammals. In most cases, rabies is diagnosed primarily on the basis of clinical symptoms and signs, and a corroborative history of or evidence of an animal bite, death of an animal and incomplete or no vaccination following exposure. The facility for laboratory diagnosis and confirmation of rabies is available in only a few institutions in India. Diagnostic tests using conventional assays like fluorescent antibody test (FAT) are unreliable at times, despite the clinical diagnosis. Currently, there are a number of molecular tests that can be used to complement conventional tests in rabies diagnosis. We have developed and evaluated an RT-PCR-ELISA using a panel of brain tissue samples from rabies suspected animals of various species. This assay was able to detect rabies virus genome in all the 43 samples that were previously tested positive for rabies. Moreover this assay was shown to be 100 % sensitive and specific in detecting the rabies virus genome in post-mortem brain tissue samples from different species of animals. Our pilot study shows the potential of this assay as an alternative diagnostic test when the samples are unsuitable for use in FAT and also a supplementary test to FAT. In addition, the region of nucleoprotein gene amplified using this assay can be used for the molecular investigation of geographical origin of the field strains.

  18. Development of a GeXP Based Multiplex RT-PCR Assay for Simultaneous Detection of Eight Arboviruses Related to Encephalitis%8种脑炎相关虫媒病毒GeXP检测方法的初步建立

    Institute of Scientific and Technical Information of China (English)

    何玢; 王环宇; 张晨; 王淼; 秦萌; 王克霞; 马学军

    2012-01-01

    利用GeXP多重基因表达遗传分析系统,建立一种多重逆转录-聚合酶链反应( mRT-PCR)方法,同时检测与病毒性脑炎相关的乙型脑炎病毒(Japanese encephalitis virus,JEV)等8种虫媒病毒.优化多重反应体系及反应条件,分别以病毒分离培养物和阳性标本来验证多重反应体系的特异性,以克隆质粒体外转录的RNA梯度稀释液来检测多重检测体系的灵敏度.结果表明,优化后的多重检测体系,可扩增出各病毒对应的特异片段,并可在102拷贝/μL水平同时并特异地检测出8种(共13个特异片段)脑炎相关病毒RNA.该方法具有高通量、灵敏度高、特异性强且快速等优点,对病毒性脑炎的分子诊断及流行病学调查具有重要意义.%Multiplex reverse transcription-polymerase chain reaction (mRT-PCR) is currently available in virus detection and defined as the simultaneous amplification of two or more DNA/RNA targets in a single reaction vessel. In this study, we attempted to modify the conventional mRT-PCR technique on a basis of GenomeLab Genetic Analysis System (GeXP). Initially, we optimized the analytical validation of the GeXP analyzer and its design of workflow and simultaneously detected eight arboviruses that related to epidemic encephalitis by verifying the specificity of mRT-PCR with Japanese encephalitis virus(JEV) cell cultures and positive strains identified previously and determining the sensitivity with in vitro-transcribed RNA of serial dilutions. The GeXP system after optimization could amplify the specific fragments related to the viruses and exposed specifically a total of 13 target genes out of eight types of arboviruses at the level of 102 copies/μL, and the findings suggest that the novel protocol we developed can be high-throughput and highly specific and sensitive as well as quickness in screening of the encephalitis viruses, and is promising in detection of encephalitis-associated viruses for molecular

  19. 猪瘟病毒Taqman实时定量RT-PCR检测方法的建立和临床应用%Development and clinical application of Taqman real-time RT-PCR assay for detection of classical swine fever virus

    Institute of Scientific and Technical Information of China (English)

    毛立; 李文良; 李彬; 江杰元

    2012-01-01

    According to the conservative sequences located on the 5' untranslated region (5'-UTR) of classical swine fever virus(CSFV) ,a pair of specific primers and Taqman probe were designed and synthesized respectively, and a Taqman real-time fluorescent quantitative reserve-transcribed polymerase chain reaction (real-lime RT-PCR) for detecting the CSFV was established in this study. Test results showed that the method had a detection limit of 10 copies of target RNA per reaction, and there was a good linear relationship between Ct value and copy numbers in diluted samples. The variation between batches was less than 1% . The RNA of porcine reproductive and respiratory syndrome virus,bovine viral diarrhea virus were detected by the Taqman RT-PCR,and the results were all negative. The CSFV-positive rate was 71. 9% in 192 samples collected from Jiangsu and Xinjiang areas. Real-time RT-PCR detection showed that the different organs of swine including hearts, lungs, livers, kidneys, brains, spleens,lymph nodes and ascites were CSFV-positive, indicating thai the method were more sensitive and effective than traditional RT-PCR.%根据猪瘟病毒5’非编码区(5’-UTR)设计特异性引物和Taqman探针,建立Taqman实时定量RT-PCR检测猪瘟病毒法.检测结果显示,该方法的灵敏度为1μl 10拷贝,在病毒拷贝数为1μl 108~101时,循环数(Ct)值与拷贝数对数呈现较好的线性关系,且重复性好,批间变异系数小于1%.用该方法检测猪繁殖与呼吸综合征病毒、牛病毒性腹泻病毒,结果均为阴性.用该方法检测采集自江苏和新疆的192份组织和血清样品,猪瘟病毒阳性率为71.9%;检测感染猪的不同脏器,发现在心、肺、肝、肾、脑、脾脏、淋巴结、腹水中均可以检测到猪瘟病毒,与常规RT-PCR方法相比,该方法敏感性更高.该方法的建立为猪瘟病毒的流行病学调查和定量提供了有效手段.

  20. Multiplex RT-PCR-based detections of CEA, CK20 and EGFR in colorectal cancer patients

    Science.gov (United States)

    Tsouma, Aikaterini; Aggeli, Chrysanthi; Lembessis, Panagiotis; Zografos, George N; Korkolis, Dimitris P; Pectasides, Dimitrios; Skondra, Maria; Pissimissis, Nikolaos; Tzonou, Anastasia; Koutsilieris, Michael

    2010-01-01

    AIM: To develop a multiplex reverse transcription polymerase chain reaction (RT-PCR) method detecting circulating tumor cells in the peripheral blood of colorectal cancer (CRC) patients. METHODS: Peripheral blood samples were collected from 88 CRC patients and 40 healthy individuals from the blood donors’ clinic and subsequently analyzed by multiplex RT-RCR for the expression of carcinoembryonic antigen (CEA), cytokeratin 20 (CK20) and epidermal growth factor receptor (EGFR) mRNA. The analysis involved determining the detection rates of CEA, CK20 and EGFR transcripts vs disease stage and overall survival. Median follow-up period was 19 mo (range 8-28 mo). RESULTS: Rates of CEA, CK20 and EGFR detection in CRC patients were 95.5%, 78.4% and 19.3%, respectively. CEA transcripts were detected in 3 healthy volunteer samples (7.5%), whereas all control samples were tested negative for CK20 and EGFR transcripts. The increasing number of positive detections for CEA, CK20 and EGFR transcripts in each blood sample was positively correlated with Astler-Coller disease stage (P < 0.001) and preoperative serum levels of CEA (P = 0.029) in CRC patients. Data analysis using Kaplan-Meier estimator documented significant differences in the overall survival of the different CRC patient groups as formed according to the increasing number of positivity for CEA, CK20 and EGFR transcripts. CONCLUSION: These data suggest that multiplex RT-PCR assay can provide useful information concerning disease stage and overall survival of CRC patients. PMID:21157973

  1. Clinical evaluation of the Serum CrossLaps One Step ELISA, a new assay measuring the serum concentration of bone-derived degradation products of type I collagen C-telopeptides

    DEFF Research Database (Denmark)

    Christgau, S; Rosenquist, C; Alexandersen, P

    1998-01-01

    The Serum CrossLaps One Step ELISA is a sandwich assay using two monoclonal antibodies specific for a beta-aspartate form of the epitope EKAHDGGR derived from the carboxy-terminal telopeptide region of type I collagen alpha1-chain. Our objective was to assess the clinical value of the Serum Cross......Laps assay for monitoring antiresorptive therapy in osteoporosis treatment. Samples obtained from postmenopausal women treated with different doses of cyclic or continuous hormone replacement therapy (HRT) with an estrogen analog (tibolone) or with a bisphosphonate (ibandronate) were measured in the Serum...

  2. Detection of cancer cells in peripheral blood with nested RT-PCR and itssignificance in patients with gastric carcinomas

    Institute of Scientific and Technical Information of China (English)

    Jia Zeng Xia; Hao Ran Yin; Zheng Gang Zhu; Min yan

    2000-01-01

    AIM To study the detection of micrometastasis in peripheral blood of patients with gastric carcinomas andits clinical significance.METHODS A cytokeratin 19 (CK19)-specific nested reverse transcriptase-polimerase chain reaction (RT-PCR) assay was developed to detect CK19 expressing cancer cells, the sensitivity was determined by serialdilution method using CK19 expressing gastric cancer cells, the specificity was assessed by examining 12negative controls and 12 positive controls. Then pre-operative peripheral blood from 42 patients with gastriccancer was detected and the relationship between positive results and biological behavior was studied.RESULTS CK19mRNA was expressed in all the 12 gastric cancer tissues but not in peripheral blood from12 healthy individuals;sensitivity of nested RT-PCR amplification for CK19mRNA was confirmed to be 1/106 by serial dilution method using human gastric cancer line SGC-7901; micrometastases in pre-operativeperipheral blood were detected in 13 (30,9%) patients with gastric carcinomas, the frequency ofmicrometastasis in peripheral blood was significantly correlated with tumor size,depth of invasion and TNMstage (x2 test, P<0.05).CONCLUSION Nested RT-PCR amplification for CK19mRNA is a sensitive and specific method for thedetection of micrometastases in peripheral blood in gastric cancer patients; pre-operative detection ofmicrometastasis in peripheral blood may be helpful in the prediction of tumor progression.

  3. Quality control of RNA preservation and extraction from paraffin-embedded tissue: implications for RT-PCR and microarray analysis.

    Science.gov (United States)

    Kashofer, Karl; Viertler, Christian; Pichler, Martin; Zatloukal, Kurt

    2013-01-01

    Analysis of RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathological diagnostics. We have performed a comprehensive and systematic investigation of the impact of factors in the pre-analytical workflow, such as different fixatives, fixation time, RNA extraction method and storage of tissues in paraffin blocks, on several downstream reactions including complementary DNA (cDNA) synthesis, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microarray hybridization. We compared the effects of routine formalin fixation with the non-crosslinking, alcohol-based Tissue Tek Xpress Molecular Fixative (TTXMF, Sakura Finetek), and cryopreservation as gold standard for molecular analyses. Formalin fixation introduced major changes into microarray gene expression data and led to marked gene-to-gene variations in delta-ct values of qRT-PCR. We found that qRT-PCR efficiency and gene-to-gene variations were mainly attributed to differences in the efficiency of cDNA synthesis as the most sensitive step. These differences could not be reliably detected by quality assessment of total RNA isolated from formalin-fixed tissues by electrophoresis or spectrophotometry. Although RNA from TTXMF fixed samples was as fragmented as RNA from formalin fixed samples, much higher cDNA yield and lower ct-values were obtained in qRT-PCR underlining the negative impact of crosslinking by formalin. In order to better estimate the impact of pre-analytical procedures such as fixation on the reliability of downstream analysis, we applied a qRT-PCR-based assay using amplicons of different length and an assay measuring the efficiency of cDNA generation. Together these two assays allowed better quality assessment of RNA extracted from fixed and paraffin-embedded tissues and should be used to supplement quality scores derived from automated electrophoresis. A better standardization of the pre-analytical workflow, application

  4. CM Affi-Gel Blue chromatography of human urine: a simple one-step procedure for obtaining erythropoietin suitable for in vitro erythropoietic progenitor assays.

    Science.gov (United States)

    Krystal, G; Eaves, C J; Eaves, A C

    1984-11-01

    A method for both concentrating and purifying human urinary erythropoietin (Ep) using CM Affi-Gel Blue is described. We have found that up to 40 litres of urine can be processed on a 1 litre gel bed of this material. This gives a 25-50-fold purification of Ep with an apparent Ep recovery in excess of 100%. The high recovery of Ep is probably due, in part, to the removal of inhibitors present in the initial urine. By selecting urine that contains high levels of Ep (greater than 0.5 units/ml), it is possible with this method routinely to obtain preparations with specific activities of 100-300 units of Ep per mg protein. Such preparations are noninhibitory when assayed in either short-term suspension cultures or in longer-term methylcellulose cultures at concentrations up to 5-10 units/ml. Similar tests with these same bioassay systems have shown that other non-Ep stimulating factors (i.e. erythroblast enhancing factor (EEF), granulocyte/macrophage colony stimulating factor (GM-CSF) and burst promoting activity (BPA) ) are also not present at detectable levels. In this study we also show that the loss of biological activity which often occurs when partially purified Ep preparations are stored in solution is markedly reduced in the presence of either 1% bovine serum albumin or 0.1% sodium dodecyl sulphate.

  5. Development and Application of a Multiplex RT-PCR Assay for Detecting Three Sweet Cherry Virus Species%3种甜樱桃病毒PNRSV、PDV及LChV-2的多重RT-PCR检测方法的建立与应用

    Institute of Scientific and Technical Information of China (English)

    宗晓娟; 王文文; 魏海蓉; 王甲威; 陈新; 徐丽; 刘庆忠

    2014-01-01

    【目的】建立可同时检测李属坏死环斑病毒(Prunus necrotic ringspot virus,PNRSV)、李矮缩病毒(Prune dwarf virus,PDV)、樱桃小果病毒2(Little cherry virus-2,LChV-2)的多重RT-PCR检测方法。【方法】以复合感染3种病毒的甜樱桃病株叶片为材料,采用CTAB法提取样本总RNA,选用随机六聚体引物对植物样本总RNA进行反转录,所得cDNA作为多重RT-PCR的扩增模板。根据GenBank中PNRSV、PDV、LChV-2基因组序列共设计6对特异引物,分别通过单一RT-PCR和多重RT-PCR筛选出可用于同时检测3种甜樱桃病毒的引物组合。对多重RT-PCR的退火温度及循环数进行优化,以筛选出各引物组合的最适扩增条件。分别以单一感染PNRSV、PDV、LChV-2、复合感染3种病毒、单一感染樱桃病毒 A(Cherry virus A,CVA)及甜樱桃无毒苗为样本,对多重RT-PCR引物的特异性进行分析。选取复合感染3种病毒的甜樱桃总RNA的反转录产物为初始模板,按照梯度稀释法依次将模板稀释为2、22、23、24、25倍,在相同 PCR反应体系及反应条件下分别对各引物组合的灵敏度进行分析。多重 RT-PCR 的扩增条带经凝胶回收试剂盒回收纯化后连接至 pMD18-T vector,克隆测序,以验证多重RT-PCR检测的准确性。并应用该方法对山东泰安地区甜樱桃生产园中间隔栽培的中国樱桃进行检测。【结果】筛选到2个可以应用的引物组合,组合1“PNRSV-S1/A1、PDV-S2/A2、LChV2-S1/A1”可分别特异性地扩增733、467、337 bp的片段。组合2“PNRSV-S1/A1、PDV-S3/A3、LChV2-S1/A1”可分别扩增得到733、265、337 bp的片段。扩增产物大小与预期相符。多重RT-PCR反应条件优化结果显示,在退火温度52℃、35个循环条件下,2个引物组合的检测效果均较为理想。特异性分析结果显示,2个引物组合均能特异性检测其各自的靶病毒。

  6. A broad spectrum, one-step reverse-transcription PCR amplification of the neuraminidase gene from multiple subtypes of influenza A virus

    Directory of Open Access Journals (Sweden)

    Chen Wenbin

    2008-07-01

    Full Text Available Abstract Background The emergence of high pathogenicity strains of Influenza A virus in a variety of human and animal hosts, with wide geographic distribution, has highlighted the importance of rapid identification and subtyping of the virus for outbreak management and treatment. Type A virus can be classified into subtypes according to the viral envelope glycoproteins, hemagglutinin and neuraminidase. Here we review the existing specificity and amplification of published primers to subtype neuraminidase genes and describe a new broad spectrum primer pair that can detect all 9 neuraminidase subtypes. Results Bioinformatic analysis of 3,337 full-length influenza A neuraminidase segments in the NCBI database revealed semi-conserved regions not previously targeted by primers. Two degenerate primers with M13 tags, NA8F-M13 and NA10R-M13 were designed from these regions and used to generate a 253 bp cDNA product. One-step RT-PCR testing was successful in 31/32 (97% cases using a touchdown protocol with RNA from over 32 different cultured influenza A virus strains representing the 9 neuraminidase subtypes. Frozen blinded clinical nasopharyngeal aspirates were also assayed and were mostly of subtype N2. The region amplified was direct sequenced and then used in database searches to confirm the identity of the template RNA. The RT-PCR fragment generated includes one of the mutation sites related to oseltamivir resistance, H274Y. Conclusion Our one-step RT-PCR assay followed by sequencing is a rapid, accurate, and specific method for detection and subtyping of different neuraminidase subtypes from a range of host species and from different geographical locations.

  7. TaqMan real-time RT-PCR detection of infectious salmon anaemia virus (ISAV) from formalin-fixed paraffin-embedded Atlantic salmon Salmo salar tissues.

    Science.gov (United States)

    Godoy, M G; Kibenge, F S; Kibenge, M J; Olmos, P; Ovalle, L; Yañez, A J; Avendaño-Herrera, R

    2010-05-18

    The objective of this study was to evaluate the application of a TaqMan real-time reverse transcriptase PCR (RT-PCR) assay for the detection of infectious salmon anaemia virus (ISAV) in formalin-fixed paraffin-embedded (FFPE) fish tissues from Atlantic salmon Salmo salar with and without clinical signs of infection, and to compare it with histological and immunohistochemical (IHC) techniques. Sixteen fish samples obtained in 2007 and 2008 from 4 different farms in Chile were examined. The real-time RT-PCR allowed the detection of ISAV in FFPE samples from 9 of 16 fish, regardless of the organs analyzed, whereas 4 of the real-time RT-PCR negative fish were positive as indicated by histological examination and 3 of the real-time RT-PCR positive fish were negative as indicated by immunohistochemistry evaluation. The presence of ISAV in RT-PCR positive samples was confirmed by amplicon sequencing. This work constitutes the first report on the use of real-time RT-PCR for the detection of ISAV in FFPE sections. The assay is very useful for the examination of archival wax-embedded tissues, and allows for both prospective and retrospective evaluation of tissue samples for the presence of ISAV. However, the method only confirms the presence of the pathogen and should be used in combination with histopathology, which is a more precise tool. The combination of both techniques would be invaluable for confirmatory diagnosis of infectious salmon anaemia (ISA), which is essential for solving salmon farm problems.

  8. Gastroenteritis outbreaks associated with Norwalk-like viruses and their investigation by nested RT-PCR

    Directory of Open Access Journals (Sweden)

    Mitchell Frederick

    2001-08-01

    Full Text Available Abstract Background Norwalk-like viruses are the most common cause of gastroenteritis outbreaks and sporadic cases of vomiting and diarrhoea. In healthy individuals infection is often mild and short-lived but in debilitated patients infection can be severe. It is essential that the virus laboratory can offer a sensitive and specific test, delivered in a timely manner. Methods We have developed a nested reverse transcriptase PCR based on published primers against the RNA polymerase gene and after comparison with electronmicroscopy used the assay to investigate 31 outbreaks of gastroenteritis. These were in diverse situations including nursing homes, small district hospitals, large general hospitals, a ferry ship, hotels, restaurants and staff canteens. Results A positive diagnosis was made in 30/31 outbreaks investigated giving an overall outbreak positive detection rate of 97%. At an individual patient level there was a positive diagnostic rate of 11.5% in a large hospital environment to 100% in smaller outbreak situations. The average patient positive rate was 34%. In addition we investigated 532 control faecal specimens from adults. Of these 530 were negative and 2 were repeatedly positive. Conclusions It is essential that insensitive electronmicroscopy is replaced with the more sensitive reverse transcription PCR assays. These tests should be made available "on call" at weekends and public holidays. It is also important that outbreaks of NLV infection are monitored using sensitive RT-PCR assays so that the laboratory information can be used in ascertaining the spread and duration of the outbreak

  9. Hepatitis C Virus RNA Real-Time Quantitative RT-PCR Method Based on a New Primer Design Strategy.

    Science.gov (United States)

    Chen, Lida; Li, Wenli; Zhang, Kuo; Zhang, Rui; Lu, Tian; Hao, Mingju; Jia, Tingting; Sun, Yu; Lin, Guigao; Wang, Lunan; Li, Jinming

    2016-01-01

    Viral nucleic acids are unstable when improperly collected, handled, and stored, resulting in decreased sensitivity of currently available commercial quantitative nucleic acid testing kits. Using known unstable hepatitis C virus RNA, we developed a quantitative RT-PCR method based on a new primer design strategy to reduce the impact of nucleic acid instability on nucleic acid testing. The performance of the method was evaluated for linearity, limit of detection, precision, specificity, and agreement with commercial hepatitis C virus assays. Its clinical application was compared to that of two commercial kits--Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) and Kehua. The quantitative RT-PCR method delivered a good performance, with a linearity of R(2) = 0.99, a total limit of detection (genotypes 1 to 6) of 42.6 IU/mL (95% CI, 32.84 to 67.76 IU/mL), a CV of 1.06% to 3.34%, a specificity of 100%, and a high concordance with the CAP/CTM assay (R(2) = 0.97), with a means ± SD value of -0.06 ± 1.96 log IU/mL (range, -0.38 to 0.25 log IU/mL). The method was superior to commercial assays in detecting unstable hepatitis C virus RNA (P quantitative RT-PCR method can effectively eliminate the influence of RNA instability on nucleic acid testing. The principle of primer design strategy may be applied to the detection of other RNA or DNA viruses.

  10. RT-PCR and Electrospray Ionization Mass Spectrometry (RT-PCR/ESI-MS) for Identifying Acute Viral Upper Respiratory Tract Infections

    Science.gov (United States)

    Chen, Kuan-Fu; Blyn, Lawrence; Rothman, Richard E.; Ramachandran, Padmini; Valsamakis, Alexandra; Ecker, David; Sampath, Rangarajan; Gaydos, Charlotte A.

    2010-01-01

    Diagnosis of respiratory viruses traditionally relies on culture or antigen detection.We aimed to demonstrate capacity of the RT-PCR/ESI-MS platform to identify clinical relevant respiratory viruses in nasopharyngeal aspirate (NPA) samples and compare the diagnostic performance characteristics relative to conventional culture- and antigen-based methods. A RT-PCR/ESI-MS respiratory virus surveillance kit designed to detect respiratory syncytial virus, influenza A and B, parainfluenza types 1-4, adenoviridae types A-F, coronaviridae, human bocavirus, and human metapneumovirus was evaluated using both mock-ups and frozen archived NPA (N=280), 95 of which were positive by clinical virology methods. RT-PCR/ESI-MS detected 74/95 (77.9%) known positive samples and identified an additional 13/185 (7%) from culture negative samples. Viruses that are non-detectable with conventional methods were also identified. Viral load was semi-quantifiable and ranged from 2,400 to >320,000copies/ml. Time to results was 8hrs. RT-PCR/ESI-MS showed promise in rapid detection of respiratory viruses, merits further evaluation for use in clinical settings. PMID:21251562

  11. Detecciones de la expresion de citoquinas mediante RT-PCR en tiempo real en ganado ovino

    National Research Council Canada - National Science Library

    Perez de Diego Camacho, Ana Cristina; Sanchez Matamoros, Almudena; Sanchez-Vizcaino Rodriguez, Jose Manuel

    2009-01-01

    .... Existen diferentes tecnicas para estudiar la produccion y accion de estas en los individuos. Este estudio consiste en la puesta a punto de una tecnica de Retrotranscripcion- Reaccion en cadena de la polimerasa (RT-PCR...

  12. Detecting Newcastle disease virus in combination of RT-PCR with red blood cell absorption

    Directory of Open Access Journals (Sweden)

    Liu Chengqian

    2011-05-01

    Full Text Available Abstract Reverse transcription-polymerase chain reaction (RT-PCR has limited sensitivity when treating complicated samples, such as feces, waste-water in farms, and nucleic acids, protein rich tissue samples, all the factors may interfere with the sensitivity of PCR test or generate false results. In this study, we developed a sensitive RT-PCR, combination of red blood cell adsorption, for detecting Newcastle disease virus (NDV. One pair of primers which was highly homologous to three NDV pathotypes was designed according to the consensus nucleocapsid protein (NP gene sequence. To eliminate the interfere of microbes and toxic substances, we concentrated and purified NDV from varied samples utilizing the ability of NDV binding red blood cells (RBCs. The RT-PCR coupled with red blood cell adsorption was much more sensitive in comparison with regular RT-PCR. The approach could also be used to detect other viruses with the property of hemagglutination, such as influenza viruses.

  13. Evaluation of four different systems for extraction of RNA from stool suspensions using MS-2 coliphage as an exogenous control for RT-PCR inhibition.

    Directory of Open Access Journals (Sweden)

    Lester M Shulman

    Full Text Available Knowing when, and to what extent co-extracted inhibitors interfere with molecular RNA diagnostic assays is of utmost importance. The QIAamp Viral RNA Mini Kit (A; MagNA Pure LC2.0 Automatic extractor (B; KingFisher (C; and NucliSENS EasyMag (D RNA extraction systems were evaluated for extraction efficiency and co-purification of inhibitors from stool suspensions. Real-Time Reverse Transcriptase Polymerase Chain Reaction (rRT-PCR of MS-2 coliphage spiked into each system's lysis buffer served as an external control for both. Cycle thresholds (Cts of the MS2 were determined for RNA extracted from stool suspensions containing unknown (n = 93 or varying amounts of inhibitors (n = 92. Stool suspensions from the latter group were also used to determine whether MS-2 and enterovirus rRT-PCR inhibitions were correlated. Specifically 23 RNA extracts from stool suspensions were spiked with enterovirus RNA after extraction and 13 of these stool suspension were spiked with intact enterovirus before extraction. MS2 rRT-PCR inhibition varied for RNAs extracted by the different systems. Inhibition was noted in 12 (13.0%, 26 (28.3%, 7 (7.6%, and 7 (7.6% of the first 93 RNA extracts, and 58 (63.0%, 55 (59.8%, 37 (40.2% and 30 (32.6% of the second 92 extracts for A, B, C, and D, respectively. Furthermore, enterovirus rRT-PCR inhibition correlated with MS2 rRT-PCR inhibition for added enterovirus RNA or virus particles. In conclusion, rRT-PCR for MS-2 RNA is a good predictor of inhibition of enterovirus RNA extracted from stool suspensions. EasyMag performed the best, however all four extraction methods were suitable provided that external controls identified problematic samples.

  14. Investigation of Reference Genes in Vibrio parahaemolyticus for Gene Expression Analysis Using Quantitative RT-PCR

    OpenAIRE

    Yue-Jiao Ma; Xiao-Hong Sun; Xiao-Yan Xu; Yong Zhao; Ying-Jie Pan; Cheng-An Hwang; Wu, Vivian C. H.

    2015-01-01

    Vibrio parahaemolyticus is a significant human pathogen capable of causing foodborne gastroenteritis associated with the consumption of contaminated raw or undercooked seafood. Quantitative RT-PCR (qRT-PCR) is a useful tool for studying gene expression in V. parahaemolyticus to characterize its virulence factors and understand the effect of environmental conditions on its pathogenicity. However, there is not a stable gene in V. parahaemolyticus that has been identified for use as a reference ...

  15. Detection and surveillance of viral hemorrhagic septicemia virus using real-time RT-PCR. II. Diagnostic evaluation of two protocols.

    Science.gov (United States)

    Warg, Janet V; Clement, Travis; Cornwell, Emily R; Cruz, Angela; Getchell, Rodman G; Giray, Cem; Goodwin, Andrew E; Groocock, Geoffrey H; Faisal, Mohamed; Kim, Robert; Merry, Gwenn E; Phelps, Nicholas B D; Reising, Monica M; Standish, Isaac; Zhang, Yan; Toohey-Kurth, Kathy

    2014-08-21

    Two real-time reverse transcription polymerase chain reaction (rRT-PCR) assays under consideration for deployment to multiple testing laboratories across the USA were evaluated for diagnostic sensitivity and specificity on tissue homogenates obtained from natural and experimental viral hemorrhagic septicemia (VHS)-infected fish. Estimates for diagnostic specificity using virus isolation as the reference method were similar between laboratories regardless of the assay. Diagnostic sensitivity estimates of 0.96 (95% CI: 0.95, 0.97) for Jonstrup et al. (2013)'s assay (J Fish Dis 36:9-23) exceeded the diagnostic sensitivity of 0.85 (95% CI: 0.83, 0.87) for Phelps et al. (2012)'s assay (J Aquat Anim Health 24:238-243). The Jonstrup rRT-PCR assay is robust as demonstrated by high sensitivity and specificity estimates across laboratories and can be used as a valuable tool for targeted surveillance and for testing of suspect VHSV samples.

  16. TaqMan荧光定量RT-PCR检测猪脂蛋白酯酶mRNA方法的建立%Establishment of Real-time TaqMan-Fluorescence Quantitative RT-PCR Assay for Detection and Quantification of Porcine Lipoprotein Lipase mRNA

    Institute of Scientific and Technical Information of China (English)

    廉红霞; 卢德勋; 高民

    2008-01-01

    [目的]克隆猪cDNA,作为猪LPL mRNA定量检测的标准品,建立检测方法.[方法]用RT-PCR,从猪背最长肌的总RNA中逆转录扩增LPL的cDNA,将纯化的LPL cDNA与pGM-T载体进行连接,转化宿主菌TOP10,提取重组质粒DNA,PCR鉴定并测序分析,对质粒标准进行实时荧光定量PCR检测.纯化质粒并检测260 nm吸光度,确定原液的重组质粒拷贝浓度并以此制备荧光定量PCR梯度浓度标准品.[结果]建立了猪LPL mRNA实时定量PCR检测方法,特异性好,检测灵敏度达103拷贝,线性范围为1×103~1×1010拷贝,阈值与PCR体系中起始模板量的对数值之间有着良好的线性关系(R2=0.9871).[结论]成功克隆了LPL实时荧光PCR定量标准,且TaqMan荧光定量RT-PCR的方法可对猪背最长肌LPL mRNA的表达进行准确定量.

  17. Application of Multiplex RT-PCR Assay for Screening Rare or Cryptic Chromosome Translocations in De Novo Patients with Acute Myeloid Leukemia%应用多重RT-PCR在初发急性髓系白血病患者中筛查少见或难以识别的染色体易位

    Institute of Scientific and Technical Information of China (English)

    陈海敏; 员海洋; 樊星; 何海燕; 陈冰; 施静艺; 朱勇梅

    2010-01-01

    本研究探讨应用多重逆转录聚合酶链反应(reverse transcription polymerase chain reaction,RT-PCR)筛查少见/难以识别的染色体易位患者的临床应用可行性.建立了3组多重RT.PCR以检测常规核型分析检出率较低的6种mll相关基因重排(mll/af10、mll/af17、mll/ell、mll/af9、mll/af6和mll/enl)和4种少见融合基因(dek/can、tls/erg、amll/mds(evil)和npm/mlfl).结果表明:126例标本中共检测出11例阳性标本,涉及5种分子异常,其中10例为M5亚型(16.67%),1例为M4亚型(1.51%).在这11例阳性标本中,2例患者核型分析见到标志染色体,1例患者仅有1个分裂相以致无法分析核型,其余8例用R-显带核型分析均未发现异常.结论:本研究设计的多重RT-PCR可便捷地、有效和准确地识别多种少见/难以发现的染色体易位.

  18. Establishment of RT-PCR assay for detection of bovine viral diarrhea virus genotype 2%基因2型牛病毒性腹泻病毒RT-PCR检测方法的建立和初步应用

    Institute of Scientific and Technical Information of China (English)

    任敏; 焦海宏; 蒋颖; 刘振华; 王传彬; 朱国强

    2008-01-01

    根据已发表的牛病毒性腹泻病毒基因1型(BVDV-1)和2型(BVDV-2)5非编码区(5-UTR)的序列,分别设计了针对BVDV-1型、BVDV-2型以及针对BVDV-1和BVDV-2两型的特异性引物和通用引物,以标准参考株BVDV-1 NADL株和BVDV-2890 株为对照,建立了分别能检测BVDV-1、BVDV-2和BVDV-1/BVDV-2的RT-PCR 检测方法.在此基础上,进一步对疑似BVDV-2感染牛的临床病料组织(脾、淋巴结、心肌、血液等)检测结果表明,在所检测的18份样品中,BVDV-2阳性8份(44%).经RT-PCR鉴定为阳性的组织病料,进一步通过MDBK 细胞进行病毒分离,病毒分离率为100%;结合感染细胞病变观察,间接免疫荧光试验RT-PCR和序列测定鉴定,所分离的病毒均为BVDV-2.上述研究表明,该RT-PCR检测方法敏感、特异;并证实我国牛群已存在BVDV-2的污染或感染.

  19. Cytokine mRNA quantification in histologically normal canine duodenal mucosa by real-time RT-PCR.

    Science.gov (United States)

    Peters, I R; Helps, C R; Calvert, E L; Hall, E J; Day, M J

    2005-01-10

    CD4(+) T helper cells are important for the regulation of immune responses in the intestinal mucosa and they exert their effects through the secretion of pro-inflammatory and immunomodulatory cytokines. Human patients with inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis have alterations in the normal intestinal cytokine profile. These cytokine abnormalities have been shown at both the protein and messenger RNA (mRNA) level. The role that mucosal cytokines play in the pathogenesis of canine IBD has only been investigated using semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis of gut tissue, as cytokine antisera are not available for this species. Real-time RT-PCR has been recognised to be a more accurate and sensitive method of quantifying mRNA transcripts, so in this study TaqMan real-time RT-PCR assays for the quantification of mRNA encoding IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, IFN-gamma, TNF-alpha and TGF-beta in canine intestinal mucosa were developed. The amount of these templates was quantified in normal canine duodenal mucosa (n = 8). IL-18, TGF-beta and TNF-alpha were found to be the most abundant transcripts, with IL-10 and IFN-gamma present at levels approximately 10-fold less. IL-2, IL-4, IL-5, IL-6 and IL-12 were the least abundant templates, with some RNA samples having no detectable mRNA copies. The methods developed in this study will form the basis of further work investigating the expression of mRNA encoding cytokines in mucosa from dogs with chronic enteropathies. In addition, these real-time PCR assays can also be used for the quantification of canine cytokine mRNA in other diseases.

  20. RT-PCR using glycoprotein target is more sensitive for the detection of Ebola virus in clinical samples.

    Science.gov (United States)

    Yang, Mingjuan; Ke, Yuehua; Zhang, Wenyi; Liu, Chao; Yang, Ruifu; Chen, Zeliang

    2017-03-01

    The recent largest ever Ebola virus disease (EVD) outbreak in West Africa has been of worldwide concern, causing huge economic losses and constituting serious threat to the local residents and health care workers. Rapid detection of Ebola virus (EBOV) using RT-PCR has been suggested to be of great value in stopping the outbreak, because it is highly sensitive and specific and can return results within hours. In this study, 210 clinical samples, including 109 blood and 101 nasopharyngeal swab samples were used to compare the performance of glycoprotein (GP) and nucleoprotein (NP) gene targets for the detection of EBOV. The analytical sensitivity of both assays were 10 molecules/μL. For clinical samples, the sensitivity of the assay targeting GP gene is higher than that of NP gene (respectively 98% and 94%) and the specificities for both targets were 100%. In addition, the positive samples in the RT-PCR assay targeting GP showed lower cycle threshold values and higher virus loads than NP gene.

  1. Microdroplet sandwich real-time rt-PCR for detection of pandemic and seasonal influenza subtypes.

    Directory of Open Access Journals (Sweden)

    Stephanie L Angione

    Full Text Available As demonstrated by the recent 2012/2013 flu epidemic, the continual emergence of new viral strains highlights the need for accurate medical diagnostics in multiple community settings. If rapid, robust, and sensitive diagnostics for influenza subtyping were available, it would help identify epidemics, facilitate appropriate antiviral usage, decrease inappropriate antibiotic usage, and eliminate the extra cost of unnecessary laboratory testing and treatment. Here, we describe a droplet sandwich platform that can detect influenza subtypes using real-time reverse-transcription polymerase chain reaction (rtRT-PCR. Using clinical samples collected during the 2010/11 season, we effectively differentiate between H1N1p (swine pandemic, H1N1s (seasonal, and H3N2 with an overall assay sensitivity was 96%, with 100% specificity for each subtype. Additionally, we demonstrate the ability to detect viral loads as low as 10(4 copies/mL, which is two orders of magnitude lower than viral loads in typical infected patients. This platform performs diagnostics in a miniaturized format without sacrificing any sensitivity, and can thus be easily developed into devices which are ideal for small clinics and pharmacies.

  2. Subtyping Animal Influenza Virus with General Multiplex RT-PCR and Liquichip High Throughput (GMPLex)

    Institute of Scientific and Technical Information of China (English)

    Zhi-feng Qin; Bing Cheng; Zhou-xi Ruan; Ying-zuo Bi; Joseph J Giambrone; Hong-zhuan Wu; Jie Sun; Ti-kang Lu; Shao-ling Zeng; Qun-yi Hua; Qing-yan Ling; Shu-kun Chen; Jian-qiang Lv; Cai-hong Zhang

    2012-01-01

    This study developed a multiplex RT-PCR integrated with luminex technology to rapidly subtype simultaneously multiple influenza viruses.Primers and probes were designed to amplify NS and M genes of influenza A viruses HA gene of H1,H3,H5,H7,H9 subtypes,and NA gene of the N1 and N2 subtypes.Universal super primers were introduced to establish a multiplex RT-PCR (GM RT-PCR).It included three stages of RT-PCR amplification,and then the RT-PCR products were further tested by LiquiChip probe,combined to give an influenza virus (Ⅳ) rapid high throughput subtyping test,designated as GMPLex.The IV GMPLex rapid high throughput subtyping test presents the following features:high throughput,able to determine the subtypes of 9 target genes in H1,H3,H5,H7,H9,N1,and N2 subtypes of the influenza A virus at one time; rapid,completing the influenza subtyping within 6 hours; high specificity,ensured the specificity of the different subtypes by using two nested degenerate primers and one probe,no cross reaction occurring between the subtypes,no non-specific reactions with other pathogens and high sensitivity.When used separately to detect the product of single GM RT-PCR for single H5 or N1 gene,the GMPLex test showed a sensitivity of 105(=280ELD50) forboth tests and the Luminex qualitative ratio results were 3.08 and 3.12,respectively.When used to detect the product of GM RT-PCR for HSN1 strain at the same time,both showed a sensitivity of 10-4(=2800 ELD50).The GMPLex rapid high throughput subtyping test can satisfy the needs of influenza rapid testing.

  3. Role of RT-PCR and FISH in diagnosis and monitoring of acute promyelocytic leukemia

    Directory of Open Access Journals (Sweden)

    S Polampalli

    2011-01-01

    Full Text Available Background: Patients with a presence of Promyelocytic Leukemia-Retinoic Acid Receptor Alpha (PML-RARA genes rearrangement predict a favorable response to all-trans retinoic acid (ATRA, and a significant improvement in survival. Therefore, establishing the presence of PML-RARA rearrangement is important for optimal patient management. Aim: The objective of this study is to compare and assess the role of fluorescent in situ hybridization (FISH and reverse transcriptase polymerase chain reaction (RT-PCR in the diagnosis and long-term monitoring of Acute Promyelocytic Leukemia (APL. Materials and Methods: We compared 145 samples received at different interval of times to analyze the sensitivity of RT-PCR and FISH. Results: The failure rate for RT-PCR was 4% at baseline, 13% at induction, and 0% at the end of consolidation. And for FISH it was 8% at baseline, 38% at induction, and 66% at the end of consolidation. The predictive values of relapse in the patients who were positive and negative by RT-PCR, at the end of induction, were 60 % and 3%, respectively, and at end of consolidation it was 67 % and 4%, respectively. On the other hand the predictive values of relapse in patients who were positive and negative by FISH at end of induction were 57 % and 6%, respectively; while at end of consolidation it was 14% who were negative by FISH. Conclusion: Both RT-PCR and FISH are important for the diagnosis of APL cases, as both techniques complement each other in the absence or failure of any one of them. However, RT-PCR is more sensitive than FISH for the detection of minimal residual disease in the long-term monitoring of these patients. The present study shows that the predictive value of relapse is more associated with minimal residual disease (MRD results by RT-PCR than that by FISH.

  4. Simultaneous detection and identification of four cherry viruses by two step multiplex RT-PCR with an internal control of plant nad5 mRNA.

    Science.gov (United States)

    Noorani, Md Salik; Awasthi, Prachi; Sharma, Maheshwar Prasad; Ram, Raja; Zaidi, Aijaz Asgar; Hallan, Vipin

    2013-10-01

    A multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed and standardized for the simultaneous detection of four cherry viruses: Cherry virus A (CVA, Genus; Capillovirus), Cherry necrotic rusty mottle virus (CNRMV, unassigned species of the Betaflexiviridae), Little cherry virus 1 (LChV-1, Genus; Closterovirus) and Prunus necrotic ringspot virus (PNRSV, Genus; Ilarvirus) with nad5 as plant internal control. A reliable and quick method for total plant RNA extraction from pome and stone fruit trees was also developed. To minimize primer dimer formation, a single antisense primer for CVA and CNRMV was used. A mixture of random hexamer and oligo (dT) primer was used for cDNA synthesis, which was highly suited and economic for multiplexing. All four viruses were detected successfully by mRT-PCR in artificially created viral RNA mixture and field samples of sweet cherry. The identity of the viruses was confirmed by sequencing. The assay could detect above viruses in diluted cDNA (10(-4)) and RNA (10(-3), except PNRSV which was detected only till ten times lesser dilution). The developed mRT-PCR will not only be useful for the detection of viruses from single or multiple infections of sweet cherry plants but also for other stone and pome fruits. The developed method will be therefore quite helpful for virus indexing, plant quarantine and certification programs. This is the first report for the simultaneous detection of four cherry viruses by mRT-PCR.

  5. Detection limits of several commercial reverse transcriptase enzymes: impact on the low- and high-abundance transcript levels assessed by quantitative RT-PCR

    Directory of Open Access Journals (Sweden)

    Delbecchi Louis

    2007-10-01

    Full Text Available Abstract Background In functional genomics, transcript measurement is of fundamental importance. Quantitative reverse transcription polymerase chain reaction (qRT-PCR assays are the most popular technology and depend on the initial molecular step, the reverse transcription (RT. This study provides a complex overview of the influence of elements such as RT systems, amount of background RNA, and transcript abundance on the efficiency of qRT-PCR. Using qRT-PCR, we compared the efficiency of some commonly used RT systems and measured the production of PCR-amplifiable products and the influence of PCR inhibitor contents. Results The qRT-PCR assays were conducted using the TaqMan system, although we also tested the SYBR Green I chemistry, which is not compatible with all the RT systems. When dealing with low-abundance transcripts, the SuperScript II system generated more detectable molecules than the four other systems tested: Sensiscript, Omniscript, SuperScript III and PowerScript (P P Conclusion This study provides a complex overview of the influence of elements such as RT systems, qRTPCR chemistry, amount of background RNA, and transcript abundance on the efficiency of qRT-PCR. Whereas the most significant influencing factor is the presence of inhibitors in the RT systems, total background RNA is also a major influencing component that affects the PCR reaction. Whenever the aim of a study is to obtain a precise gene expression measurement or to profile the global transcriptome (e.g. microarray, the RT step is critical and should be examined with care.

  6. Modeling qRT-PCR dynamics with application to cancer biomarker quantification.

    Science.gov (United States)

    Chervoneva, Inna; Freydin, Boris; Hyslop, Terry; Waldman, Scott A

    2017-01-01

    Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is widely used for molecular diagnostics and evaluating prognosis in cancer. The utility of mRNA expression biomarkers relies heavily on the accuracy and precision of quantification, which is still challenging for low abundance transcripts. The critical step for quantification is accurate estimation of efficiency needed for computing a relative qRT-PCR expression. We propose a new approach to estimating qRT-PCR efficiency based on modeling dynamics of polymerase chain reaction amplification. In contrast, only models for fluorescence intensity as a function of polymerase chain reaction cycle have been used so far for quantification. The dynamics of qRT-PCR efficiency is modeled using an ordinary differential equation model, and the fitted ordinary differential equation model is used to obtain effective polymerase chain reaction efficiency estimates needed for efficiency-adjusted quantification. The proposed new qRT-PCR efficiency estimates were used to quantify GUCY2C (Guanylate Cyclase 2C) mRNA expression in the blood of colorectal cancer patients. Time to recurrence and GUCY2C expression ratios were analyzed in a joint model for survival and longitudinal outcomes. The joint model with GUCY2C quantified using the proposed polymerase chain reaction efficiency estimates provided clinically meaningful results for association between time to recurrence and longitudinal trends in GUCY2C expression.

  7. THE DETECTION OF MDR1 GENE EXPRESSION USING FLUOROGENIC PROBE QUANTITATIVE RT-PCR METHOD

    Institute of Scientific and Technical Information of China (English)

    高劲松; 马刚; 仝明; 陈佩毅; 王传华; 何蕴韶

    2001-01-01

    Objective: To establish a fluoregenic probe quantitative RT-PCR (FQ-RT-PCR) method for detection of the expression of MDR1 gene in tumor cells and to investigate the expression of MDR1 gene in patients with lung cancer. Methods: The fluorogenic quantitative RT-PCR method for detection of the expression of MDR1 gene was established. K562/ADM and K562 cell lines or 45 tumor tissues from patients with lung cancer were examined on PE Applied Biosystems 7700 Sequence Detection machine. Results: the average levels of MDR1 gene expression in K562/ADM cells and K562 cells were (6.86±0.65)× 107copies/mg RNA and (8.49±0.67)×105 copies/mg RNA, respectively. The former was 80.8 times greater than the latter. Each sample was measured 10 times and the coefficient variation (CV) was 9.5% and 7.9%, respectively. Various levels of MDR1 gene expression were detected in 12 of 45 patients with lung cancer. Conclusion: Quantitative detection of MDR1 gene expression in tumor cells was achieved by using FQ-RT-PCR. FQ-RT-PCR is an accurate, and sensitive method and easy to perform. Using this method, low levels of MDR1 gene expression could be detected in 24% of the patients with lung cancer.

  8. Detection of the Genomic Sequence of Yellow Fever Virus by One-step RT-PCR%黄热病毒的一步RT-PCR法检测

    Institute of Scientific and Technical Information of China (English)

    彭文明; 邓永强; 于曼; 范保昌; 祝庆余; 秦鄂德

    2003-01-01

    自不同稀释度的乳鼠脑病毒悬液中制备总RNA,在自制缓冲液条件下,进行一步RT-PCR法检测.对一步RT-PCR法与常规RT-PCR的敏感性进行比较,并且以基孔肯亚病毒和登革1-4型病毒RNA为模板进行特异性观察.结果表明,本研究建立的一步RT-PCR法可检出2.8×103PFU的病毒,敏感性比常规RT-PCR法高10倍,且与基孔肯亚病毒、登革病毒1-4型无交叉反应,具有良好的特异性和敏感性,适用于黄热病毒的病原学检测.

  9. One-step Multiplex RT-PCR Method for Simultaneous Detection of Seed Transmissible Bacteria and Viruses in Pepper and Tomato Seeds

    Directory of Open Access Journals (Sweden)

    Kyusik Jeong

    2011-04-01

    Full Text Available The aim of this study was to develop specific and sensitive PCR-based procedures for simultaneous detection of economically important plant seed infection pathogenic bacteria and virus, Xanthomonns campestris pv. vesicatoria (Xcv, Clavibacter michiganensis subsp. michiganensis (Cmm, Erwinia carotovora subsp. carotovora (Ecc, Pepper mild mottle virus (PMMoV and Tobacco mild green mosaic virus (TMGMV in pepper and tomato seeds. Most of pepper and tomato bacterial and virus diseases are responsible for germination and growth obstruction. PCR with arbitral primers: selection of specific primers, performance of PCR with specific primers and determination of the threshold level for pathogens detection. To detect simultaneously the Xcv, Cmm, Ecc, PMMoV and TMGMV in pepper and tomato seeds, five pairs (Cmm-F/R, Ecc-F/R, Xcv-F/R, PMMoV-F/R, TMGMV-F/R of specific primer were synthesized by primer-blast program. The multiplex PCR for the five pathogens in pepper and tomato seeds could detect specially without interference among primers and/or cDNA of plant seeds and other plant pathogens. The PCR result for pathogen detection using 20 commercial pepper and 10 tomato seed samples, Ecc was detected from 4 pepper and 2 tomato seed samples, PMMoV was detected from 1 pepper seed sample, and PMMoV and TMGMV were simultaneously detected from 1 pepper seed sample.

  10. Identification of nasal blood by real-time RT-PCR.

    Science.gov (United States)

    Sakurada, Koichi; Akutsu, Tomoko; Watanabe, Ken; Yoshino, Mineo

    2012-07-01

    A new approach for the identification of body fluid stains by comparing specific mRNA expression levels has been extensively studied in recent years. Here, we examine whether nasal blood, which is regarded as one of the most difficult types of blood to identify, can be identified by comparing mRNA expression levels of target genes specific to saliva, nasal secretion, and blood. The saliva-specific statherin gene (STATH) was found to be expressed at high levels in not only saliva (dCt value: 1.32±1.39, n=5), but also nasal secretions (dCt value: 0.90±1.14, n=5), while the histatin gene (HTN3) was only expressed at high levels in saliva (dCt value: 1.08±2.35, n=5). We also confirmed that the hemoglobin-beta gene (HBB) showed high expression levels in blood (dCt value: -9.51±0.40, n=5). Four nasal blood stains were found to highly express STATH (dCt value: 5.65±3.98) and HBB (dCt value: -8.79±1.67) but not HTN3, suggesting that the stain samples contained both nasal secretions and blood and can therefore be identified as nasal blood stains. Although menstrual blood showed the same expression pattern as nasal blood, the menstrual blood-specific protein matrix metallopeptidase 7 (MMP7) was not expressed in all nasal blood stain samples. Therefore, its expression levels could be used to discriminate between nasal and menstrual blood. In conclusion, real-time RT-PCR was able to identify nasal blood, although the stability of gene expression in nasal blood stains was low over time, suggesting that this assay may not be effective for older stains. Future work should examine the usefulness of this assay under various environmental conditions.

  11. Detection and Molecular Characterization of Enteroviruses in Korean Surface Water by Using Integrated Cell Culture Multiplex RT-PCR

    Institute of Scientific and Technical Information of China (English)

    GYUCHEOL LEE; CHANHEE LEE; CHANSEUNG PARK; SANGGI JEONG

    2008-01-01

    Objective To identify waterborne enteric viruses in Korean surface water. Methods Integrated cell culture(ICC)-multiplex reverse transcription-polymerase chain reaction (RT-PCR) was simultaneously designed to detect coxsackieviruses (CV), polioviruses (PV), and reoviruses (RV). ICC-multiplex RT-PCR and phylogenetic analysis were conducted using 21 total culturable virus assay (TCVA)-positive sample-inoculated cell cultures. Results CV and RV were detected in 9 samples each, and 3 samples were positive for both CV and RV. PV was not etected in any sample. Molecular phylogenetic analysis of the VPl gene sequences revealed that CV types B2 and B4 redominated in Korean surface water, and the nucleotide sequences of CV type B2 were clustered with those of CVs isolated from China and Japan. The results suggested that the evolution of these viruses occurred in a region-specific manner. Conclusion CV and RV are detectable in Korean surface water, with a predominance of CV type B2, and the evolution of CV type B2 occur in a region-specific manner.

  12. Rapid detection of BCR-ABL fusion genes using a novel combined LUX primer, in-cell RT-PCR and flow cytometric method.

    Science.gov (United States)

    Shi, Yan; Li, Li-Zhen; Sun, Jian-Zhi; Zhang, Ti; Peng, Jun; Xu, Cong-Gao

    2008-01-01

    Currently, quantitative and semiquantitative assays for minimal residual disease detection include fluorescence in situ hybridisation, multiparameter flow cytometric immunophenotyping and real-time quantitative polymerase chain reaction (RQ-PCR). We have developed a new approach to detect hybrid breakpoint cluster region and Abelson proto-oncogene (BCR-ABL) transcripts inside suspension cells using in situ RT-PCR and light upon extension (LUX) primer, followed by rapid quantitative analysis with flow cytometry. After cellular permeabilization and fixation of single cell suspension, the neoplastic mRNA was reverse transcribed and amplified by PCR with LUX primer. The results demonstrated that a strong positive yellow-green signal was observed in 99-100% cells of K562 cell line, only the red nucleus was detected in NB4 cell line and normal controls. The technique has been utilised to study 12 patients with chronic myeloid leukemia, and the results were compared with those of BCR-ABL fusion mRNA by RT-PCR and BCR-ABL fusion gene of the interphase cells by fluorescence in situ hybridization (FISH). In the five diagnosed patients, 90-98% cells were strongly positive. Four patients, including three patients treated with interferon-alpha and hydroxyurea and one patient treated with imatinib mesylate, had 26-82.5% positive cells. Three patients treated with imatinib mesylate were negative. The in situ RT-PCR results demonstrated complete concordance with the results of I-FISH and RT-PCR. A fluorescence signal was detectable at 1/10(4) cells and became negative below this threshold with flow cytometry. The results of the present study suggest that (1) LUX primers can be used to efficiently detect BCR-ABL fusion mRNA by in-cell RT-PCR; (2) the novel technique is a specific and sensitive way of detecting fusion gene with potential clinical usefulness.

  13. One-step reverse transcription loop-mediated isothermal amplification for the rapid detection of cucumber green mottle mosaic virus.

    Science.gov (United States)

    Li, Jin-yu; Wei, Qi-wei; Liu, Yong; Tan, Xin-qiu; Zhang, Wen-na; Wu, Jian-yan; Charimbu, Miriam Karwitha; Hu, Bai-shi; Cheng, Zhao-bang; Yu, Cui; Tao, Xiao-rong

    2013-11-01

    Cucumber green mottle mosaic virus (CGMMV) has caused serious damage to Cucurbitaceae crops worldwide. The virus is considered one of the most serious Cucurbitaceae quarantine causes in many countries. In this study, a highly efficient and practical one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed for the detection of CGMMV. The total RNA or crude RNA extracted from watermelon plants or seeds could be detected easily by this RT-LAMP assay. The RT-LAMP assay was conducted in isothermal (63°C) conditions within 1h. The amplified products of CGMMV could be detected as ladder-like bands using agarose gel electrophoresis or visualized in-tube under UV light with the addition of a fluorescent dye. The RT-LAMP amplification was specific to CGMMV, as no cross-reaction was observed with other viruses. The RT-LAMP assay was 100-fold more sensitive than that of reverse-transcription polymerase chain reaction (RT-PCR). This is the first report of the application of the RT-LAMP assay to detect CGMMV. The sensitive, specific and rapid RT-LAMP assay developed in this study can be applied widely in laboratories, the field and quarantine surveillance of CGMMV.

  14. The one step fermionic ladder

    Science.gov (United States)

    Das, Joy Prakash; Setlur, Girish S.

    2017-10-01

    The one step fermionic ladder refers to two parallel Luttinger Liquids (poles of the ladder) placed such that there is a finite probability of electrons hopping between the two poles at a pair of opposing points along each of the poles. The many-body Green function for such a system is calculated in presence of forward scattering interactions using the powerful non-chiral bosonization technique (NCBT). This technique is based on a non-standard harmonic analysis of the rapidly varying parts of the density fields appropriate for the study of strongly inhomogeneous ladder systems. The closed analytical expression for the correlation function obtained from NCBT is nothing but the series involving the RPA (Random Phase Approximation) diagrams in powers of the forward scattering coupling strength resummed to include only the most singular terms with the source of inhomogeneities treated exactly. Finally the correlation functions are used to study physical phenomena such as Friedel oscillations and the conductance of such systems with the potential difference applied across various ends.

  15. Evaluation of two singleplex reverse transcription-Insulated isothermal PCR tests and a duplex real-time RT-PCR test for the detection of porcine epidemic diarrhea virus and porcine deltacoronavirus.

    Science.gov (United States)

    Zhang, Jianqiang; Tsai, Yun-Long; Lee, Pei-Yu Alison; Chen, Qi; Zhang, Yan; Chiang, Cheng-Jen; Shen, Yu-Han; Li, Fu-Chun; Chang, Hsiao-Fen Grace; Gauger, Phillip C; Harmon, Karen M; Wang, Hwa-Tang Thomas

    2016-08-01

    Recent outbreaks of porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) in multiple countries have caused significant economic losses and remain a serious challenge to the swine industry. Rapid diagnosis is critical for the implementation of efficient control strategies before and during PEDV and PDCoV outbreaks. Insulated isothermal PCR (iiPCR) on the portable POCKIT™ device is user friendly for on-site pathogen detection. In the present study, a singleplex PEDV RT-iiPCR, a singleplex PDCoV RT-iiPCR, and a duplex PEDV/PDCoV real-time RT-PCR (rRT-PCR) commercial reagents targeting the M gene were compared to an N gene-based PEDV rRT-PCR and an M gene-based PDCoV rRT-PCR that were previously published and used as reference PCRs. All PCR assays were highly specific and did not cross react with other porcine enteric pathogens. Analytical sensitivities of the PEDV RT-iiPCR, PDCoV RT-iiPCR and duplex PEDV/PDCoV rRT-PCR were determined using in vitro transcribed RNA as well as viral RNA extracted from ten-fold serial dilutions of PEDV and PDCoV cell culture isolates. Performance of each PCR assay was further evaluated using 170 clinical samples (86 fecal swabs, 24 feces, 19 intestines, and 41 oral fluids). Compared to the reference PEDV rRT-PCR, the sensitivity, specificity and accuracy of the PEDV RT-iiPCR were 97.73%, 98.78%, and 98.24%, respectively, and those of the duplex PEDV/PDCoV rRT-PCR were 98.86%, 96.34%, and 97.65%, respectively. Compared to the reference PDCoV rRT-PCR, the sensitivity, specificity and accuracy of the PDCoV RT-iiPCR were 100%, 100%, and 100%, respectively, and those of the PEDV/PDCoV duplex rRT-PCR were 96.34%, 100%, and 98.24%, respectively. Overall, all three new PCR assays were comparable to the reference rRT-PCRs for detection of PEDV and/or PDCoV. The PEDV and PDCoV RT-iiPCRs are potentially useful tools for on-site detection and the duplex PEDV/PDCoV rRT-PCR provides a convenient method to simultaneously detect

  16. 禽肺炎病毒实时荧光定量 RT-PCR 检测方法的建立%Development of Real-time RT-PCR for Detection of Avian Pneumovirus in Chickens

    Institute of Scientific and Technical Information of China (English)

    谢志勤; 谢芝勋; 刘加波; 庞耀珊; 邓显文; 谢丽基; 范晴; 罗思思

    2014-01-01

    A SYBR Green Ⅰ real-time RT-PCR was developed and applied to detect avian pneumovirus subtype C.A pair of specific primers were designed and synthesized according to the fusion gene of avian pneumovirus subtype C in GenBank.The reaction parameters,such as the concentration of primers,and the reaction buffer,were optimized to develop a SYBR Green Ⅰ real-time RT-PCR for the rapid detection of avian pneumovirus subtype C.The sensitivity and specificity were tested by this method.The results in-dicated that only avian pneumovirus strain had the positive curve in this assay.As limit as 10 copies of plas-mid DNA isolated from competent cells that transferred avian pneumovirus gene was detected in this test. 54 of clinical samples collected from chicken lungs and trachea were detected by this method.It showed that all of clinical samples were negative for avian pneumovirus subtype C.It indicated that this SYBR Green Ⅰ real-time RT-PCR was a good method for detection of avian pneumovirus.It was a quick,sensi-tive,specific and quantitative method for identification of avian pneumovirus in chickens in the future.%根据 C 亚型禽肺炎病毒 F 基因序列,设计了一对针对 C 亚型禽肺炎病毒的特异性引物,应用该引物建立了 SYBR Green Ⅰ实时荧光定量 RT-PCR 检测 C 亚型禽肺炎病毒的方法,并对建立的方法进行特异性、敏感性测定和临床样品检验。经检验该方法能特异性地鉴别检测禽肺炎病毒,特异性好;敏感性可检测到10个拷贝的禽肺炎病毒质粒 DNA 模板;对收集的54份鸡肺病料样品进行检测,没有检测到 C 亚型禽肺炎病毒阳性病料。建立的 SYBR Green Ⅰ实时荧光定量 RT-PCR 方法检测 C 亚型禽肺炎病毒具有特异、敏感、快速、定量等优点,用该方法检测证实,采集的临床样品中还没有 C 亚型禽肺炎病毒的存在。

  17. Use of an internal standard in a closed one-tube RT-PCR for the detection of equine arteritis virus RNA with fluorescent probes

    OpenAIRE

    Westcott, David; King, Donald; Drew, Trevor; Nowotny, Norbert; Kindermann, Johanna; Hannant, Duncan; Beláke, Sándor; Paton, David

    2003-01-01

    International audience; Routine detection of equine arteritis virus (EAV) can be achieved by virus isolation (VI) in cell culture, or by the amplification of viral genome by molecular methods. To simplify molecular diagnosis, a number of different Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and RT-nested PCR (RT-nPCR) assays were compared, and a one-tube method was developed and optimised utilizing a fluorogenic probe (TaqMan®). An artificial RNA template (Mimic) and associated p...

  18. Identification and characterization of a novel tospovirus species using a new RT-PCR approach

    NARCIS (Netherlands)

    Cortez, I.; Saaijer, J.; Wonjkaew, K.S.; Pereira, A.M.; Goldbach, R.W.; Peters, D.; Kormelink, R.

    2001-01-01

    Summary. A novel tospovirus serologically distinct from all established tospo- virus species was found in Thailand in Physalis minima L. The S RNA of this virus was cloned by a new RT-PCR approach revealing a nucleotide sequence of 3257 nucleotides. The ambisense RNA segment encoded a nonstructural

  19. Detecting the Presence of Nora Virus in "Drosophila" Utilizing Single Fly RT-PCR

    Science.gov (United States)

    Munn, Bethany; Ericson, Brad; Carlson, Darby J.; Carlson, Kimberly A.

    2015-01-01

    A single fly RT-PCR protocol has recently been developed to detect the presence of the persistent, horizontally transmitted Nora virus in "Drosophila." Wild-caught flies from Ohio were tested for the presence of the virus, with nearly one-fifth testing positive. The investigation presented can serve as an ideal project for biology…

  20. Generic RT-PCR tests for detection and identification of tospoviruses.

    Science.gov (United States)

    Hassani-Mehraban, A; Westenberg, M; Verhoeven, J T J; van de Vossenberg, B T L H; Kormelink, R; Roenhorst, J W

    2016-07-01

    A set of tests for generic detection and identification of tospoviruses has been developed. Based on a multiple sequence alignment of the nucleocapsid gene and its 5' upstream untranslated region sequence from 28 different species, primers were designed for RT-PCR detection of tospoviruses from all recognized clades, i.e. the American, Asian and Eurasian clades, and from the small group of distinct and floating species. Pilot experiments on isolates from twenty different species showed that the designed primer sets successfully detected all species by RT-PCR, as confirmed by nucleotide sequence analysis of the amplicons. In a final optimized design, the primers were applied in a setting of five RT-PCR tests. Seven different tospoviruses were successfully identified from diagnostic samples and in addition a non-described tospovirus species from alstroemeria plants. The results demonstrate that the newly developed generic RT-PCR tests provide a relevant tool for broad detection and identification of tospoviruses in plant quarantine and diagnostic laboratories.

  1. Salmonella detection from chicken rinsate with surface enhanced Raman spectroscopy and RT-PCR validation

    Science.gov (United States)

    Optical detection of bacteria has been approached in recent years as a bacteria detection method that can counter time restraints of traditional plating or the high reoccurring cost of real-time polymerase chain reaction (RT-PCR). The goal of optical detection is to identify bacteria with spectral s...

  2. Validation of Zebrafish (Danio rerio) Reference Genes for Quantitative Real-time RT-PCR Normalization

    Institute of Scientific and Technical Information of China (English)

    Rongying TANG; Andrew DODD; Daniel LAI; Warren C.MCNABB; Donald R.LOVE

    2007-01-01

    The normalization of quantitative real time RT-PCR (qRT-PCR) is important to obtain accurate gene expression data. The most common method for qRT-PCR normalization is to use reference, or housekeeping genes. However, there is emerging evidence that even reference genes can be regulated under different conditions, qRT-PCR has only recently been used in terms of zebrafish gene expression studies and there is no validated set of reference genes. This study characterizes the expression of nine possible reference genes during zebrafish embryonic development and in a zebrafish tissue panel. All nine reference genes exhibited variable expression. The β-actin, EF1α and Rpl13α genes comprise a validated reference gene panel for zebrafish developmental time course studies, and the EF1α, Rpl13α and 18S rRNA genes are more suitable as a reference gene panel for zebrafish tissue analysis. Importantly, the zebrafish GAPDH gene appears unsuitable as reference gene for both types of studies.

  3. Thermostable DNA polymerase from a viral metagenome is a potent RT-PCR enzyme.

    Directory of Open Access Journals (Sweden)

    Michael J Moser

    Full Text Available Viral metagenomic libraries are a promising but previously untapped source of new reagent enzymes. Deep sequencing and functional screening of viral metagenomic DNA from a near-boiling thermal pool identified clones expressing thermostable DNA polymerase (Pol activity. Among these, 3173 Pol demonstrated both high thermostability and innate reverse transcriptase (RT activity. We describe the biochemistry of 3173 Pol and report its use in single-enzyme reverse transcription PCR (RT-PCR. Wild-type 3173 Pol contains a proofreading 3'-5' exonuclease domain that confers high fidelity in PCR. An easier-to-use exonuclease-deficient derivative was incorporated into a PyroScript RT-PCR master mix and compared to one-enzyme (Tth and two-enzyme (MMLV RT/Taq RT-PCR systems for quantitative detection of MS2 RNA, influenza A RNA, and mRNA targets. Specificity and sensitivity of 3173 Pol-based RT-PCR were higher than Tth Pol and comparable to three common two-enzyme systems. The performance and simplified set-up make this enzyme a potential alternative for research and molecular diagnostics.

  4. Diagnostic accuracy of FISH and RT-PCR in 50 routinely processed synovial sarcomas

    NARCIS (Netherlands)

    Ten Heuvel, Suzan E.; Hoekstra, Harald J.; Suurmeijer, Albert J. H.

    Background: Molecular detection of SYT-SSX fusion genes is the most reliable tool for diagnosing synovial sarcoma (SS). The objective of this study was to investigate the accuracy of reverse transcription-polymerase chain reaction (RT-PCR) and a commercially available fluorescence in situ

  5. [Detection of tobacco mosaic virus (TMV) in Rehmannia glutinosa f. hueichingensis by IC-RT-PCR].

    Science.gov (United States)

    Du, Lin; Xiang, Jin-Le; Fan, Jin-Ling; Li, Xin; Luo, Lei

    2013-07-01

    To establish a rapid, sensitive and efficient detection method for tobacco mosaic virus (TMV), and provide technical support of TMV detection of Rehmannia glutinosa f. hueichingensis. The virus-free plantlets could be produced on a large scale to ameliorate breed degeneration caused by viral disease. Specific primers were designed based on the conserved region of coat protein(CP) gene of TMV. Immunocapture RT-PCR (IC-RT-PCR) was employed to detect TMV and the sequence of the products was detected. The expected nucleotide acid fragments were amplified by IC-RT-PCR. The homology of nucleotide acid sequence and amino acid sequence were 95.29% and 96.7% between the PCR products and the CP gene of TMV (accession number AY555269). The method was established for the detection of TMV in R. glutinosa f. hueichingensis by IC-RT-PCR. This detection combined molecular biology technology with immunology, was convenient for a quick, sensitive and simple detection of TMV.

  6. Detection of circulating tumor cells by nested RT-PCR targeting ...

    African Journals Online (AJOL)

    Salwa H. Teama

    cancer compared with that of known markers of circulating cancer cells CEA and CK20. Subjects ..... Greene MI. ErbB receptors: from oncogenes to targeted cancer therapies. ... and prostate stem cell antigen RT-PCR in blood of patients with.

  7. Direct recovery of infectious Pestivirus from a full-length RT-PCR amplicon

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Reimann, Ilona; Hoffmann, Bernd;

    2008-01-01

    , and the resulting RNA transcripts were electroporated into ovine cells. Infectious virus was obtained after one cell culture passage. The rescued viruses had a phenotype similar to the parental Border Disease virus strain. Therefore, direct generation of infectious pestiviruses from full-length RT-PCR cDNA products...

  8. Detection and surveillance of viral hemorrhagic septicemia virus using real-time RT-PCR. I. Initial comparison of four protocols.

    Science.gov (United States)

    Warg, Janet V; Clement, Travis; Cornwell, Emily R; Cruz, Angela; Getchell, Rodman G; Giray, Cem; Goodwin, Andrew E; Groocock, Geoffrey H; Faisal, Mohamed; Kim, Robert; Merry, Gwenn E; Phelps, Nicholas B D; Reising, Monica M; Standish, Isaac; Zhang, Yan; Toohey-Kurth, Kathy

    2014-08-21

    Eight laboratories worked collectively to evaluate 4 real-time RT-PCR (rRT-PCR) protocols targeting viral hemorrhagic septicemia virus (VHSV) being considered for deployment to a USA laboratory testing network. The protocols utilized previously published primers and probe sets developed for detection and surveillance of VHSV. All participating laboratories received and followed a standard operating protocol for extraction and for each of the rRT-PCR assays. Performance measures specifically evaluated included limit of detection (defined as the smallest amount of analyte in which 95% of the samples are classified as positive), analytical specificity, assay efficiency across genotype representatives, within- and between-plate variation within a laboratory, and variation between laboratories using the same platform, between platforms, and between software versions. This evaluation clearly demonstrated that the TaqMan®-based assay developed by Jonstrup et al. (2013; J Fish Dis 36:9-23) produced the most consistent analytical performance characteristics for detecting all genotypes of VHSV across the 8 participating laboratories.

  9. 弹回引物鉴定军团菌属与嗜肺军团菌%A snapback primer mediated one-step PCR assay for the identification of Legionella and Legionella pneumophila strains

    Institute of Scientific and Technical Information of China (English)

    柳朔怡; 屈平华; 顾全

    2015-01-01

    目的:设计一种弹回引物荧光PCR方法鉴定军团菌属和嗜肺军团菌。方法通过分析军团菌16S rRNA基因序列,采用生物信息学方法设计引物,优化实验条件,对方法的特异性、灵敏度进行评估,并对186株环境军团菌分离株与15份环境水样进行鉴定。结果经弹回引物检测,军团菌属菌株在85℃~86℃处有扩增子熔解峰,嗜肺军团菌在71℃处有探针结合区熔解峰,非军团菌未检测到熔解峰。弹回引物对标准菌株DNA与模拟水样灵敏度分别为1 ng/μl与(1×103~1×104)/ml。弹回引物对环境分离株验证实验中,成功鉴定了186株军团菌和44株嗜肺军团菌;对15份环境水样直接检测,检出12份军团菌属阳性水样与4份嗜肺军团菌阳性水样。结论弹回引物荧光PCR方法可用于军团菌属和嗜肺军团菌的鉴定,具有较高的特异性与灵敏度。%Objective To test a snapback primer for the identification of Legionella and Legionella pneumophila ( L.pneumophila) strains in a one-step real-time PCR assay.Methods A novel primer was designed with a pair of genus-specific primers of Legionella strains.The species-specific probe sequences of L.pneumophila strains were linked at the 5′end of the reverse primer.The sensitivity and specificity of the novel PCR assay were tested with 43 types of Legionella and non-Legionella strains.The established PCR as-say was used to identify 186 wild Legionella strains isolated from 11 provinces of China and 15 environmental water samples.Results The amplicon melting peak of Legionella strains was detected at 85-86℃.The snapback melting peak of L.pneumophila was detected at 71℃.No melting peak of non-Legionella strains was detected.The sensitivity of the standard strains and simulated water samples were 1 ng/μl DNA tem-plates and (1×103-1×104 )/ml, respectively.186 Legionella strains and 44 L.pneumophila strains isolated from environmental water

  10. Development of a novel RT-PCR and IC-RT-PCR method for detecting Squash mosaic virus%应用RT-PCR和IC-RT-PCR方法检测南瓜花叶病毒

    Institute of Scientific and Technical Information of China (English)

    廖富荣; 叶志红; 陈青; 吴媛; 陈红运; 林石明

    2013-01-01

    南瓜花叶病毒(Squash mosaic virus,SqMV)是葫芦科作物上一种重要的种传病毒.本研究根据其外壳蛋白(CP)基因设计一对特异引物,建立了SqMV的RT-PCR和IC-RT-PCR检测方法.特异性检测结果表明,所建立的方法可将SqMV与同一个病毒属的其他病毒及健康寄主植物准确区分开来,具有良好的特异性.在此基础上,建立了SqMV的IC-RT-PCR检测方法,其灵敏度则比DAS-ELISA方法提高10倍以上.所建立的方法具有良好的特异性和灵敏度,适用于进出境植物繁殖材料的快速检测.%A novel reverse transcription - polymerase chain reaction ( RT - PCR) method was developed to detect Squash mosaic virus (SqMV). The assay specificity was evaluated on other comoviruses and heathy host plants. The IC - RT - PCR assay was also developed and it was up to 10 times more sensitive than DAS - ELISA in detecting SqMV. The method in this study is suitable for fast, specific and sensitive detection of SqMV.

  11. One-step real-time fluorescent quantitative PCR assay for mumps virus in clinical saliva specimens%腮腺炎病毒临床口漱液样本的一步法实时荧光定量PCR检测

    Institute of Scientific and Technical Information of China (English)

    施海晶; 王晶晶; 陈俊英; 陈巍; 潘明; 赵钢; 孙强明

    2013-01-01

    Objective To develop an one-step real-time fluorescent quantitative PCR assay for mumps virus (MuV) in clinical saliva specimens and provide a basis for clinical diagnosis,prevention and control of mumps.Methods A total of 101 clinical saliva specimens collected from suspected patients with mumps in Yunnan and Sichuan Province as well as Guangxi Zhuang Autonomous Region,China were determined by cell culture-MuV specific nested PCR and real-time PCR with TaqMan probe respectively,based on which the sensitivities of the two methods were compared.Results Thirty-one MuV-positive specimens were found by cell culture-MuV specific nested PCR,all of which belonged to genotype F as proved by phylogenetic tree.However,41 MuV-positive specimens were found by real-time PCR with TaqMan probe,including the 31 positive specimens proved by cell culture-MuV specific nested PCR,indicating that the positive rate of Muv by real-time RT-PCR with TaqMan probe (40.59%) was higher than that by traditional cell culture-nested PCR (30.69%).Conclusion The sensitivity,specificity and simplicity of real-time PCR with TaqMan probe were higher than those of traditional cell culture-MuV specific nested PCR,which might be used for the clinical diagnosis,prevention and control of mumps.%目的 建立腮腺炎病毒(mumps virus,MuV)临床口漱液样本一步法荧光定量PCR检测方法,为腮腺炎的临床诊断及免疫防控提供依据.方法 对101份采集自中国南部3个省、自治区(云南、四川、广西)的临床疑似腮腺炎患者口漱液样本,分别采用细胞培养-MuV特异的巢式PCR法及TaqMan探针Real-time PCR法进行检测,比较两种方法检测MuV的灵敏性.结果 细胞培养-MuV特异的巢式PCR法共检出31份MuV阳性样本,系统进化分析表明,该31株MuV均属于F基因亚型;TaqMan探针Real-time PCR法共检出41份MuV阳性样本,其中包含了细胞培养-MuV特异的巢式PCR法检出的31份阳性样本,TaqMan探针Real-time RT

  12. Application of real-time RT-PCR quantification to evaluate differential expres sion of Arabidopsis Aux/IAAgenes

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The molecular techniques including Northern blot, dot blot, in situ hybridization, etc. have been success fully used to estimate semi-quantitatively mRNA levels in plant samples. In this study, we employed a real-time reverse transcription-PCR (RT-PCR) assay using SYBR Green Ifluorescence methodology to evaluate accurate quant itation and sequence specific detection of Aux/IAA mRNA levels in Arabidopsis. Results obtained indicate a linear dynamic range of 102-106 Aux/IAA mRNA copies with standard de viations of generally less than 15%. As a model experiment,the outcome of analysis of expression patterns of five Aux/IAA genes in Arabidopsis under various chemical and temperature treatments is presented. The method presented here provides a sensitive and rapid technique to evaluate plant Aux/IAA mRNA expression levels in nanogram order.

  13. Novel Rotavirus VP7 Typing Assay Using a One-Step Reverse Transcriptase PCR Protocol and Product Sequencing and Utility of the Assay for Epidemiological Studies and Strain Characterization, Including Serotype Subgroup Analysis

    Science.gov (United States)

    DiStefano, Daniel J.; Kraiouchkine, Nikolai; Mallette, Laura; Maliga, Marianne; Kulnis, Gregory; Keller, Paul M.; Clark, H. Fred; Shaw, Alan R.

    2005-01-01

    Rotavirus is the most common cause of severe dehydrating gastroenteritis in infants. To date, 10 different serotypes of rotavirus have been identified in human stools. While four or five serotypes dominate, serotype circulation varies with season and geography. Since our laboratory has been involved in the development of a multivalent rotavirus vaccine, it is important to identify the serotypes of rotavirus encountered during our clinical trials. We have developed methodologies for the molecular identification of rotavirus strains based on VP7 gene segment sequence. A 365-bp reverse transcriptase PCR product was generated from the VP7 gene segment using a pair of novel degenerate primers. All serotypes tested (both animal and human) yielded an identically sized product after amplification. Sequencing of these products is performed using truncated versions of the original primers. The sequence generated is compared against a database of rotavirus VP7 sequences, with the G type determined, based on the sequence homology. Using this assay, we have correctly identified human VP7 strains from a panel of available serotypes, as well as numerous animal strains. The assay was qualified using rotavirus positive stool samples, negative stool samples, and rotavirus-spiked stool samples. In addition, samples from cases of acute gastroenteritis collected at Children's Hospital of Philadelphia have been evaluated and indicate that the assay is able to discriminate subtle differences within serotypes. The assay has been utilized in the testing of >3,000 antigen-positive (enzyme immunoassay) samples collected during clinical trials of a rotavirus vaccine (RotaTeq) and identified a serotype in ∼92% of samples (3, 17, 19). PMID:16333070

  14. A real-time RT-PCR for detection of clade 1 and 2 H5N1 Influenza A virus using Locked Nucleic Acid (LNA) TaqMan probes

    NARCIS (Netherlands)

    Thanh, T.T.; Pawestri, H.A.; Ngoc, N.M.; Hien, V.M.; Syahrial, H.; Trung, N.V.; van Doorn, R.H.; Wertheim, H.F.L.; Chau, N.V.V.; Ha, D.Q.; Farrar, J.J.; Hien, T.T.; Sedyaningsih, E.R.; de Jong, M.D.

    2010-01-01

    ABSTRACT: BACKGROUND: The emergence and co-circulation of two different clades (clade 1 and 2) of H5N1 influenza viruses in Vietnam necessitates the availability of a diagnostic assay that can detect both variants. RESULTS: We developed a single real-time RT-PCR assay for detection of both clades of

  15. Detection of poliovirus type 2 in oysters by using cell culture and RT-PCR Detecção de poliovírus tipo 2 em ostras através de cultura celular e RT-PCR

    Directory of Open Access Journals (Sweden)

    Cecília E.B. Vinatea

    2006-03-01

    Full Text Available Shellfish are readily contaminated with viruses present in water containing sewage due to the concentrating effect of filter feeding. Enteroviruses are generally used as a model for the detection of viruses from shellfish due to their public health significance. In the present work, oysters were placed in glass aquaria containing seawater plus unicellular algae. Two experiments were performed: 1 oysters bioaccumulating four different poliovirus type 2 concentrations: 5 x 10(4, 2.5 x 10(4, 5 x 10³ and 5 x 10² PFU/mL during 20h; 2 oyster tissues directly inoculated with 6.0 x 10(5 and 1.0 x 10(5 PFU/mL. After viruses seeding, tissue samples were processed by an adsorption-elution-precipitation method. Positive controls were performed by seeding 6.0 x 10(5 PFU/mL of poliovirus type 2 directly on the final oyster tissue extracts. Oyster extracts were assayed for viruses recovery by plaque assay, RT-PCR and integrated cell culture-PCR methodologies (ICC/PCR. The last one was based on the inoculation of the samples onto VERO cell monolayer followed by RT-PCR analysis of the infected cell fluid. In the first experiment (20h bioaccumulation until 5 x 10³ PFU were detected after 24 and 48h growth on VERO cells. Direct RT-PCR and ICC/PCR were able to detect 3 and 0.04 PFU of poliovirus, respectively, when bioaccumulation assay was used. When direct tissue virus seeding was performed, the plaque assays showed that polioviruses were recovered in all tested concentrations. Based on these results, it is possible to conclude that viable polioviruses can be detected in oysters after bioaccumulation and these techniques can be directly applied for monitoring virus contamination in environmental samples.Devido ao hábito alimentar filtrante, os moluscos bivalves são contaminados por vírus presentes em águas contaminadas por esgoto. Os enterovírus são geralmente usados como modelos para a detecção de vírus em moluscos bivalves devido a sua import

  16. A FQ-RT-PCR method for quantitative determination of IL-2 mRNA and IL-4 mRNA and its application

    Institute of Scientific and Technical Information of China (English)

    QING HUI ZHU; QING LU; GU SHENG ZHANG

    2006-01-01

    The purpose of the study is to establish a fluorescence quantitative reverse transcription polymerase chain response (FQ-RT-PCR) method for the quantitative determination of IL-2 mRNA and IL-4mRNA in Th cells, with which the Th cells status of the patients with gynaecological tumors and chronic renal failure (CRF) can be analyzed. IL-2 cDNA and IL-4 cDNA were prepared, and the plasmid pMD18 carrying IL-2 cDNA or IL-4 cDNA fragment was constructed and cloned as the template for quantitative determination. The primers and probes labelled with 6-carboxy-fluorescein (FAM) and 6-carboxytetramethylrhodamine (TAMRA) were prepared, and the experimental conditions were optimized to set up the FQ-RT-PCR method for quantitative determination of IL-2 mRNA and IL-4 mRNA. Th cells enriched from peripheral blood mononuclear cells (PBMCs) of 20 healthy volunteers (HVs), 16 gynaecological benign (GB) cases, 18 gynaecological malignant (GM) tumor cases and 16 chronic renal failure (CRF) patients were tested for IL-2 mRNA and IL-4 mRNA by FQ-RT-PCR. The house-keeping gene 3-actin was used as the internal control gene of the experiment. The standard curve for log concentration of series of quantitative templates vs threshold cycle (CT) was established by linear regression, and the linear range was 102-107 copies/μl. The imprecision test showed the CV of inter-assay and intra-assay of a high content sample by FQ-RT-PCR were 7.8% and 12.5%, respectively. The CV of inter-assay and intra-assay of a low content sample were 10.8% and 19.5%, respectively. The IL-2 mRNA expressions in Th of the patients with gynaecological malignant tumor (compared with the HVs and the patients with gynaecological benign disease) and in Th of the CRF patients (compared with the HVs) were declined significantly and at the same time the IL-4 mRNA expression increased significantly ( P < 0. 001 ). A simple, sensitive and accurate FQ-RT-PCR method for the quantitative detection of IL-2 mRNA and IL-4 mRNA has

  17. Use of proteinase K for RT-PCR of cytokine mRNA in formalin fixed tissue

    DEFF Research Database (Denmark)

    Davies, G N; Bevan, I S; Banner, Jytte;

    1996-01-01

    formalin fixed, paraffin wax embedded material of sufficient purity for reverse transcription (RT)-PCR is described. Proteinase K treatment of formalin fixed, wax embedded tissue followed by RNA STAT-60 extraction was successful in isolating mRNA suitable for RT-PCR. Interleukin (IL)-1alpha, IL-6...

  18. A novel real-time RT-PCR with TaqMan-MGB probes and its application in detecting BVDV infections in dairy farms

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yong-qiang; WANG Zhi-liang; LIU Hai-sheng; WU Xiao-dong; WANG Xiao-zhen; LI Jin-ming; ZHAO Yong-gang; L Yan; REN Wei-jie; GE Sheng-qiang

    2015-01-01

    A real-time RT-PCR assay using TaqMan-MGB probes was developed to detect and type the bovine viral diarrhea virus (BVDV) in cattle. Universal primers and TaqMan-MGB probes were designed from the 5´-untranslated region of known pestiviral sequences. Prior to optimizing the assay, cRNAs were transcribed in vitro from the BVDV 1 and BVDV 2 RT-PCR products to make standard curves. The detection limit of the assay was 1.72×102 copies for BVDV 1 and 2.14×102 copies for BVDV 2. The speciifcity of the assay evaluated on several BVDV strains including bovine herpesvirus 1 (BHV 1), foot and mouth disease virus (FMDV) and several classical swine fever virus (CSFV) strains showed speciifc detection of the positive virus over 40 cycles. The assay was highly reproducible with the coefifcient of variance ranging from 1.04 to 1.33%for BVDV 1 and from 0.83 to 1.48%for BVDV 2, respectively. Using this method, we tested a total of 2 327 cattle from three dairy farms for the presence of BVDV persistently infected (PI) animals. In this assay, each RT-PCR template contained a mixture of ten samples from different animals. The occurrence rate of PI cattle in three farms ranging from 0.9 to 2.54%could represent partly the PI rates in cattle farm in China. In conclusion, using our real-time PCR assay, we could effectively detect and type BVDV and identify PI cattle in a rapid and cost-effective manner.

  19. Diferenciação de estirpes de Potato virus Y (PVY por RT-PCR RT-PCR For differentiation of Potato virus Y strains in potato

    Directory of Open Access Journals (Sweden)

    Leonardo N. Fonseca

    2005-12-01

    Full Text Available O Potato virus Y (PVY tornou-se o maior problema nas áreas de produção de batata semente do Brasil. Somente as estirpes comum (PVYº e necrótica (PVY N eram detectadas infectando batata no Brasil. Esta situação mudou drasticamente a partir de 1997 quando um surto epidêmico de uma variante da estirpe necrótica de PVY causando arcos e anéis necróticos na superfície do tubérculo (PVY NTN foi observado no país. Este estudo visou avaliar e validar uma metodologia de diferenciação de estirpes que causam necrose em tubérculos, proposta por Weilguny e Singh (1998. Vinte e oito isolados de PVY originários de tubérculos infectados de batata, provenientes de quatro estados brasileiros, foram analisados em sua reação em plantas de fumo, por Elisa, utilizando anti-soro policlonal e pelo método 3-primer RT-PCR. Quatro isolados induziram clareamento de nervuras e manchas peroladas em folhas de Nicotiana tabacum conforme esperado para a estirpe comum. Os 24 isolados restantes induziram necrose de nervuras neste hospedeiro portanto, classificados como da estirpe necrótica. Todos os 28 isolados de PVY reagiram positivamente contra o anti-soro policlonal de PVY por Elisa. Três métodos de extração de RNA foram testados, sendo que o método de hidrocloreto de guanidina mostrou-se o mais eficiente e de menor custo. Dos 28 isolados submetidos ao RT-PCR, um isolado de Santa Catarina e três do Rio Grande do Sul foram diferenciados como PVYº, confirmando os resultados do teste biológico. Um isolado de PVY N foi detectado no Estado de Santa Catarina e quatro no Rio Grande do Sul. O PVY NTN foi detectado em Minas Gerais (seis isolados, Santa Catarina (três isolados e São Paulo (dez isolados. Estes resultados confirmam a presença dessa variante necrótica, PVY NTN, nas principais regiões produtoras de batata do Brasil.The Potato virus Y (PVY has become the major virus problem in seed potato growing areas of Brazil. Only necrotic and ordinary

  20. Multiplex RT-PCR-based detections of CEA, CK20 and EGFR in colorectal cancer patients

    Institute of Scientific and Technical Information of China (English)

    Aikaterini; Tsouma; Chrysanthi; Aggeli; Panagiotis; Lembessis; George; N; Zografos; Dimitris; P; Korkolis; Dimitrios; Pectasides; Maria; Skondra; Nikolaos; Pissimissis; Anastasia; Tzonou; Michael; Koutsilieris

    2010-01-01

    AIM: To develop a multiplex reverse transcription polymerase chain reaction (RT-PCR) method detecting cir-culating tumor cells in the peripheral blood of colorectal cancer (CRC) patients. METHODS: Peripheral blood samples were collected from 88 CRC patients and 40 healthy individuals from the blood donors' clinic and subsequently analyzed by multiplex RT-RCR for the expression of carcinoembryonic antigen (CEA), cytokeratin 20 (CK20) and epidermal growth factor receptor (EGFR) mRNA. The analysis involved det...

  1. EXPRESSION OF MOUSE Tbx2 GENE IN NORMAL AND MALIGNANTMELANOPHORES BY RT-PCR

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To observe the expression of mouse Tbx2 gene in normal and malignant melanophore. Methods: The normal and malignant cells were used to extract total RNA. The expression of the Tbx2 gene was detected by RT-PCR. Results: No expression of the Tbx2 gene in the normal melanocytes was noted, but all malignant cells showed expression of the Tbx2 gene. Conclusion: Tbx2 plays a critical role during the development of the malignant cells.

  2. Detection of Schmallenberg virus in different Culicoides spp. by real-time RT-PCR.

    Science.gov (United States)

    De Regge, N; Deblauwe, I; De Deken, R; Vantieghem, P; Madder, M; Geysen, D; Smeets, F; Losson, B; van den Berg, T; Cay, A B

    2012-12-01

    To identify possible vectors of Schmallenberg virus (SBV), we tested pools containing heads of biting midges (Culicoides) that were caught during the summer and early autumn of 2011 at several places in Belgium by real-time RT-PCR. Pools of heads originating from following species: C. obsoletus complex, C. dewulfi and C. chiopterus were found positive, strongly indicating that these species are relevant vectors for SBV.

  3. Mass scale screening of common arboviral infections by an affordable, cost effective RT-PCR method

    Institute of Scientific and Technical Information of China (English)

    Debjani Taraphdar; Arindam Sarkar; Shyamalendu Chatterjee

    2012-01-01

    Objective:To develop a rapid, cost effective RT-PCR method for the mass scale diagnosis of such diseases at the viremia stage to find out the actual disease burden in that area. Methods:For this purpose, cases with the history of only short febrile illness were considered. Thus 157 samples with the history of dengue/chikungunya like illness and only 58 samples with a history of acute encephalitis syndrome (AES) were selected. Results:Out of 157 samples, 42 and 74 were detected as dengue and chikungunya, respectively and out of 58 AES cases only 23 could be detected as Japanese encephalitis by this RT-PCR method. Conclusions:This cost effective RT-PCR method can detect the total positive cases that remain undetected by ELISA method. Moreover, this method is capable to detect the viral RNA from patients’ sera even after the appearance of IgM antibody at one fifth costs as compared with the other commercially available kits.

  4. Validation of reference genes for real-time quantitative RT-PCR studies in Talaromyces marneffei.

    Science.gov (United States)

    Dankai, Wiyada; Pongpom, Monsicha; Vanittanakom, Nongnuch

    2015-11-01

    Talaromyces marneffei (or Penicillium marneffei) is an opportunistic pathogen that can cause disseminated disease in human immunodeficiency virus (HIV)-infected patients, especially in Southeast Asia. T. marneffei is a thermally dimorphic fungus. Typically, T. marneffei has an adaptive morphology. It grows in a filamentous form (mould) at 25°C and can differentiate to produce asexual spores (conidia). In contrast, at 37°C, it grows as yeast cells that divide by fission. This study aimed to validate a quantitative reverse-transcription polymerase chain reaction (qRT-PCR) for gene expression analysis in T. marneffei. Analysis of relative gene expression by qRT-PCR requires normalization of data using a proper reference gene. However, suitable reference genes have not been identified in gene expression studies across different cell types or under different experimental conditions in T. marneffei. In this study, four housekeeping genes were selected for analysis: β-actin (act); glyceraldehyde-3-phosphate dehydrogenase (gapdh); β-tubulin (benA) and 18S rRNA. Two analysis programs; BestKeeper and geNorm software tools were used to validate the expression of the candidate normalized genes. The results indicated that the actin gene was the one which was most stably expressed and was recommended for use as the endogenous control for gene expression analysis of all growth forms in T. marneffei by qRT-PCR under normal and stress conditions.

  5. How Many Microorganisms Are Present? Quantitative Reverse Transcription PCR (qRT-PCR)

    Science.gov (United States)

    Price, Andy; Álvarez, Laura Acuña; Whitby, Corinne; Larsen, Jan

    Quantitative reverse transcription PCR (qRT-PCR) is a variation of conventional quantitative or real-time PCR, whereby mRNA is first converted into the complementary DNA (cDNA) by reverse transcription, the cDNA is then subsequently quantified by qPCR. The use of mRNA as the initial template allows the quantification of gene transcripts, rather than gene copy numbers. mRNA is only produced by actively metabolising cells and is produced by its corresponding gene to provide a 'blueprint' in order for a cell to manufacture a specific protein. Conventional qPCR detects not only DNA present in actively metabolising cells but also inactive and dead cells. qRT-PCR has the advantage that only actively metabolising cells are detected, hence provides a more reliable measure of microbial activity in oilfield samples. When qRT-PCR is combined with primers and probes for specific genes, the activity of microbial processes important in the oilfield, such as sulphate reduction, methanogenesis and nitrate reduction can be monitored.

  6. Investigation of Reference Genes in Vibrio parahaemolyticus for Gene Expression Analysis Using Quantitative RT-PCR.

    Directory of Open Access Journals (Sweden)

    Yue-Jiao Ma

    Full Text Available Vibrio parahaemolyticus is a significant human pathogen capable of causing foodborne gastroenteritis associated with the consumption of contaminated raw or undercooked seafood. Quantitative RT-PCR (qRT-PCR is a useful tool for studying gene expression in V. parahaemolyticus to characterize its virulence factors and understand the effect of environmental conditions on its pathogenicity. However, there is not a stable gene in V. parahaemolyticus that has been identified for use as a reference gene for qRT-PCR. This study evaluated the stability of 6 reference genes (16S rRNA, recA, rpoS, pvsA, pvuA, and gapdh in 5 V. parahaemolyticus strains (O3:K6-clinical strain-tdh+, ATCC33846-tdh+, ATCC33847-tdh+, ATCC17802-trh+, and F13-environmental strain-tdh+ cultured at 4 different temperatures (15, 25, 37 and 42°C. Stability values were calculated using GeNorm, NormFinder, BestKeeper, and Delta CT algorithms. The results indicated that recA was the most stably expressed gene in the V. parahaemolyticus strains cultured at different temperatures. This study examined multiple V. parahaemolyticus strains and growth temperatures, hence the finding provided stronger evidence that recA can be used as a reference gene for gene expression studies in V. parahaemolyticus.

  7. Investigation of Reference Genes in Vibrio parahaemolyticus for Gene Expression Analysis Using Quantitative RT-PCR.

    Science.gov (United States)

    Ma, Yue-Jiao; Sun, Xiao-Hong; Xu, Xiao-Yan; Zhao, Yong; Pan, Ying-Jie; Hwang, Cheng-An; Wu, Vivian C H

    2015-01-01

    Vibrio parahaemolyticus is a significant human pathogen capable of causing foodborne gastroenteritis associated with the consumption of contaminated raw or undercooked seafood. Quantitative RT-PCR (qRT-PCR) is a useful tool for studying gene expression in V. parahaemolyticus to characterize its virulence factors and understand the effect of environmental conditions on its pathogenicity. However, there is not a stable gene in V. parahaemolyticus that has been identified for use as a reference gene for qRT-PCR. This study evaluated the stability of 6 reference genes (16S rRNA, recA, rpoS, pvsA, pvuA, and gapdh) in 5 V. parahaemolyticus strains (O3:K6-clinical strain-tdh+, ATCC33846-tdh+, ATCC33847-tdh+, ATCC17802-trh+, and F13-environmental strain-tdh+) cultured at 4 different temperatures (15, 25, 37 and 42°C). Stability values were calculated using GeNorm, NormFinder, BestKeeper, and Delta CT algorithms. The results indicated that recA was the most stably expressed gene in the V. parahaemolyticus strains cultured at different temperatures. This study examined multiple V. parahaemolyticus strains and growth temperatures, hence the finding provided stronger evidence that recA can be used as a reference gene for gene expression studies in V. parahaemolyticus.

  8. Evaluation of reference genes for gene expression analysis using quantitative RT-PCR in Azospirillum brasilense.

    Directory of Open Access Journals (Sweden)

    Mary McMillan

    Full Text Available Azospirillum brasilense is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions A. brasilense exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for A. brasilense. Here we evaluated the expression of ten candidate reference genes (16S rRNA, gapB, glyA, gyrA, proC, pykA, recA, recF, rpoD, and tpiA in wild-type and mutant A. brasilense strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that gyrA, glyA and recA are the most stably expressed reference genes in A. brasilense. The results also suggested that the use of two reference genes (gyrA and glyA is sufficient for effective normalisation of qRT-PCR data.

  9. Evaluation of reference genes for gene expression analysis using quantitative RT-PCR in Azospirillum brasilense.

    Science.gov (United States)

    McMillan, Mary; Pereg, Lily

    2014-01-01

    Azospirillum brasilense is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions A. brasilense exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for A. brasilense. Here we evaluated the expression of ten candidate reference genes (16S rRNA, gapB, glyA, gyrA, proC, pykA, recA, recF, rpoD, and tpiA) in wild-type and mutant A. brasilense strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that gyrA, glyA and recA are the most stably expressed reference genes in A. brasilense. The results also suggested that the use of two reference genes (gyrA and glyA) is sufficient for effective normalisation of qRT-PCR data.

  10. Multiplex RT-PCR detection of Cucumber mosaic virus subgroups and Tobamoviruses infecting Tomato using 18S rRNA as an internal control.

    Science.gov (United States)

    Chen, Shaoning; Gu, Hao; Wang, Xiaoming; Chen, Jishuang; Zhu, Weimin

    2011-06-01

    A multiplex reverse-transcription polymerase chain reaction (RT-PCR) protocol was developed for simultaneous detection and discrimination of subgroups of Cucumber mosaic virus (CMV), including its satellite RNA, Tomato mosaic virus (ToMV) and Tobacco mosaic virus (TMV), using 18S rRNA as an internal control. Species- and subgroups-specific primers designed to differentiate CMV subgroups I and II, ToMV and TMV, were assessed using the cDNA clones of viral genomes, CMV satellite RNA and 18S rRNA gene from tomato (Solanum lycopersicum L.) or tobacco (Nicotiana tobacum). Using total RNA extracted from artificial mixture of tomato leaf tissues infected by each virus, the reaction components and cycling parameters were optimized and a multiplex RT-PCR procedure was established. Six fragments of 704, 593, 512, 421, 385, 255 bp, specific to CMV subgroup II, CMV subgroup I, ToMV, TMV, satellite RNA and 18S rRNA, respectively, were simultaneously amplified. The sensitivity of the multiplex RT-PCR method for detecting CMV was 100 times higher than that of double-antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA). This method was successfully used for field detection. Among 141 samples collected from East China through tomato growth seasons, 106 single infections with one of the above isolates were detected and 13 mixed infections were found. The results showed the potential use of this method for investigating the epidemiology of viral diseases infecting tomato.

  11. An Evidence-Based Approach to Plum Pox Virus Detection by DASI-ELISA and RT-PCR in Dormant Period

    Directory of Open Access Journals (Sweden)

    Antonio Olmos

    2008-01-01

    Full Text Available An evidence-based approach, such as those developed in clinical and veterinary medicine, was applied to the detection of Plum pox virus (PPV during the dormant period. A standardized methodology was used for the calculation of parameters of the operational capacity of DASI-ELISA and RT-PCR in wintertime. These methods are routinely handled to test the sanitary status of plants in national or international trading and in those cases concerning export-import of plant materials. Diagnosis often has to be performed during the dormant period, when plant material is commercialized. Some guidelines to interpret diagnostic results of wintertime are provided in an attempt to minimize risks associated with the methods and over-reliance on the binary outcome of a single assay. In order to evaluate if a complementary test increased the confidence of PPV diagnosis when discordant results between DASI-ELISA and RT-PCR are obtained, NASBA-FH also was included. Likelihood ratios of each method were estimated based on the sensitivity and specificity obtained in wintertime. Subsequently, a Bayesian approach was performed to calculate post-test probability of PPV infection in spring. Results of evidence-based approach show that different PPV prevalences require different screening tests. Thus, at very low PPV prevalence levels DASI-ELISA should be used as the election method, whilst at the highest PPV prevalence levels RT-PCR should be performed. NASBA-FH could be used at medium prevalences to clarify discordances between DASIELISA and RT-PCR.

  12. Real Time Polymerase Chain Reaction (rt-PCR): A New Patent to Diagnostic Purposes for Paracoccidioidomycosis.

    Science.gov (United States)

    Rocha-Silva, Fabiana; Gomes, Luciana I; Gracielle-Melo, Cidiane; Goes, Alfredo M; Caligiorne, Rachel B

    2017-01-01

    Paracoccidioidomycosis (PCM) is a systemic mycosis caused by dimorphic fungi Paracoccidioides brasiliensis and Paracoccidioides lutzii. It is prevalent in Latin American, mainly in Brazil. Therefore, PCM has fundamental impact on the Brazilian global economy, especially in public health system, since it is affecting economical active population in different country regions. The present study aimed to standardize the Real Time-Polymerase Chain Reaction (rt-PCR) for an efficient and safe PCM diagnosis amplifying the recombinant protein PB27 gene, only expressed by specimens of Paracoccidioides genus. To standardize a methodology of rt-PCR using species-specific primers and probe designed for annealing in this specific region of the fungi´s genome, amplifying the recombinant protein PB27 gene, only expressed by specimens of Paracoccidioides genus. Followed by design in silico, experiments were performed in vitro to determine rt-PCR specificity, efficiency and genome detection limit. The primers and probe sequences were deposited in Brazilian Coordination of Technological Innovation and Transfer (CTIT), under patent reference number BR1020160078830. The present study demonstrated the rt-PCR applicability for support on diagnosis of paracoccidioidomycosis, presenting low cost, which makes it affordable for public health services in developing countries as Brazil. It is noteworthy that it is necessary to validate this methodology using clinical samples before to use as a safe method of diagnosis. A review of all patents related to this topic was performed and it was shown that, to date, there are no records of patent on kits for paracoccidioidomycosis´s diagnostic. Indeed, there is still a lot to go to reach this goal. The reaction developed was standardized and patented, opening perspectives to molecular diagnosis development for paracoccidioidomycosis, since rt-PCR can be applied to a broad spectrum of infectious diseases. It would need to be tested in biological

  13. Selection of housekeeping genes as internal controls for quantitative RT-PCR analysis of the veined rapa whelk (Rapana venosa).

    Science.gov (United States)

    Song, Hao; Dang, Xin; He, Yuan-Qiu; Zhang, Tao; Wang, Hai-Yan

    2017-01-01

    The veined rapa whelk Rapana venosa is an important commercial shellfish in China and quantitative real-time PCR (qRT-PCR) has become the standard method to study gene expression in R. venosa. For accurate and reliable gene expression results, qRT-PCR assays require housekeeping genes as internal controls, which display highly uniform expression in different tissues or stages of development. However, to date no studies have validated housekeeping genes in R. venosa for use as internal controls for qRT-PCR. In this study, we selected the following 13 candidate genes for suitability as internal controls: elongation factor-1α (EF-1α), α-actin (ACT), cytochrome c oxidase subunit 1 (COX1), nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) 1α subcomplex subunit 7 (NDUFA7), 60S ribosomal protein L5 (RL5), 60S ribosomal protein L28 (RL28), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-tubulin (TUBB), 40S ribosomal protein S25 (RS25), 40S ribosomal protein S8 (RS8), ubiquitin-conjugating enzyme E2 (UBE2), histone H3 (HH3), and peptidyl-prolyl cis-trans isomerase A (PPIA). We measured the expression levels of these 13 candidate internal controls in eight different tissues and twelve larvae developmental stages by qRT-PCR. Further analysis of the expression stability of the tested genes was performed using GeNorm and RefFinder algorithms. Of the 13 candidate genes tested, we found that EF-1α was the most stable internal control gene in almost all adult tissue samples investigated with RL5 and RL28 as secondary choices. For the normalization of a single specific tissue, we suggested that EF-1α and NDUFA7 are the best combination in gonad, as well as COX1 and RL28 for intestine, EF-1α and RL5 for kidney, EF-1α and COX1 for gill, EF-1α and RL28 for Leiblein and mantle, EF-1α, RL5, and NDUFA7 for liver, GAPDH, PPIA, and RL28 for hemocyte. From a developmental perspective, we found that RL28 was the most stable gene in all developmental stages measured

  14. Optimization of diagnostic RT-PCR protocols and sampling procedures for the reliable and cost-effective detection of Cassava brown streak virus.

    Science.gov (United States)

    Abarshi, M M; Mohammed, I U; Wasswa, P; Hillocks, R J; Holt, J; Legg, J P; Seal, S E; Maruthi, M N

    2010-02-01

    Sampling procedures and diagnostic protocols were optimized for accurate diagnosis of Cassava brown streak virus (CBSV) (genus Ipomovirus, family Potyviridae). A cetyl trimethyl ammonium bromide (CTAB) method was optimized for sample preparation from infected cassava plants and compared with the RNeasy plant mini kit (Qiagen) for sensitivity, reproducibility and costs. CBSV was detectable readily in total RNAs extracted using either method. The major difference between the two methods was in the cost of consumables, with the CTAB 10x cheaper (0.53 pounds sterling=US$0.80 per sample) than the RNeasy method (5.91 pounds sterling=US$8.86 per sample). A two-step RT-PCR (1.34 pounds sterling=US$2.01 per sample), although less sensitive, was at least 3-times cheaper than a one-step RT-PCR (4.48 pounds sterling=US$6.72). The two RT-PCR tests revealed consistently the presence of CBSV both in symptomatic and asymptomatic leaves and indicated that asymptomatic leaves can be used reliably for virus diagnosis. Depending on the accuracy required, sampling 100-400 plants per field is an appropriate recommendation for CBSD diagnosis, giving a 99.9% probability of detecting a disease incidence of 6.7-1.7%, respectively. CBSV was detected at 10(-4)-fold dilutions in composite sampling, indicating that the most efficient way to index many samples for CBSV will be to screen pooled samples. The diagnostic protocols described below are reliable and the most cost-effective methods available currently for detecting CBSV.

  15. Development and implementation of the quality control panel of RT-PCR and real-time RT-PCR for avian influenza A (H5N1 surveillance network in mainland China

    Directory of Open Access Journals (Sweden)

    Wang Wei

    2011-03-01

    Full Text Available Abstract Background Reverse transcription PCR (RT-PCR and real time RT-PCR (rRT-PCR have been indispensable methods for influenza surveillance, especially for determination of avian influenza. The movement of testing beyond reference lab introduced the need of quality control, including the implementation of an evaluation system for validating personal training and sample proficiency testing. Methods We developed a panel with lysates of seasonal influenza virus (H1N1, H3N2 and B, serials of diluted H5N1 virus lysates, and in-vitro transcribed H5 hemaglutinin (HA and an artificial gene RNAs for RT-PCR and rRT-PCR quality control assessment. The validations of stability and reproducibility were performed on the panel. Additionally, the panel was implemented to assess the detection capability of Chinese human avian influenza networks. Results The panel has relatively high stability and good reproducibility demonstrated by kappa's tests. In the implementation of panel on Chinese human avian influenza networks, the results suggested that there were a relatively low number of discrepancies for both concise and reproducibility in Chinese avian influenza virus net works. Conclusions A quality control panel of RT-PCR and real-time RT-PCR for avian influenza A (H5N1 surveillance network was developed. An availably statistical data, which are used to assess the detection capability of networks on avian influenza virus (H5N1, can be obtained relatively easily through implementation of the panel on networks.

  16. Screening of dengue virus in field-caught Aedes aegypti and Aedes albopictus (Diptera: Culicidae) by one-step SYBR green-based reverse transcriptase-polymerase chain reaction assay during 2004-2007 in Southern Taiwan.

    Science.gov (United States)

    Chen, Chien-Fu; Shu, Pei-Yun; Teng, Hwa-Jen; Su, Chien-Ling; Wu, Jhy-Wen; Wang, Jen-Hsin; Lin, Ting-Hsiang; Huang, Jyh-Hsiung; Wu, Ho-Sheng

    2010-12-01

    We carried out virological surveillance of dengue virus (DENV) in field-caught Aedes mosquitoes during 2004-2007 to estimate the monthly prevalence of infected females in dengue high-risk areas of Taiwan. A total of 92,892 Aedes aegypti (43,133 females and 49,759 males) and 79,315 Aedes albopictus (57,319 females and 21,996 males) adults were collected, grouped into 25,654 pools, and processed for virus detection using a one-step SYBR Green-based real-time reverse transcriptase-polymerase chain reaction assay. DENVs were periodically and sympatrically detected in Ae. aegypti females in accordance with major dengue outbreaks and the corresponding dengue serotypes. Only 0.2% of 7628 pools of Ae. aegypti females were positive for DENVs. This resulted in an overall estimated infection rate (maximum likelihood estimation) of 0.970 per 1000 mosquitoes (95% confidence interval [CI] = 0.53-1.65). The total monthly infection rates ranged from 0.50 to 2.23 per 1000 mosquitoes (95% CI = 0.03-10.71). When sampling areas were scaled down to the city level, monthly infection rates increased to 0.73-12.59 (95% CI = 0.06-59.19). Monthly infection rates over all sampling areas and at the city level increased significantly by month. All positive pools were collected in July (one pool), August (two pools), September (one pool), October (three pools), November (four pools), and December (one pool). All four virus serotypes were detected in mosquitoes, which were consistent with dengue serotypes infecting humans in 2004 (DENV-4), 2005 and 2006 (DENV-2 and DENV-3), and 2007 (DENV-1). Our results provide supporting evidence that, in general, DENV infection rates were low in local Aedes mosquito population during 2004-2007 and that transovarial transmission may not be occurring or is occurring at much lower rates than evidenced in some endemic countries.

  17. Immunocapture RT-PCR detection of Bean common mosaic virus and strain blackeye cowpea mosaic in common bean and black gram in India

    DEFF Research Database (Denmark)

    Udayashankar, A.C.; Nayaka, S. Chandra; Niranjana, S.R.

    2012-01-01

    plant test, an electron microscopic observations, an enzyme linked immunosorbent assay and an immunocapture RT-PCR. The incidence of the two tested viruses in common bean and black gram seed samples was 1–6% and 0.5–3.5%, respectively in growing-on test evaluations. Electron microscopic observations...... revealed filamentous virion particles from the leaves of plants showing characteristic virus disease symptoms in growing-on and host inoculation tests. The identity of the strains was confirmed by immunocapture RT-PCR, with a final amplification product of approximately 700 bp for BCMV and BCMV...

  18. Ring test evaluation of the detection of influenza A virus in swine oral fluids by real-time, reverse transcription polymerase chain reaction (rRT-PCR) and virus isolation

    Science.gov (United States)

    The probability of detecting influenza A virus (IAV) in oral fluid (OF) specimens was calculated for each of 13 real-time, reverse transcription polymerase chain reaction (rRT-PCR) and 7 virus isolation (VI) assays. To conduct the study, OF was inoculated with H1N1 or H3N2 IAV and serially 10-fold d...

  19. Detection and Comparison of Pathogen of Virus Disease in Pumpkin by RT-PCR and IC-PCR

    Institute of Scientific and Technical Information of China (English)

    YANG Guohui; ZHANG Zhongkai; CUI Chongshi

    2006-01-01

    Two kinds of methods RT-PCR and IC-PCR were used to detect pathogen of virus disease of pumpkin and the sensitivity of the two methods was compared. The results showed that PRSV-W and CMV were detected in diseased samples gathered in Yunnan Province, while WMV and CMV were detected in diseased samples gathered in Heilongjiang Province. The sensitivity of RT-PCR is higher than that of IC-PCR, but the effect of IC-PCR in the specialization of bonding reaction and requisition for experiment material is better than that of RT-PCR.

  20. Biosurveillance of avian influenza and Newcastle disease viruses in the Barda region of Azerbaijan using real time RT-PCR and hemagglutination inhibition

    Directory of Open Access Journals (Sweden)

    Shalala eZeynalova

    2015-11-01

    Full Text Available The Azerbaijan State Veterinary Control Service (SVCS has conducted active serological surveillance for avian influenza (AI in poultry since 2006, when the first outbreak of AI H5N1 occurred in Azerbaijan. Samples are collected from September to May annually and tested using a hemagglutination inhibition (HI assay to detect antibodies against H5 AI viruses. HI testing is also performed for Newcastle disease virus (NDV upon request, but since this method cannot distinguish between natural infections and immune responses to vaccination, all positive results require follow-up epidemiological investigations. Furthermore, blood collection for the surveillance program is time-intensive and can be stressful to birds. In order to improve the national surveillance program, alternative sampling and testing methodologies were applied among a population of birds in the Barda region and compared with results of the national surveillance program. Tracheal and cloacal swabs were collected instead of blood. Rather than testing individual samples, RNA was pooled to conserve resources and time, and pools were tested by real-time reverse transcription PCR (rRT-PCR. Environmental sampling at a live bird market was also introduced as another surveillance mechanism. A total of 1,030 swabs were collected, comprising tracheal and cloacal samples from 441 birds and 148 environmental surface samples from farms or the live bird market. During the same time, 3,890 blood samples were collected nationally for the surveillance program; 400 of these samples originated in the Barda region. Birds sampled for rRT-PCR were likely different than those tested as part of national surveillance. All swab samples tested negative by rRT-PCR for both AI and NDV. All blood samples tested negative for H5 by HI, while 6.2% of all samples and 5% of the Barda samples tested positive for exposure to NDV. Follow-up investigations found that positive samples were from birds vaccinated in the

  1. Biosurveillance of avian influenza and Newcastle disease viruses in the Barda region of Azerbaijan using real time RT-PCR and hemagglutination inhibition.

    Science.gov (United States)

    Zeynalova, Shalala; Guliyev, Fizuli; Vatani, Mahira; Abbasov, Bahruz

    2015-01-01

    The Azerbaijan State Veterinary Control Service (SVCS) has conducted active serological surveillance for avian influenza (AI) in poultry since 2006, when the first outbreak of AI H5N1 occurred in Azerbaijan. Samples are collected from September to May annually and tested using a hemagglutination inhibition (HI) assay to detect antibodies against H5 AI viruses. HI testing is also performed for Newcastle disease virus (NDV) upon request, but since this method cannot distinguish between natural infections and immune responses to vaccination, all positive results require follow-up epidemiological investigations. Furthermore, blood collection for the surveillance program is time-intensive and can be stressful to birds. In order to improve the national surveillance program, alternative sampling and testing methodologies were applied among a population of birds in the Barda region and compared with results of the national surveillance program. Tracheal and cloacal swabs were collected instead of blood. Rather than testing individual samples, RNA was pooled to conserve resources and time, and pools were tested by real-time reverse transcription polymerase chain reaction (rRT-PCR). Environmental sampling at a live bird market was also introduced as another surveillance mechanism. A total of 1,030 swabs were collected, comprising tracheal, and cloacal samples from 441 birds and 148 environmental surface samples from farms or the live bird market. During the same time, 3,890 blood samples were collected nationally for the surveillance program; 400 of these samples originated in the Barda region. Birds sampled for rRT-PCR were likely different than those tested as part of national surveillance. All swab samples tested negative by rRT-PCR for both AI and NDV. All blood samples tested negative for H5 by HI, while 6.2% of all samples and 5% of the Barda samples tested positive for exposure to NDV. Follow-up investigations found that positive samples were from birds vaccinated in

  2. Development and application of an RT-PCR test for detecting avian nephritis virus.

    Science.gov (United States)

    Todd, D; Trudgett, J; McNeilly, F; McBride, N; Donnelly, B; Smyth, V J; Jewhurst, H L; Adair, B M

    2010-06-01

    The development of a reverse transcriptase-polymerase chain reaction (RT-PCR) test for detecting avian nephritis virus (ANV) is described. Primers, which amplified a fragment of 182 base pairs (bp), were located in the conserved 3' untranslated region (UTR) of the genome. The limit of detection of the test was estimated to be approximately 18 viral copies using a 10-fold dilution series of in vitro transcribed RNA. Positive signals were produced with representative ANV samples, some of which were not detected by previously described RT-PCR tests for detecting ANV, but other avian astroviruses including chicken astrovirus isolates and duck hepatitis virus types 2 and 3 tested negative. When applied to gut content samples from UK, German and US broiler flocks with enteritis/growth problems, ANVs were detected by RT-PCR in 82/82 (100%) samples. ANVs were also detected in 80/96 (83%) pooled gut content samples from longitudinal surveys of four broiler flocks displaying below-average performance. Whereas all samples collected on day 0 from the surveys were negative for ANV, all samples collected at days 4/5, 7, 10, 14, 21 and 28 tested positive. Sequence determinations performed with amplicons produced with 14 field samples confirmed the ANV specificity of the test, while comparative and phylogenetic analyses based on 109-nucleotide 3'-UTR sequences demonstrated that the majority of ANVs investigated were more closely related to the serotype 2 ANV (accession number AB 046864) than to the serotype 1 ANV (accession number NC 003790).

  3. RT-PCR test for detecting porcine sapovirus in weanling piglets in Hunan Province, China.

    Science.gov (United States)

    Liu, Guo-Hua; Li, Run-Cheng; Huang, Ze-Bin; Yang, Jun; Xiao, Chao-Ting; Li, Jing; Li, Man-Xiang; Yan, Yun-Qiu; Yu, Xing-Long

    2012-10-01

    The prevalence of porcine sapovirus infection in weanling pigs was investigated in Hunan Province, China, between August 2006 and October 2007. A total of 153 diarrheic fecal samples from ten intensive pig farms from ten representative administrative regions in Hunan province were examined for porcine sapoviruses using RT-PCR. Twenty-two of 153 (14.37 %) samples were found to contain porcine sapoviruses. Phylogenetic analysis showed that all the porcine sapovirus isolates in Hunan Province belonged to the porcine sapovirus genogroup III. The results of the present investigation have implications for the control of porcine sapovirus infection in pigs in Hunan Province, China.

  4. Identification of rat genes by TWINSCAN gene prediction, RT-PCR, and direct sequencing

    DEFF Research Database (Denmark)

    Wu, Jia Qian; Shteynberg, David; Arumugam, Manimozhiyan

    2004-01-01

    The publication of a draft sequence of a third mammalian genome--that of the rat--suggests a need to rethink genome annotation. New mammalian sequences will not receive the kind of labor-intensive annotation efforts that are currently being devoted to human. In this paper, we demonstrate...... an alternative approach: reverse transcription-polymerase chain reaction (RT-PCR) and direct sequencing based on dual-genome de novo predictions from TWINSCAN. We tested 444 TWINSCAN-predicted rat genes that showed significant homology to known human genes implicated in disease but that were partially...

  5. Comparision of clinical diagnostic value between PCR and TaqMan RT-PCR for Mycoplasma pneumoniae in throat swabs%PCR及RT-PCR检测咽拭子标本肺炎支原体的比较

    Institute of Scientific and Technical Information of China (English)

    李淳; 吴移谋; 朱翠明; 钟礼立; 陈丹; 吕建华

    2012-01-01

    To compare the clinical diagnostic value between PCR and RT PCR for M. Pneumoniae in swab, we per formed both PCR and RT PCR assays analysis for M. Pneumoniae DNA on a total of 566 samples of throat swab from 106 ped iatric children with M. Pneumoniae and in whom M. Pneumoniae was suspected. Among the 566 pediatric children, there were 45(7. 95%) PCR positive specimens and 175(30. 92%) RT PCR positive specimens. In the 106 pediatric children with M. Pneumoniae, 5 were positive for PCR, and 95 were positive for RT PCR. In the 460 pediatric children with symptom of M. Pneumoniae, 40 were positive for PCR, and 80 were positive for RT PCR. The sensitivy of Rt PCR for M. Pneumoniae detec tion appeared to be better than that of PCR. (sensitivity . RT PCR 89. 62 % , PCR 4. 72 % , x2=146. 322, P = 0. 000), but there was no significant difference in the specificity between RT PCR and PCR (specifitivity: RT PCR 82. 60 %, PCR 91. 30%,x2 - 3. 331, P - 0. 068). It is concluded that the TaqMan based RT PCR assay is a rapid, sensitive and specific meth od for the detection of M. Pneumoniae in throat swabs of children in early period of diagnosis.%目的 应用聚合酶链反应(PCR)与实时taqMan荧光定量PCR(RT-PCR)检测咽拭子标本中的肺炎支原体DNA(Mp-DNA),比较2种方法检测结果的临床诊断价值.方法 随机选取I临床儿科门诊患儿566例,包括临床治诊Mp感染患儿106例和临床疑似Mp感染忠儿460例,分别采用PCR法和RT-PCR法检测,以临床治诊Mp作为参照标准,采用x2检验评定2种检测方法诊断的灵敏度和特异度,比较2种检测方法对Mp的诊断价值.结果 566份受检患儿的咽拭子标本中,PCR法检测阳性45例(7.95%)(临床治诊Mp感染患儿5例,临床疑似Mp感染患儿40例),RT-PCR法检测阳性175例(30.92%)(临床治诊Mp感染患儿95例,临床疑似Mp感染患儿80例).RT-PCR法检测咽拭子Mp-DNA诊断Mp感染的敏感度显著高于PCR法(敏感度RT-PCR 89.62%,PCR 4.72%,x2=146.322,P

  6. Detection and identification of dengue virus isolates from Brazil by a simplified reverse transcription-polymerase chain reaction (RT-PCR) method.

    Science.gov (United States)

    Figueiredo, L T; Batista, W C; Igarashi, A

    1997-01-01

    We show here a simplified RT-PCR for identification of dengue virus types 1 and 2. Five dengue virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD as a negative control, were used in this study. C6/36 cells were infected and supernatants were collected after 7 days. The RT-PCR, done in a single reaction vessel, was carried out following a 1/10 dilution of virus in distilled water or in a detergent mixture containing Nonidet P40. The 50 microliters assay reaction mixture included 50 pmol of specific primers amplifying a 482 base pair sequence for dengue type 1 and 210 base pair sequence for dengue type 2. In other assays, we used dengue virus consensus primers having maximum sequence similarity to the four serotypes, amplifying a 511 base pair sequence. The reaction mixture also contained 0.1 mM of the four deoxynucleoside triphosphates, 7.5 U of reverse transcriptase, 1U of thermostable Taq DNA polymerase. The mixture was incubated for 5 minutes at 37 degrees C for reverse transcription followed by 30 cycles of two-step PCR amplification (92 degrees C for 60 seconds, 53 degrees C for 60 seconds) with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized by UV light after staining with ethidium bromide solution. Low virus titer around 10(3, 6) TCID50/ml was detected by RT-PCR for dengue type 1. Specific DNA amplification was observed with all the Brazilian dengue strains by using dengue virus consensus primers. As compared to other RT-PCRs, this assay is less laborious, done in a shorter time, and has reduced risk of contamination.

  7. Detection and identification of dengue virus isolates from Brazil by a simplified reverse transcription - polymerase chain reaction (RT-PCR method

    Directory of Open Access Journals (Sweden)

    FIGUEIREDO Luiz Tadeu Moraes

    1997-01-01

    Full Text Available We show here a simplified RT-PCR for identification of dengue virus types 1 and 2. Five dengue virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD as a negative control, were used in this study. C6/36 cells were infected and supernatants were collected after 7 days. The RT-PCR, done in a single reaction vessel, was carried out following a 1/10 dilution of virus in distilled water or in a detergent mixture containing Nonidet P40. The 50 µl assay reaction mixture included 50 pmol of specific primers amplifying a 482 base pair sequence for dengue type 1 and 210 base pair sequence for dengue type 2. In other assays, we used dengue virus consensus primers having maximum sequence similarity to the four serotypes, amplifying a 511 base pair sequence. The reaction mixture also contained 0.1 mM of the four deoxynucleoside triphosphates, 7.5 U of reverse transcriptase, 1U of thermostable Taq DNA polymerase. The mixture was incubated for 5 minutes at 37ºC for reverse transcription followed by 30 cycles of two-step PCR amplification (92ºC for 60 seconds, 53ºC for 60 seconds with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized by UV light after staining with ethidium bromide solution. Low virus titer around 10 3, 6 TCID50/ml was detected by RT-PCR for dengue type 1. Specific DNA amplification was observed with all the Brazilian dengue strains by using dengue virus consensus primers. As compared to other RT-PCRs, this assay is less laborious, done in a shorter time, and has reduced risk of contamination

  8. Citrus psorosis virus: nucleotide sequencing of the coat protein gene and detection by hybridization and RT-PCR.

    Science.gov (United States)

    Barthe, G A; Ceccardi, T L; Manjunath, K L; Derrick, K S

    1998-06-01

    Citrus psorosis virus (CPV) is a multicomponent ssRNA virus with a coat protein of approximately 48 kDa. The viral genome is encapsidated in short and long particles that are readily separated by sucrose density-gradient centrifugation. CPV particles are spiral filaments that are referred to as spiroviruses (SV). A cDNA library of purified short particles from isolate CPV-4 was prepared in a Lambda vector and screened for expression of the coat protein gene (CPG) with a monoclonal antibody to the coat protein. Sequencing of immunopositive clones indicated a single ORF encoding a 49 kDa protein. This ORF, when expressed in E. coli, gave a protein identical in size and immunoreactivity to the CPV coat protein. A full-length clone of the CPG was transcribed and used in Northern hybridization assays to establish that short particle RNA of CPV is negative sense and contains the CPG. Moreover, the CPG was not found on RNA extracted from long particles or on the sedimentable dsRNA from CPV infected tissue. RT-PCR assays were developed for the amplification of a 600 bp fragment of CPG and for the complete CPG (1317 bp). The 600 bp fragment from a biologically and serologically different isolate, CPV-6, was cloned, sequenced and found to share 86% (nucleotide) and 96% (amino acid) identity with CPV-4. BLAST analysis of sequences from CPV-4 and CPV-6 detected no significant nucleic acid or protein similarity with any known viral sequences.

  9. Multiplex qRT-PCR for the Detection of Western Equine Encephalomyelitis, St. Louis Encephalitis, and West Nile Viral RNA in Mosquito Pools (Diptera: Culicidae).

    Science.gov (United States)

    Brault, Aaron C; Fang, Ying; Reisen, William K

    2015-05-01

    Following the introduction of West Nile virus into California during the summer of 2003, public health and vector control programs expanded surveillance efforts and were in need of diagnostics capable of rapid, sensitive, and specific detection of arbovirus infections of mosquitoes to inform decision support for intervention. Development of a multiplex TaqMan or real-time semiquantitative reverse transcription polymerase chain reaction (RT-PCR) assay in which three virus specific primer-probe sets were used in the same reaction is described herein for the detection of western equine encephalomyelitis, St. Louis encephalitis and West Nile viral RNA. Laboratory validation and field data from 10 transmission seasons are reported. The comparative sensitivity and specificity of this multiplex assay to singleplex RT-PCR as well as an antigen detection (rapid analyte measurement platform) and standard plaque assays indicate this assay to be rapid and useful in providing mosquito infection data to estimate outbreak risk. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  10. Identification of Candidate Reference Genes in Perennial Ryegrass for Quantitative RT-PCR under Various Abiotic Stress Conditions

    OpenAIRE

    2014-01-01

    BACKGROUND: Quantitative real-time reverse-transcriptase PCR (qRT-PCR) is an important technique for analyzing differences in gene expression due to its sensitivity, accuracy and specificity. However, the stability of the expression of reference genes is necessary to ensure accurate qRT-PCR assessment of expression in genes of interest. Perennial ryegrass (Lolium perenne L.) is important forage and turf grass species in temperate regions, but the expression stability of its reference genes un...

  11. Improved Serotype-Specific Dengue Virus Detection in Trinidad and Tobago using a Multiplex, Real-Time RT-PCR

    Science.gov (United States)

    Waggoner, Jesse J.; Sahadeo, Nikita S. D.; Brown, Arianne; Mohamed-Hadley, Alisha; Hadley, Dexter; Carrington, Leslie; Carrington, Christine V. F.; Pinsky, Benjamin A.

    2014-01-01

    Dengue virus (DENV) transmission occurs throughout the Caribbean, though laboratory confirmation and epidemiologic surveillance is limited by the availability of serotype-specific molecular diagnostics. In this study, we show that a serotype-specific DENV multiplex, real-time RT-PCR detected DENV RNA in significantly more samples (82/182) than a reference hemi-nested RT-PCR (57/182; p=0.01). PMID:25533614

  12. Application of in situ reverse trancriptase-polymerase chain reaction (RT-PCR to tissue microarrays

    Directory of Open Access Journals (Sweden)

    Terrett Jonathan A

    2003-05-01

    Full Text Available Abstract Detection of disease-associated gene transcripts in primary disease tissues is frequently confounded by the presence of non-involved cell types. Alternative methods of detecting gene expression directly within tissues involve either the generation of antibodies, which can be a lengthy process and may suffer from lack of specificity, or amplification of reverse-transcribed cDNA in tissue sections (in situ RT-PCR. The latter method is highly specific and enables detection of transcripts in the cells originally responsible for their synthesis, but is highly destructive of tissue structures and can be carried out on only one or a few sections per experiment, resulting in low reproducibility. In this study, in situ RT-PCR was applied for the first time to commercially available tissue section microarrays enabling the examination of up to 70 different samples simultaneously. Modifications to the technique are detailed that preserved visible tissue and cellular structures and improved transcript detection whilst preventing significant generation of artefacts.

  13. Quantification of llama inflammatory cytokine mRNAs by real-time RT-PCR.

    Science.gov (United States)

    Odbileg, Raadan; Konnai, Satoru; Usui, Tatsufumi; Ohashi, Kazuhiko; Onuma, Misao

    2005-02-01

    We have developed a method by which llama cytokine mRNAs can be quantified using real-time reverse transcription polymerase chain reaction (RT-PCR). Total RNA was extracted from lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) of llama, reverse transcribed to cDNA, and cytokine profiles for interleukin (IL)-1alpha, IL-1beta, IL-6 and tumor necrosis factor (TNF) alpha were quantified by real-time PCR. The expressions of mRNAs of inflammatory cytokines IL-1alpha, IL-1beta, IL-6 and TNFalpha were upregulated upon stimulation with LPS in a dose- and time-dependent manner. Incubation of PBMCs with 100 and 1,000 pg/ml of LPS for 3 to 6 hr resulted in the acceleration of the mRNA levels of inflammatory cytokines. Here, we describe a highly sensitive and reproducible method to quantify the transcription of llama cytokine mRNAs by real-time RT-PCR with the double-stranded DNA-binding dye SYBR Green I.

  14. A simple RT-PCR-based strategy for screening connexin identity

    Directory of Open Access Journals (Sweden)

    M. Urban

    1999-08-01

    Full Text Available Vertebrate gap junctions are aggregates of transmembrane channels which are composed of connexin (Cx proteins encoded by at least fourteen distinct genes in mammals. Since the same Cx type can be expressed in different tissues and more than one Cx type can be expressed by the same cell, the thorough identification of which connexin is in which cell type and how connexin expression changes after experimental manipulation has become quite laborious. Here we describe an efficient, rapid and simple method by which connexin type(s can be identified in mammalian tissue and cultured cells using endonuclease cleavage of RT-PCR products generated from "multi primers" (sense primer, degenerate oligonucleotide corresponding to a region of the first extracellular domain; antisense primer, degenerate oligonucleotide complementary to the second extracellular domain that amplify the cytoplasmic loop regions of all known connexins except Cx36. In addition, we provide sequence information on RT-PCR primers used in our laboratory to screen individual connexins and predictions of extension of the "multi primer" method to several human connexins.

  15. Highly Specific Detection of Five Exotic Quarantine Plant Viruses using RT-PCR

    Directory of Open Access Journals (Sweden)

    Hoseong Choi

    2013-03-01

    Full Text Available To detect five plant viruses (Beet black scorch virus, Beet necrotic yellow vein virus, Eggplant mottled dwarf virus, Pelargonium zonate spot virus, and Rice yellow mottle virus for quarantine purposes, we designed 15 RT-PCR primer sets. Primer design was based on the nucleotide sequence of the coat protein gene, which is highly conserved within species. All but one primer set successfully amplified the targets, and gradient PCRs indicated that the optimal temperature for the 14 useful primer sets was 51.9°C. Some primer sets worked well regardless of annealing temperature while others required a very specific annealing temperature. A primer specificity test using plant total RNAs and cDNAs of other plant virus-infected samples demonstrated that the designed primer sets were highly specific and generated reproducible results. The newly developed RT-PCR primer sets would be useful for quarantine inspections aimed at preventing the entry of exotic plant viruses into Korea.

  16. Development of a real-time quantitative RT-PCR to detect REV contamination in live vaccine.

    Science.gov (United States)

    Luan, Huaibiao; Wang, Yixin; Li, Yang; Cui, Zhizhong; Chang, Shuang; Zhao, Peng

    2016-09-01

    Based on the published Avian reticuloendotheliosis virus (REV) whole genome sequence, primers and TaqMan probes were designed and synthesized, and the TaqMan probe fluorescence real-time quantitative RT-PCR (qRT-PCR) method for detecting the REV pol gene was established by optimizing the reaction conditions. Sensitivity analysis showed that the qRT-PCR method had a sensitivity that was 1,000-fold higher than conventional PCR. Additionally, no amplification signals were obtained when we attempted to detect DNA or cDNA of ALV-A/B/J, MDV, CIAV, IBDV, ARV, NDV, AIV, or other viruses, suggesting a high specificity for our method. Various titers of REV were artificially "spiked" into the FPV and MDV vaccines to simulate REV contamination in attenuated vaccines to validate this qRT-PCR method. Our findings indicated that this qRT-PCR method could detect REV contamination at a dose of 1 TCID50/1,000 feathers, which was 10,000-fold more sensitive than the regular RT-PCR detection (10(4) TCID50/1000 feathers).

  17. Análise de citocinas pela RT-PCR em pacientes com rinite alérgica RT-PCR cytokine study in patients with allergic rhinitis

    Directory of Open Access Journals (Sweden)

    Tarcimara Moreira da Silva

    2009-02-01

    Full Text Available Rinite alérgica é uma doença que decorre de um processo inflamatório da mucosa nasal conseqüente à reação de hipersensibilidade a alérgenos inalatórios e, eventualmente, alimentares. É mediada por IgE, envolvendo diferentes células, mediadores e citocinas. OBJETIVO: Avaliar as transcrições para as seguintes citocinas: IL-4, IL-5, IL-8 e IFN-gama, particularmente importantes no processo alérgico nasal, principalmente IL-4 e IL-5. Neste estudo, optou-se por avaliar os pacientes atópicos fora das crises alérgicas, com a finalidade de se conhecer as expressões das citocinas neste período. MATERIAL E MÉTODO: Realizou-se um estudo transversal e prospectivo, selecionando-se 30 pacientes, sendo 13 pacientes portadores de rinite alérgica paucissintomáticos e 17 pacientes não-atópicos. Os grupos foram selecionados através da história, do exame clínico otorrinolaringológico e do teste alérgico cutâneo - Prick Test. O perfil das citocinas foi pesquisado nos fragmentos de mucosa nasal, através da RT-PCR semiquantitativa, escolhida por apresentar boa reprodutibilidade e especificidade, utilizando-se como referência o gene da Beta-actina. RESULTADOS: Os valores de IL-5, IL-8, IFN-gama mantiveram-se homogêneos em relação ao grupo controle. A IL-4 apresentou diferença com significância estatística. CONCLUSÃO: Os pacientes alérgicos paucissintomáticos apresentaram normalização da expressão das citocinas na mucosa nasal à exceção de IL-4.Allergic rhinitis is an inflammatory reaction of the nasal mucosa, in consequence of an IgE mediated hypersensitive reaction to inhaling allergens, involving different mediators and cytokine cells. AIM: The purpose of this study was to evaluate the transcriptions for IL-4, IL-5, IL-8 and IFN-gama, particularly important in the nasal allergy process, especially IL-4 and IL-5. For this study we decided to evaluate atopic patients who were free from allergic crises, with the purpose of

  18. QUANTIFICATION OF P4HA2 mRNA OF FIBROBLASTS WITH SYBR GREEN BASED RT-PCR FOR CORRECTING CMV INACTIVATION EFFICIENCY IN DONOR BLOOD

    Institute of Scientific and Technical Information of China (English)

    FANG Feng-qin; ZHANG Yue; LU Ping; ZHANG Li; JI Yu-hua

    2009-01-01

    Objective To quantify proline 4-hydroxylase, alpha polypeptide Ⅱ (P4HA2) mRNA of human embryo lung fibroblast (HELF) with SYBR green based reversed transcript PCR (RT-PCR) for correcting cytomegalovirus (CMV) inactivation or clearance efficiency in donor blood.Methods A pair of specific primers of exon 12a of P4HA2 was designed, and the related PCR-reaction system and condition were optimized. Then the recombinant plasmid containing the target fragment was constructed for making standard curve with SYBR green based real-time RT-PCR. Finally, the sensitivity, reproducibility, and specificity of this method were fully estimated.Results The sensitivity of the method was 1.5E+04 copies/mL of P4HA2 mRNA, corresponding to 103 fibroblasts. In addition, existence of 8.67E+06 leukocytes could not interfere with the accurate quantification of HELF in the large dynamic range. The intra-assay variability and inter-assay variability both varied in different concentrations, being higher in low concentrations and lower in high concentrations. But all of them were below 13.76% in variation, which showed acceptable stability of this method.Conclusion SYBR green and specific primer based real-time RT-PCR show up a good quality for quantifying HELF P4HA2 mRNA with good specificity, stability, and high sensitivity. Approximate 10 copies of P4HA2 mRNA per cell in average can be detected by the method. Therefore, this method can be used to deduct fibroblast-associated CMV for correcting CMV inactivation efficiency in leukocytes.

  19. Detección y caracterización del virus de bronquitis infecciosa aviaria en Chile mediante RT-PCR y análisis secuencial Detection and characterization of infectious bronchitis virus in Chile by RT-PCR and sequence analysis

    Directory of Open Access Journals (Sweden)

    J C Lopez

    2006-01-01

    Full Text Available Una técnica de reacción en cadena de la polimerasa transcriptasa reversa (RT-PCR junto a una secuenciación fue usada para detectar y caracterizar genéticamente virus diferentes de bronquitis infecciosa aviar (VBIA aislados en Chile. El procedimiento de RT-PCR incluyó el uso de los partidores NT1 y NT2, los cuales se localizaron cerca del término N del gen S1 y cubrieron la región hipervariable. La secuencia amplificada fue alineada y analizada con el programa computacional DNAman, y comparada con secuencias reportadas en GenBank. El nivel de detección de la técnica de RT-PCR fue equivalente al aislamiento viral en huevos cuando se usaron directamente tejidos, pero el ensayo fue más sensitivo cuando fue usado para detectar virus almacenados en fluido alantoideo. Los amplificados de todos los aislados históricos de Chile fueron idénticos en tamaño (193pb y exhibieron entre ellos, al analizar la secuencia una similitud del 71 al 96%. Estos aislados mostraron entre 68 y 97% de similitud con cepas de Estados Unidos, Europa, Asia, Nueva Zelandia y Australia.A reverse transcriptase-polymerase chain reaction (RT-PCR assay, coupled with sequencing, was used to detect and genetically characterize different infectious bronchitis virus (IBV isolates in Chile. The RT-PCR procedure included the use of the primers NT1 and NT2 that were located close to the N-terminus of the S1 gene and bracketed the hypervariable region, and the amplified sequences were aligned and analyzed with DNAman software, and compared with sequences from GenBank. The level of detection of the RTPCR assay was equivalent to virus isolation in eggs when testing tissues directly, but the assay was more sensitive when used to detect virus stored in allantoic fluid. The amplimers from all historical Chilean isolates were identical in size (193 bp and exhibited 71-96% similarity on sequence analysis. These isolates showed between 68-97% similarity to strains from North America

  20. Increased urothelial cell detection in the primary bladder smooth muscle cell cultures with dual MACS/qRT-PCR approach.

    Science.gov (United States)

    Genheimer, Chistopher W; Guthrie, Kelly I; Shokes, Jacob E; Bruce, Andrew T; Quinlan, Sarah F; Sangha, Namrata; Ilagan, Roger M; Basu, Joydeep; Burnette, Teresa; Ludlow, John W

    2011-03-01

    Bladder tissue has been regenerated in humans with neurogenic bladder using an implant produced from autologous urothelial (UC) and smooth muscle cells (SMC) expanded from bladder biopsies seeded onto a biodegradable synthetic scaffold. As the majority of bladder cancers are urothelial carcinomas (aka, transitional cell carcinoma), this 2-cell type autologous sourcing strategy presents significant challenges to product development. Entire bladders have been regenerated in cystectomized animals using a single-cell-type sourcing strategy: implants were seeded with bladder-derived SMC-only. Applying the bladder SMC-only sourcing strategy to produce clinical implants for bladder replacement or urinary diversion in bladder cancer patients requires methods for screening SMC cultures for the presence of potentially cancerous UC cells to provide evidence of SMC culture purity before seeding the scaffold. In this report, we show a 10-fold to 100-fold improvement in the sensitivity of qualitative and quantitative reverse-transcription PCR (qRT-PCR)-based assays for detecting UC positive for Cytokeratin 5 (CK5) in mixed SMC/UC cultures when the cell population was first subjected to magnetic activated cell sorting to enrich for cells expressing the epithelial cell adhesion molecule (known as EPCAM or CD326), a marker known to be present in normal UC and upregulated in the cancerous UC.

  1. RT-PCR em pools de soros sangüíneos para o diagnóstico da infecção aguda e de animais persistentemente infectados pelo vírus da diarréia viral bovina RT-PCR in pools of bovine blood serum to detect acute infection and persistently infected animals with bovine viral diarrhea virus

    Directory of Open Access Journals (Sweden)

    D. Pilz

    2007-02-01

    Full Text Available Utilizou-se a técnica da RT-PCR para a detecção da região 5' UTR do genoma do vírus da diarréia viral bovina (BVDV em pools de soros sangüíneos provenientes de um rebanho, constituído por 226 animais, que apresentava distúrbios da reprodução. A partir das amostras individuais de soro e de acordo com a categoria dos animais e o número de animais por categoria foram formados 10 pools (A a J de soros. A primeira avaliação revelou a amplificação de um produto com 290pb nas reações referentes aos grupos D (35 vacas e H (25 bezerros lactentes que, após o desmembramento em amostras individuais, resultou na identificação de 11 vacas lactantes e 12 bezerros em amamentação positivos. Para a identificação de animais persistentemente infectados (PI entre os 23 positivos na primeira avaliação, realizou-se a segunda colheita de soros sangüíneos, três meses após. A RT-PCR das amostras individuais de soro revelou resultado positivo em cinco bezerros. Em dois, foi possível isolar o BVDV em cultivo de células MDBK. A especificidade das reações da RT-PCR foi confirmada pelo seqüenciamento dos produtos amplificados a partir do soro de uma vaca com infecção aguda, de um bezerro PI e das duas amostras do BVDV isoladas em cultivo celular. A utilização da RT-PCR em pools de soros sangüíneos demonstrou ser uma estratégia rápida de diagnóstico etiológico e de baixo custo tanto para a detecção de infecção aguda quanto de animais PI.The 5' untranslated region of the bovine viral diarrhea virus (BVDV genome was detected by RT-PCR assay in pools of blood sera samples collected from a cattle herd (n=226 animals with reproductive failures. Based on the classes of animal and the number of animals per class, the individual blood serum samples were distributed in 10 sera pools (A to J. During the first evaluation a 290bp amplicon was amplified in reactions from groups D (35 cows and H (25 sucking calves. The individual analysis

  2. Webtag: a new web tool providing tags/anchors for RT-PCR experiments with prokaryotes

    Directory of Open Access Journals (Sweden)

    Lindblad Peter

    2007-10-01

    Full Text Available Abstract Background Webtag is a tool providing oligonucleotide sequences (usually called tags or anchors that are absent from a specified genome. These tags/anchors can be appended to gene specific primers for reverse transcriptase polymerase chain reaction experiments, circumventing genomic DNA contamination. Results The use of a relational database, in conjunction with a series of scripts written in PHP and Perl, allows the user to rapidly obtain tags that are: 1 suitable for a specific organism, and 2 compatible with other oligonucleotides to be used in the experimental procedures. Conclusion This new web tool allows scientists to easily and rapidly obtain suitable tags for RT-PCR experiments, and is available at http://www.egs.uu.se/software/webtag/.

  3. Detection of bacterial species involved in perimplantitis concerned with cultural and RT-PCR

    Directory of Open Access Journals (Sweden)

    Marcello Gatti

    2010-06-01

    Full Text Available Dental implants offer new treatment options for edentulous either partially or completely, now represent a viable alternative to conventional fixed protheses. Dental implants are colonized by a flora dominated by Gram-positive facultative aerobic, while in patients with bone loss and formation of pockets peri-implant diseases was found a significant difference in the composition of microflora, bacteria, Gram-negative anaerobes in particular Fusobacterium spp., Treponema denticola (Spirochetes, Tannerella forsythensis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia as interim black-pigmented bacteria, Porphyromonas gingivalis, often in high concentrations. Aims. The purpose of this study was to identify those at risk of perimplantitis using 2 techniques: RT-PCR examination of trade and culture. The results were compared taking into consideration the advantages and disadvantages of both methods. Materials and methods.We studied 24 patients (14 women and 10 men, aged, women between 43 and 76 years, with an average of 63.8 + / - 10.9 years, men between 45 and 88 years with a average of 64.3 years + / - 12.5 years. Was performed a double levy of sub-gingival plaque at multiple sites that had an implant CAL (clinical attachment level> 4mm in order to assess the microbiological identification with the two techniques: Examining culture and Real-Time PCR of Commerce ( Gum-Sunstar that identifies 4 bacterial species: A. actinomycetemcomitans (A.a., P.gingivalis (P.g., T.forsythensis (T.f., and T.denticola (T.d.. Results. All patients studied were positive to both tests with charger high: the consideration of tenure, with CFU / ml > 105, was positive in 66.6% of samples by:T.f., and P.g., in 12.5% for A.a., while T.d. not been sought by examining culture, the RT-PCR was positive, with high loads, in 95.8% of samples for T.f., in 79.1% for P.g., in 12.5% for A.a. and 20.8% for T.d.The test crop showed the presence of even P.intermedia in 91

  4. Seasonal variation in transcript abundance in cork tissue analyzed by real time RT-PCR.

    Science.gov (United States)

    Soler, Marçal; Serra, Olga; Molinas, Marisa; García-Berthou, Emili; Caritat, Antònia; Figueras, Mercè

    2008-05-01

    The molecular processes underlying cork biosynthesis and differentiation are mostly unknown. Recently, a list of candidate genes for cork biosynthesis and regulation was made available opening new possibilities for molecular studies in cork oak (Quercus suber L.). Based on this list, we analyzed the seasonal variation in mRNA abundance in cork tissue of selected genes by real time reverse-transcriptase polymerase chain reaction (RT-PCR). Relative transcript abundance was evaluated by principal component analysis and genes were clustered in several functional subgroups. Structural genes of suberin pathways such as CYP86A1, GPAT and HCBT, and regulatory genes of the NAM and WRKY families showed highest transcript accumulation in June, a crucial month for cork development. Other cork structural genes, such as FAT and F5H, were significantly correlated with temperature and relative humidity. The stress genes HSP17.4 and ANN were strongly positively correlated to temperature, in accord with their protective role.

  5. Evaluation of disinfectant efficacy against hepatitis C virus using a RT-PCR-based method.

    Science.gov (United States)

    Charrel, R N; de Chesse, R; Decaudin, A; De Micco, P; de Lamballerie, X

    2001-10-01

    The methods traditionally used to evaluate the antiviral activity of antiseptics and disinfectants are based on cell cultures. However, such methods are not applicable to non-cultivable viruses such as hepatitis C (HCV). Therefore, in this case, virucidal activity is normally tested using surrogate viruses able to grow in cell culture. This paper describes a RT-PCR method for testing antiseptic/disinfectant activity against HCV, as a model for non-cultivable viruses. A chlorine-based agent used for skin and tissues, and a 2% glutaraldehyde solution used for endoscope disinfection, were the test materials. The results are discussed in the light of the use of these agents. The method is simple, fast and inexpensive, and could be used for tests on other viruses with minor modification.

  6. Multiplex RT-PCR for rapid detection of viruses commonly causing diarrhea in pediatric patients.

    Science.gov (United States)

    Thongprachum, Aksara; Khamrin, Pattara; Pham, Ngan Thi Kim; Takanashi, Sayaka; Okitsu, Shoko; Shimizu, Hiroyuki; Maneekarn, Niwat; Hayakawa, Satoshi; Ushijima, Hiroshi

    2017-05-01

    Multiplex RT-PCR method using five sets of panel primers was developed for the detection of diarrheal viruses, including rotavirus A, B, and C, adenovirus, astrovirus, norovirus GI and GII, sapovirus, Aichi virus, parechovirus, enterovirus, cosavirus, bocavirus, and Saffold virus. The sensitivity of the method was evaluated and tested with 751 fecal specimens collected from Japanese children with acute diarrhea. Several kinds of viruses were detected in 528 out of 751 (70.3%) fecal specimens. Mixed-infection with different viruses in clinical specimens could also be effectively detected. The method proved to be reliable with highly sensitive and specific and useful for routine diagnosis. J. Med. Virol. 89:818-824, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  7. [RT-PCR-based methods for identification and typing of infectious hemopoietic necrosis virus in salmons].

    Science.gov (United States)

    Popova, A G; Oreshkova, S F; Zhchelkunov, I S; Rudakova, S L; Zhchelkunova, T I; Tikunova, N V; Blinova, N N; Il'ichev, A A

    2008-01-01

    A RT-PCR method has been developed to diagnose infectious hemopoietic necrosis virus (IHNV) in salmons. The authors show it possible to use the method for viral shedding in both a cell culture and a clinical sample from infected fishes. Genotyping of IHNV strains originating from North America, Europe, and Russia, by using the restriction fragment length polymerase analysis, has revealed that 10 of them belong to 3 existing genogroups (U, M, and L). Three Russian isolates are assigned into a separate subgroup. Phylogenetic analysis of several isolates has confirmed that viral strains from Katchatka belong to the North American U-genogroup whereas 3 Russian isolates from the continental zone of the country make up a separate subgroup within the same genogroup.

  8. Construction of an adult barnacle (Balanus amphitrite cDNA library and selection of reference genes for quantitative RT-PCR studies

    Directory of Open Access Journals (Sweden)

    Burgess J Grant

    2009-06-01

    Full Text Available Abstract Background Balanus amphitrite is a barnacle commonly used in biofouling research. Although many aspects of its biology have been elucidated, the lack of genetic information is impeding a molecular understanding of its life cycle. As part of a wider multidisciplinary approach to reveal the biogenic cues influencing barnacle settlement and metamorphosis, we have sequenced and annotated the first cDNA library for B. amphitrite. We also present a systematic validation of potential reference genes for normalization of quantitative real-time PCR (qRT-PCR data obtained from different developmental stages of this animal. Results We generated a cDNA library containing expressed sequence tags (ESTs from adult B. amphitrite. A total of 609 unique sequences (comprising 79 assembled clusters and 530 singlets were derived from 905 reliable unidirectionally sequenced ESTs. Bioinformatics tools such as BLAST, HMMer and InterPro were employed to allow functional annotation of the ESTs. Based on these analyses, we selected 11 genes to study their ability to normalize qRT-PCR data. Total RNA extracted from 7 developmental stages was reverse transcribed and the expression stability of the selected genes was compared using geNorm, BestKeeper and NormFinder. These software programs produced highly comparable results, with the most stable gene being mt-cyb, while tuba, tubb and cp1 were clearly unsuitable for data normalization. Conclusion The collection of B. amphitrite ESTs and their annotation has been made publically available representing an important resource for both basic and applied research on this species. We developed a qRT-PCR assay to determine the most reliable reference genes. Transcripts encoding cytochrome b and NADH dehydrogenase subunit 1 were expressed most stably, although other genes also performed well and could prove useful to normalize gene expression studies.

  9. No control genes required: Bayesian analysis of qRT-PCR data.

    Directory of Open Access Journals (Sweden)

    Mikhail V Matz

    Full Text Available BACKGROUND: Model-based analysis of data from quantitative reverse-transcription PCR (qRT-PCR is potentially more powerful and versatile than traditional methods. Yet existing model-based approaches cannot properly deal with the higher sampling variances associated with low-abundant targets, nor do they provide a natural way to incorporate assumptions about the stability of control genes directly into the model-fitting process. RESULTS: In our method, raw qPCR data are represented as molecule counts, and described using generalized linear mixed models under Poisson-lognormal error. A Markov Chain Monte Carlo (MCMC algorithm is used to sample from the joint posterior distribution over all model parameters, thereby estimating the effects of all experimental factors on the expression of every gene. The Poisson-based model allows for the correct specification of the mean-variance relationship of the PCR amplification process, and can also glean information from instances of no amplification (zero counts. Our method is very flexible with respect to control genes: any prior knowledge about the expected degree of their stability can be directly incorporated into the model. Yet the method provides sensible answers without such assumptions, or even in the complete absence of control genes. We also present a natural Bayesian analogue of the "classic" analysis, which uses standard data pre-processing steps (logarithmic transformation and multi-gene normalization but estimates all gene expression changes jointly within a single model. The new methods are considerably more flexible and powerful than the standard delta-delta Ct analysis based on pairwise t-tests. CONCLUSIONS: Our methodology expands the applicability of the relative-quantification analysis protocol all the way to the lowest-abundance targets, and provides a novel opportunity to analyze qRT-PCR data without making any assumptions concerning target stability. These procedures have been

  10. K-19 mRNA RT-PCR in detecting micrometastasis in regional lymph nodes of gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Jian Suo; Quan Wang; Hong-Juan Jin; Hong Li; Hang Zhao

    2006-01-01

    AIM: To investigate the value and prospect of RT-PCR in detecting micrometastasis in regional lymph nodes of gastric cancer.METHODS: Histopathology was used and K19 mRNA expression was detected by RT-PCR in tumor tissues and lymph nodes from gastric cancer patients undergoing radical resection of gastric carcinoma. RESULTS: K19 mRNA was expressed in all tumor specimens of 30 cases; of the 126 lymph nodes, 26 were histopathologically positive (20.6%), and 42 positive (33.3%) by RT-PCR. Amplification fragments of 460 and 540 bp were shown in all the tumor tissues and metastatic lymph nodes after K19 and 3-actin RT-PCR, while only a 540 bp fragment appeared in the lymph nodes of non-tumor patients.CONCLUSION: K19 mRNA RT-PCR is sensitive and specific in testing micrometastasis in regional lymph nodes of gastric cancer, and it is superior to routine histopathology.

  11. Application of reference gene for real-time RT-PCR%内参照基因在实时荧光定量RT-PCR检测中的应用

    Institute of Scientific and Technical Information of China (English)

    陈瑾歆; 陈建业; 李云祥

    2011-01-01

    目的 建立测定内参照β-actin表达量的实时荧光定量RT-PCR两步法检测方法.方法 根据Genbank 中人β-actin保守区域序列设计荧光PCR适用的引物和探针,构建质粒标准品建立标准曲线用于荧光PCR相对定量,检测荧光PCR方法的特异性和重复性.结果 建立了人β-actin实时荧光RT-PCR检测方法.结论 本实验建立的人β-actin表达实时荧光RT-PCR两步法检测方法特异性和重复性较好,为β-actin作为定量RT-PCR中内参照基因进行人其他功能基因和病原基因表达的定量分析奠定了基础.%Objective To establish a Taqman real-time RT-PCR 2-step assay for quantitative detection of the expression of human β-actin. Methods Specific primers and probes were designed for real-time RT-PCR according to β-actin cDNA sequence. Plasmid standard preparations were constructed by T-A clone, extracted and prepared for establishing standard curve used for relative quantification real-time RT-PCR. The expression levels of β-actin were measured by real-time RT-PCR in HepG2 cells to detect the specificity and reproducibility of real-time RT-PCR assay. Results An effective real-time RT-PCR assay was established for detecting β-actin mRNA expression levels.Conclusions The real-time PCR assay for the expression of β-actin is a sensitive, specific tool for quantitative assay of mRNA expression levels of other gene when using β-actin as reference genes.

  12. RT-PCR for mammaglobin genes, MGB1 and MGB2, identifies breast cancer micrometastases in sentinel lymph nodes.

    Science.gov (United States)

    Ouellette, Rodney J; Richard, Dominique; Maïcas, Emmanuel

    2004-05-01

    In the present study, we examined the expression of the mammaglobin genes, MGB1 and MGB2, in the sentinel lymph nodes (SLNs) of patients with breast cancer and compared our results with the histologic status of the same SLNs. Compared with immunohistochemical staining for cytokeratin 8, which detected metastases in 17 of 42 patients, reverse transcription-polymerase chain reaction (RT-PCR) for MGB1 or MGB2 genes was positive in 22 patients. The concordance between the expression of any mammaglobin and histologic status was 79% (33/42), with a sensitivity of 88% and specificity of 72%. The detection of patients with metastases was more sensitive when testing for both MGB1 and MGB2 (P MGB2 (P < .0005) or MGB1 (P < .05) alone. The increased detection rate relative to histologic examination suggests that using RT-PCR for the mammaglobin genes might identify patients at higher risk compared with patients with negative RT-PCR results.

  13. Comparación de dos kits de RT-PCR en la detección de ARNm de dos genes endógenos de papa (Solanum tuberosum spp. Andígena

    Directory of Open Access Journals (Sweden)

    Milet Zabaleta Vanegas

    2009-01-01

    Full Text Available RT-PCR es una técnica en la que usando ARN mensajero como molde, se obtiene complementario o cADN por transcripción inversa, y luego se amplifica uno de los cADN por PCR, mediante el uso de primers específicos. Esta técnica permite realizar estudios de expresión, a nivel de ARN mensajero. Con el propósito de implementar la técnica en papa (Solanum tuberosum spp. Andígena, se utilizaron plántulas cultivadas in vitro de la variedad Pastusa Suprema. Inicialmente se establecieron las condiciones para la extracción de ARN total usando el kit TRizol® Reagent de InvitrogenTM, con el que se obtuvieron excelentes resultados. Este ARN se usó como molde para evaluar dos kits: “ONE Step superScriptTM” y “SuperScriptâ„¢ First Strand Syntesis For RT-PCR SSâ„¢ II RT”, de InvitrogenTM. Se usaron primers específicos para dos genes endógenos: cox y actina. El primero es un gen mi­tocondrial y el segundo es un gen nuclear. Se observaron señales claras y diferenciables de amplificación para cox, utilizando el kit “ONE Step superScriptTM”, con un tamaño esperado de 96 pb. Para actina, se observó una señal clara de amplificación de 300 pb, con el kit “SuperScriptâ„¢ First Strand Syntesis For RT-PCR SSâ„¢ II RT”.  Palabras clave: Solanum tuberosum; cox, actina; RT-PCR; papa; Solanum tuberosum; cox; actin; RT-PCR; potato.

  14. Atrophic dermatofibrosarcoma protuberans with the fusion gene COL1A1-PDGFB detected by RT-PCR using only a single primer pair.

    Science.gov (United States)

    Xu, Wen-Jun; Wang, Ju-Sheng

    2015-01-01

    Dermatofibrosarcoma protuberans (DFSPs) is an uncommon dermal tumor of intermediate to low-grade malignancy. A few patients have clinically persistent plaques that might be atrophic, and they are difficult to be diagnosed clinically. With the development of cytogenetic and molecular biology techniques, the detection of fusion transcripts of the collagen type 1a1 (COL1A1) and platelet-derived growth factor-BB (PDGFB) genes has been recognized as a reliable and valuable molecular tool for the diagnosis of DFSPs. We reported a 24-year-old woman who had a 2 years history of atrophic DFSPs, and detected the gene fusion between COL1A1 to PDGFB by one-step method of RT-PCR using only a single primer pair. The gene fusion detected by this rapid and efficient one-step method in our patient appears to be the first report of atrophic DFSPs, and we detected a novel COL1A1 breakpoint between exon 2 and exon 3.

  15. Genome-Wide Identification and Validation of Reference Genes in Infected Tomato Leaves for Quantitative RT-PCR Analyses.

    Directory of Open Access Journals (Sweden)

    Oliver A Müller

    Full Text Available The Gram-negative bacterium Xanthomonas campestris pv. vesicatoria (Xcv causes bacterial spot disease of pepper and tomato by direct translocation of type III effector proteins into the plant cell cytosol. Once in the plant cell the effectors interfere with host cell processes and manipulate the plant transcriptome. Quantitative RT-PCR (qRT-PCR is usually the method of choice to analyze transcriptional changes of selected plant genes. Reliable results depend, however, on measuring stably expressed reference genes that serve as internal normalization controls. We identified the most stably expressed tomato genes based on microarray analyses of Xcv-infected tomato leaves and evaluated the reliability of 11 genes for qRT-PCR studies in comparison to four traditionally employed reference genes. Three different statistical algorithms, geNorm, NormFinder and BestKeeper, concordantly determined the superiority of the newly identified reference genes. The most suitable reference genes encode proteins with homology to PHD finger family proteins and the U6 snRNA-associated protein LSm7. In addition, we identified pepper orthologs and validated several genes as reliable normalization controls for qRT-PCR analysis of Xcv-infected pepper plants. The newly identified reference genes will be beneficial for future qRT-PCR studies of the Xcv-tomato and Xcv-pepper pathosystems, as well as for the identification of suitable normalization controls for qRT-PCR studies of other plant-pathogen interactions, especially, if related plant species are used in combination with bacterial pathogens.

  16. Assessment of HER-2 gene overexpression in Isfahan province breast cancer patients using Real Time RT-PCR and immunohistochemistry.

    Science.gov (United States)

    Tabatabaeian, Hosein; Hojati, Zohreh

    2013-11-15

    Overexpression of proto-oncogene HER-2 is one of the main molecular markers of breast cancer involved in prognosis and diagnosis and also in trastuzumab therapy. Thus, a request for the evaluation of HER-2 status in breast cancer has been increasing. The aim of our study was assessment of HER-2 overexpression in malignant and benign breast cancer specimens by Real Time RT-PCR technique and comparison of its results with IHC outcomes. Twenty benign and sixty malignant breast cancers in addition to fifteen normal breast tissue specimens were analyzed by Real Time RT-PCR method. Fresh tissue samples were disrupted by mortar and pestle. A syringe and a needle were used for complete homogenization of the tissues. The RNA was then isolated from the samples and converted to cDNA. A standard curve was initially plotted using BioEasy SYBR Green I and then all 95 specimens were studied by Real Time RT-PCR using 2(-ΔΔCt) method. 23.3% of 60 malignant specimens showed HER-2 overexpression, while all of the benign samples represented the normal expression level of HER-2 gene. The concordance rate between the results of Real Time RT-PCR and IHC was 86.6%. Real Time RT-PCR method is an almost reliable technique and at least can be used as a complementary method for confirming IHC results. This is emanated from relatively high rate of concordance between outcomes of IHC test, as a routine method of detecting the HER-2 gene expression status, and Real Time RT-PCR technique. © 2013 Elsevier B.V. All rights reserved.

  17. Comparison of protocols for the analysis of type 1 porcine reproductive and respiratory syndrome virus by RT-PCR using oral fluids.

    Science.gov (United States)

    Gibert, Elisa; Martín-Valls, Gerard; Mateu, Enric

    2017-05-01

    The detection of porcine reproductive and respiratory syndrome virus (PRRSV) in oral fluids (OF) by quantitative real-time polymerase chain reaction (qRT-PCR) is gaining increasing popularity. However, the different steps leading to a result have not been extensively evaluated. The aim of the present study was to examine the effect on the performance of qRT-PCR with different sampling materials, conditions of storage of the OF, the need for centrifuging OF, as well as to compare RNA extraction methods and PCR mixes. For the assays, pen-based oral fluids were used, which were pooled and spiked in a serial dilution (up to genotype 10(0) TCID50/mL) of type 1 PRRSV isolate 3267. Centrifugation at 15,000g for 15min resulted in an increase in sensitivity (1-2 PCR cycles) that was significant (PPCR Kit PCR mix reagents were more sensitive for the detection of PRRSV using a purified plasmid as standard, but LSI VetMAX PRRSV EU/NA PRRSV reagents resulted in a slightly better sensitivity with OF (pPCR. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. The diagnostic significance of the detection of cytokeratin 19 mRNA by quantitative RT-PCR in benign and malignant pleural effusions

    Institute of Scientific and Technical Information of China (English)

    徐峰; 陈杰; 沈华浩; 王选锭; 单江

    2004-01-01

    Objective: To evaluate the diagnostic significance of detecting cytokeratin 19 (CK19) mRNA by quantitative reverse transcription polymerase chain reaction (RT-PCR) in benign and malignant pleural effusions. Methods: CK19mRNA was examined by quantitative RT-PCR and CK19 was detected by Enzyme-linked immunoadsorbent assay (ELISA)in 32 patients with malignant pleural effusions and 35 patients with benign pleural effusions. Results: On the threshold of 200 copies/μl, the positive rate of CK1 9 mRNA in patients with malignant pleural effusions was 62.5%. The positive rates of CK19 mRNA and CK19 in the malignant pleural effusions were significantly higher than those in the benign group (P<0.01). Furthermore, the positive rate of CK19 mRNA was higher than that of CK19 in the malignant group (P<0.05).Conclusion: Detection of CK19 mRNA can be a promising diagnostic marker in differential diagnosis of benign and malignant pleural effusions.

  19. Association of cucumovirus and potyvirus with betelvine (Piper betle L.) as evidenced by ELISA and RT-PCR.

    Science.gov (United States)

    Raj, S K; Srivastava, A; Chowdhury, M R; Johri, J K

    2003-03-01

    An attempt was made to detect various viruses of Piper betle grown at Mahoba and Banthara in India. DAC-ELISA and RT-PCR tests were performed in leaf sap samples of betelvine for detection of a cucumovirus (Cucumber mosaic virus) and potyvirus (Bean yellow mosaic virus) using specific antibodies and universal primers of respective viruses. DAC-ELISA could detect only CMV. However, RT-PCR detected both cucumovirus and potyvirus infection in betelvine samples. Association of CMV with betelvine was observed for the first time in the present study.

  20. Identification and validation of reference genes for quantitative RT-PCR normalization in wheat

    Directory of Open Access Journals (Sweden)

    Porceddu Enrico

    2009-02-01

    Full Text Available Abstract Background Usually the reference genes used in gene expression analysis have been chosen for their known or suspected housekeeping roles, however the variation observed in most of them hinders their effective use. The assessed lack of validated reference genes emphasizes the importance of a systematic study for their identification. For selecting candidate reference genes we have developed a simple in silico method based on the data publicly available in the wheat databases Unigene and TIGR. Results The expression stability of 32 genes was assessed by qRT-PCR using a set of cDNAs from 24 different plant samples, which included different tissues, developmental stages and temperature stresses. The selected sequences included 12 well-known HKGs representing different functional classes and 20 genes novel with reference to the normalization issue. The expression stability of the 32 candidate genes was tested by the computer programs geNorm and NormFinder using five different data-sets. Some discrepancies were detected in the ranking of the candidate reference genes, but there was substantial agreement between the groups of genes with the most and least stable expression. Three new identified reference genes appear more effective than the well-known and frequently used HKGs to normalize gene expression in wheat. Finally, the expression study of a gene encoding a PDI-like protein showed that its correct evaluation relies on the adoption of suitable normalization genes and can be negatively affected by the use of traditional HKGs with unstable expression, such as actin and α-tubulin. Conclusion The present research represents the first wide screening aimed to the identification of reference genes and of the corresponding primer pairs specifically designed for gene expression studies in wheat, in particular for qRT-PCR analyses. Several of the new identified reference genes outperformed the traditional HKGs in terms of expression stability

  1. Meloidogyne javanica Chorismate Mutase Transcript Expression Profile Using Real-Time Quantitative RT-PCR.

    Science.gov (United States)

    Painter, Janet E; Lambert, Kris N

    2003-03-01

    A developmental expression profile of the Meloidodgyne javanica esophageal gland gene chorismate mutase-1 (Mj-cm-1) could suggest when in the lifecycle of the nematode the Mj-cm-1 product is functional. This study used real-time quantitative RT-PCR to examine the variation in Mj-cm-1 transcript levels over six timepoints in the nematode lifecycle: egg, infective second-stage juveniles (Inf-J2), 2-day post-inoculation (pi), 7-day pi, 14-day pi, and adult. The Mj-cm-1 mRNA levels peaked at 2-day pi, about 100-fold above levels expressed at the egg and Inf-J2 stages. Some expression of Mj-cm-1 remained during the 7-day pi, 14-day pi, and adult stages. High transcript levels of the beta-actin control gene M. javanica Beta-actin-1 (Mj-ba-1) demonstrated the presence of cDNA at all timepoints. The peak in Mj-cm-1 transcript expression at 2-day pi as well as the previously shown esophageal gland localization of Mj-cm-1 mRNA suggest that the product of this gene may be involved early in the establishment of parasitism.

  2. Quantitative RT-PCR Gene Evaluation and RNA Interference in the Brown Marmorated Stink Bug.

    Directory of Open Access Journals (Sweden)

    Raman Bansal

    Full Text Available The brown marmorated stink bug (Halyomorpha halys has emerged as one of the most important invasive insect pests in the United States. Functional genomics in H. halys remains unexplored as molecular resources in this insect have recently been developed. To facilitate functional genomics research, we evaluated ten common insect housekeeping genes (RPS26, EF1A, FAU, UBE4A, ARL2, ARP8, GUS, TBP, TIF6 and RPL9 for their stability across various treatments in H. halys. Our treatments included two biotic factors (tissues and developmental stages and two stress treatments (RNAi injection and starvation. Reference gene stability was determined using three software algorithms (geNorm, NormFinder, BestKeeper and a web-based tool (RefFinder. The qRT-PCR results indicated ARP8 and UBE4A exhibit the most stable expression across tissues and developmental stages, ARL2 and FAU for dsRNA treatment and TBP and UBE4A for starvation treatment. Following the dsRNA treatment, all genes except GUS showed relatively stable expression. To demonstrate the utility of validated reference genes in accurate gene expression analysis and to explore gene silencing in H. halys, we performed RNAi by administering dsRNA of target gene (catalase through microinjection. A successful RNAi response with over 90% reduction in expression of target gene was observed.

  3. A simple DNA recombination screening method by RT-PCR as an alternative to Southern blot.

    Science.gov (United States)

    Albers, Eliene; Sbroggiò, Mauro; Martin-Gonzalez, Javier; Avram, Alexandra; Munk, Stephanie; Lopez-Contreras, Andres J

    2017-01-19

    The generation of genetically engineered mouse models (GEMMs), including knock-out (KO) and knock-in (KI) models, often requires genomic screening of many mouse ES cell (mESC) clones by Southern blot. The use of large targeting constructs facilitates the recombination of exogenous DNA in a specific genomic locus, but limits the detection of its correct genomic integration by standard PCR methods. Genomic Long Range PCR (LR-PCR), using primers adjacent to the homology arms, has been used as an alternative to radioactive-based Southern blot screenings. However, LR-PCRs are often difficult and render many false positive and false negative results. Here, we propose an alternative screening method based on the detection of a genetic modification at the mRNA level, which we successfully optimized in two mouse models. This screening method consists of a reverse-transcription PCR (RT-PCR) using primers that match exons flanking the targeting construct. The detection of the expected modification in this PCR product confirms the integration at the correct genomic location and shows that the mutant mRNA is expressed. This is a simple and sensitive strategy to screen locus-specific recombination of targeting constructs which can also be useful to screen KO and KI mutant mice or cell lines including those generated by CRISPR/Cas9.

  4. Identification of genes for normalization of real-time RT-PCR data in breast carcinomas

    DEFF Research Database (Denmark)

    Lyng, Maria B; Laenkholm, Anne-Vibeke; Pallisgaard, Niels

    2008-01-01

    of factors influencing the tissue, and that are stably expressed within the experimental milieu. In this study, we identified genes for normalization of RT-qPCR data for invasive breast cancer (IBC), with special emphasis on estrogen receptor positive (ER+) IBC, but also examined their applicability to ER......- IBC, normal breast tissue and breast cancer cell lines. METHODS: The reference genes investigated by qRT-PCR were RPLP0, TBP, PUM1, ACTB, GUS-B, ABL1, GAPDH and B2M. Biopsies of 18 surgically-excised tissue specimens (11 ER+ IBCs, 4 ER- IBCs, 3 normal breast tissues) and 3 ER+ cell lines were examined...... of human tissue samples (ER+ and ER- IBC and normal breast tissue) and for the invasive cancer samples (ER+ and ER- IBC) by GeNorm, where NormFinder consistently identified PUM1 at the single best gene for all sample combinations. CONCLUSION: The reference genes of choice when performing RT-qPCR on normal...

  5. The RT-PCR Analysis of Lignocellulytic Biodegradation-related Gene Expression of Phanerochaete chrysosporium

    Institute of Scientific and Technical Information of China (English)

    Jiang Mingfeng(江明锋); Zhang Yizheng

    2004-01-01

    Expression of lignocellulytic biodegradation-related genes of Phanerochaete chrysosporium grown in 10 kinds of defined media for 5 days and on fir wood chip for 2 to 8 weeks is analyzed by using the RT-PCR method. The result shows that an individual gene of lip gene family responds differently to different nutrient factors. The expression of lipD gene can be promoted by molecular O2 but suppressed by Mn2+. The influence of nitorgen is not the controlling factor for lipD gene expression. No clear relationship is found between nutrient factors and the expression of lipA gene which may be regulated by several nutrient factors through a complex system. Mnp3 gene is not strongly regulated by Mn2+ and other nutrient factors. It can be expressed in different media. CBHI gene family can not be expressed in the presence of glucose as the sole carbon source. Glx expression is regulated by Mn2+ and molecular O2, and depressed when Mn2+ concerntration goes up to 300 mg/L. The transcription patterns of lip gene family grown on fir wood chip are shown to be markedly different from those patterns in defined media. The expression of single lip gene changes with colonized time. No difference is observed between the expression pattern of mnp, cbh, glx gene in defined media and fir wood chips.

  6. Expression patterns of Doppel gene in golden hamster: quantification using real-time RT-PCR.

    Science.gov (United States)

    Li, Y R; Li, Q; Yang, J M; Zhou, X M; Yin, X M; Zhao, D M

    2008-08-01

    Doppel (prion-like protein, Dpl) may act as a useful molecular marker in tumor diagnosis and in tumor grade definition, as over-expression of Dpl protein has been found in tumors with different histologic origin. Accordingly, the quantitative analysis of the expression of Dpl in different tissues is essential for understanding its role in tumor progression and cancer diagnostic. Herein we report Dpl mRNA quantification in golden hamster by calibrated highly sensitive externally standardized real-time RT-PCR with LightCycler instrument. Total RNA was isolated from nine different organs of golden hamster in different stages of development: from neonatal to adult golden hamster. Highest level of Dpl mRNA was detected in the testis, and lower levels of Dpl mRNA were detected in the following tissues: spleen, heart, bone marrow, skeletal muscles and neocortex (only in neonatal hamster). The expression of Dpl was not detected in kidney, liver and lung. This is the first study to report the expression of Dpl in bone marrow of murine and the difference of expression levels of Dpl in testis between adult and neonatal hamsters.

  7. Detecting PML-RARα transcript in acute promyelocytic leukemia using real-time quantitative RT-PCR

    Institute of Scientific and Technical Information of China (English)

    ZHU Hong-hu; LIU Yan-rong; QIN Ya-zhen; JIANG Bin; SHAN Fu-xiang; WU Shu-lan; YANG Ping-di; ZHAO Jie; LU Dao-pei

    2007-01-01

    Background Real-time quantitative RT-PCR (RQ-PCR) assay has become a vital tool to monitor residual disease of leukemia. However, the complexity and standardization of RQ-PCR should never be overlooked and the results should be interpreted cautiously in clinical conditions. We aimed to assess the methodology of RQ-PCR and its clinical applications in monitoring molecular kinetics of 36 newly diagnosed cases of acute promyelocytic leukemia patients with t (15; 17) from October 2004 to December 2005.Methods All the TaqMan probe-based RQ-PCR reactions and analysis were performed on an ABI-PRISM 7500platform. The quantitation of PML-RARα transcripts was represented by the normalized quotient, that is, PML-RARα transcript copies divided by ABL transcript copies. According to induction therapy, the patients were classed into two groups: group 1 (n=23), three-drug combination including arsenics, all-trans retinoic acid and mitoxantrone; and group 2 (n=13), two-drug combination from all-trans retinoic acid, arsenics and mitoxantrone.Results The sensitivity of RQ-PCR was 1 per 105 cells and 5 copies of the PML-RARα transcript could be reproducibly detected. No false positive results occurred in 40 non-acute promyelocytic leukemia samples. Optimal amplification efficiency could be attained, which was determined by the slope of the standard curves (slope: -3.2 - -3.7). The inter-assay and intra-assay variation coefficients of the method were 1.01% and 0.56% respectively. Although the time to attain hematological complete remission was similar in both groups, the time to achieve molecular remission of group 1 was significantly shorter than that of group 2 (61 days vs 75 days, P=0.034). The rate of molecular remission within 70days was higher in group 1 than in group 2 (75.00% vs 38.46%, P=0.036). Compared with pretreatment, median reduction of the PML-RARα transcript before first consolidation therapy differed significantly between group 1 and group 2 (log scale, 3.15 vs 2

  8. One-step fabrication of multifunctional micromotors

    Science.gov (United States)

    Gao, Wenlong; Liu, Mei; Liu, Limei; Zhang, Hui; Dong, Bin; Li, Christopher Y.

    2015-08-01

    Although artificial micromotors have undergone tremendous progress in recent years, their fabrication normally requires complex steps or expensive equipment. In this paper, we report a facile one-step method based on an emulsion solvent evaporation process to fabricate multifunctional micromotors. By simultaneously incorporating various components into an oil-in-water droplet, upon emulsification and solidification, a sphere-shaped, asymmetric, and multifunctional micromotor is formed. Some of the attractive functions of this model micromotor include autonomous movement in high ionic strength solution, remote control, enzymatic disassembly and sustained release. This one-step, versatile fabrication method can be easily scaled up and therefore may have great potential in mass production of multifunctional micromotors for a wide range of practical applications.Although artificial micromotors have undergone tremendous progress in recent years, their fabrication normally requires complex steps or expensive equipment. In this paper, we report a facile one-step method based on an emulsion solvent evaporation process to fabricate multifunctional micromotors. By simultaneously incorporating various components into an oil-in-water droplet, upon emulsification and solidification, a sphere-shaped, asymmetric, and multifunctional micromotor is formed. Some of the attractive functions of this model micromotor include autonomous movement in high ionic strength solution, remote control, enzymatic disassembly and sustained release. This one-step, versatile fabrication method can be easily scaled up and therefore may have great potential in mass production of multifunctional micromotors for a wide range of practical applications. Electronic supplementary information (ESI) available: Videos S1-S4 and Fig. S1-S3. See DOI: 10.1039/c5nr03574k

  9. A review of RT-PCR technologies used in veterinary virology and disease control: sensitive and specific diagnosis of five livestock diseases notifiable to the World Organisation for Animal Health

    OpenAIRE

    Hoffmann, Bernd; Beer, Martin; Reid, Scott M.; Mertens, Peter; Oura, Chris A.L.; van Rijn, Piet A.; Slomka, Marek J.; Banks, Jill; Brown, Ian H; Alexander, Dennis J.; King, Donald P.

    2009-01-01

    Abstract Real-time, reverse transcription polymerase chain reaction (rRT-PCR) has become one of the most widely used methods in the field of molecular diagnostics and research. The potential of this format to provide sensitive, specific and swift detection and quantification of viral RNAs has made it an indispensable tool for state-of-the-art diagnostics of important human and animal viral pathogens. Integration of these assays into automated liquid handling platforms for nucleic a...

  10. Exercise induced stress in horses: Selection of the most stable reference genes for quantitative RT-PCR normalization

    Directory of Open Access Journals (Sweden)

    Silvestrelli Maurizio

    2008-05-01

    Full Text Available Abstract Background Adequate stress response is a critical factor during athlete horses' training and is central to our capacity to obtain better performances while safeguarding animal welfare. In order to investigate the molecular mechanisms underlying this process, several studies have been conducted that take advantage of microarray and quantitative real-time PCR (qRT-PCR technologies to analyse the expression of candidate genes involved in the cellular stress response. Appropriate application of qRT-PCR, however, requires the use of reference genes whose level of expression is not affected by the test, by general physiological conditions or by inter-individual variability. Results The expression of nine potential reference genes was evaluated in lymphocytes of ten endurance horses during strenuous exercise. These genes were tested by qRT-PCR and ranked according to the stability of their expression using three different methods (implemented in geNorm, NormFinder and BestKeeper. Succinate dehydrogenase complex subunit A (SDHA and hypoxanthine phosphoribosyltransferase (HPRT always ranked as the two most stably expressed genes. On the other hand, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, transferrin receptor (TFRC and ribosomal protein L32 (RPL32 were constantly classified as the less reliable controls. Conclusion This study underlines the importance of a careful selection of reference genes for qRT-PCR studies of exercise induced stress in horses. Our results, based on different algorithms and analytical procedures, clearly indicate SDHA and HPRT as the most stable reference genes of our pool.

  11. Detection of EML4-ALK in lung adenocarcinoma using pleural effusion with FISH, IHC, and RT-PCR methods.

    Directory of Open Access Journals (Sweden)

    Leilei Liu

    Full Text Available Anaplastic lymphoma kinase (ALK and echinoderm microtubule-associated protein-like 4 (EML4 gene rearrangements occur in approximately 5% of non-small-cell lung cancers (NSCLC, leading to the overexpression of anaplastic lymphoma kinase and predicting a response to the targeted inhibitor, crizotinib. Malignant pleural effusion occurs in most patients with advanced lung cancer, especially adenocarcinoma, and tissue samples are not always available from these patients. We attempted to clarify the feasibility of detecting the EML4-ALK fusion gene in pleural effusion cells using different methods. We obtained 66 samples of pleural effusion from NSCLC patients. The pleural effusion fluid was centrifuged, and the cellular components obtained were formalin fixed and paraffin embedded. The EML4-ALK fusion gene status was determined with fluorescent in situ hybridization (FISH, reverse transcription-polymerase chain reaction (RT-PCR, and immunohistochemistry (IHC. EML4-ALK was detected in three of 66 patient samples (4.5% with RT-PCR. When the RT-PCR data were used as the standard, one false positive and one false negative samples were identified with IHC; and one false negative sample was identified with FISH. These results suggest that a block of pleural effusion cells can be used to detect the EML4-ALK fusion gene. IHC had good sensitivity, but low specificity. FISH had low sensitivity, but high specificity. RT-PCR is a good candidate method for detecting EML4-ALK in blocks of pleural effusion cells from lung cancer patients.

  12. Effect of specific or random c-DNA priming on sensitivity of tyrosinase nested RT-PCR : Potential clinical relevance

    NARCIS (Netherlands)

    Calogero, A; Hospers, GAP; Timmer-Bosscha, H; Mulder, NH; Schraffordt Koops, H.

    2000-01-01

    The reverse transcriptase polymerase chain reaction (RT-PCR) can be of clinical relevance in identifying malignant melanoma cells in blood or tissues of patients at risk for disseminated melanoma. The diagnostic value of this marker however, is still controversial. The objective of this study was to

  13. Characterization of cytokine expression induced by avian influenza virus infection with real-time RT-PCR

    Science.gov (United States)

    Knowledge of how birds react to infection from avian influenza virus is critical to understanding disease pathogenesis and host response. The use of real-time (R), reverse-transcriptase (RT), PCR to measure innate immunity, including cytokine and interferon gene expression, has become a standard tec...

  14. PrimerSeq:Design and Visualization of RT-PCR Primers for Alternative Splicing Using RNA-seq Data

    Institute of Scientific and Technical Information of China (English)

    Collin Tokheim; Juw Won Park; Yi Xing

    2014-01-01

    The vast majority of multi-exon genes in higher eukaryotes are alternatively spliced and changes in alternative splicing (AS) can impact gene function or cause disease. High-throughput RNA sequencing (RNA-seq) has become a powerful technology for transcriptome-wide analysis of AS, but RT-PCR still remains the gold-standard approach for quantifying and validating exon splicing levels. We have developed PrimerSeq, a user-friendly software for systematic design and visualization of RT-PCR primers using RNA-seq data. PrimerSeq incorporates user-provided tran-scriptome profiles (i.e., RNA-seq data) in the design process, and is particularly useful for large-scale quantitative analysis of AS events discovered from RNA-seq experiments. PrimerSeq features a graphical user interface (GUI) that displays the RNA-seq data juxtaposed with the expected RT-PCR results. To enable primer design and visualization on user-provided RNA-seq data and transcript annotations, we have developed PrimerSeq as a stand-alone software that runs on local computers. PrimerSeq is freely available for Windows and Mac OS X along with source code at http://primerseq.sourceforge.net/. With the growing popularity of RNA-seq for transcriptome stud-ies, we expect PrimerSeq to help bridge the gap between high-throughput RNA-seq discovery of AS events and molecular analysis of candidate events by RT-PCR.

  15. RT-PCR and Northern blot analysis in search for a putative Paramecium beta-adrenergic receptor.

    Science.gov (United States)

    Płatek, A; Wiejak, J; Wyroba, E

    1999-01-01

    RT-PCR and Northern blot analysis were performed in order to search for a putative beta-adrenergic receptor (beta-AR) in Paramecium using several beta2-adrenergic-specific molecular probes. Under strictly defined RT-PCR conditions DNA species of expected molecular size about 360 bp were generated with the primers corresponding to the universal mammalian beta2-AR sequence tagged sites (located within the 4th and the 6th transmembrane regions of the receptor). This RT-PCR product hybridized in Southern blot analysis with the oligonucleotide probe designed to the highly conservative beta2-AR region involved in G-proteins interaction and located within the amplified region. Northern hybridization was performed on Paramecium total RNA and mRNA with human beta2-AR cDNA and two oligonucleotide probes: the first included Phe 290 involved in agonist binding (Strader et al., 1995) and the second was the backward RT-PCR primer. All these probes revealed the presence of about 2 kb mRNA which is consistent with the size of beta2-AR transcripts found in higher eukaryotes.

  16. Validation of putative reference genes for normalization of Q-RT-PCR data from paraffin-embedded lymphoid tissue

    DEFF Research Database (Denmark)

    Green, Tina Marie; de Stricker, Karin; Møller, Michael Boe

    2009-01-01

    , represented by non-neoplastic lymph nodes and diffuse large B-cell lymphomas, by using 2 statistical software applications, geNorm and NormFinder. In addition, we wanted to validate the usefulness of paraffin-embedded samples for Q-RT-PCR studies by investigating gene expressions of relevant target genes...

  17. Measurement of bacterial gene expression in vivo by laser capture microdissection and quantitative real-time RT-PCR

    DEFF Research Database (Denmark)

    Schou, Kirstine Klitgaard; Jensen, Tim Kåre; Angen, Øystein

    2007-01-01

    Due to the relative small number of bacterial pathogens present in an infected host, exploration of pathogen gene expression in vivo is challenging. This study reports the development of a protocol for quantifying bacterial gene expression in vivo in Actinobacillus pleuropneumoniae using laser ca...... capture microdissection and real-time quantitative RT-PCR....

  18. Culture and Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Proven Mycobacterium Tuberculosis Endophthalmitis: A Case Series.

    Science.gov (United States)

    Rishi, Ekta; Rishi, Pukhraj; Therese, K Lily; Ramasubban, Gayathri; Biswas, Jyotirmay; Sharma, Tarun; Bhende, Pramod; Susvar, Pradeep; Agarwal, Mamta; George, Amala Elizabeth; Delhiwala, Kushal; Sharma, Vishal Rajan

    2016-09-06

    To report early confirmation of Mycobacterium tuberculosis (MTB) endophthalmitis by detection of 85B mRNA in vitreous by a reverse transcriptase polymerase chain reaction (RT-PCR) technique. Retrospective, interventional case series of 5 patients with MTB endogenous endophthalmitis. Vitreous aspirate was subjected to Ziehl-Neelsen (ZN) staining, BACTEC MicroMGIT culture, RT-PCR targeting the 85B gene, real-time PCR targeting the IS6110 region, and nested PCR targeting the MPB64 gene and IS6110 region. Correlation between detection of MTB RNA, culture positivity, and ZN staining was studied. Five patients with endophthalmitis with no history of tuberculosis revealed acid-fast bacilli on ZN staining of vitreous. RT-PCR detected 85B RNA within 24 h. Culture for MTB was positive in 3/5 patients after 1 month. None of the eyes recovered any useful vision. RT-PCR can detect viable MTB RNA and provide evidence of active infection much earlier than culture.

  19. Quantitative real-time RT-PCR in sentinel lymph nodes from melanoma patients. Detection of melanocytic mRNA predicts disease-free survival

    DEFF Research Database (Denmark)

    Riber-Hansen, Rikke; Abrahamsen, Helene Nortvig; Sorensen, Boe Sandahl

    2008-01-01

    Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) for specific melanoma markers is more sensitive than histology for detecting cells of melanocytic origin in sentinel lymph nodes (SLNs) in cutaneous melanoma. The clinical significance of a positive qRT-PCR analysis...... that the presence of submicroscopic metastases may influence prognosis, indicating that RT-PCR detection of melanocytic cells in SLNs may be an important diagnostic marker....

  20. Quantitative RT-PCR and its application in botany research%定量RT-PCR及其在植物学研究中的应用

    Institute of Scientific and Technical Information of China (English)

    胡丹丹; 顾金刚; 姜瑞波; 董金皋

    2007-01-01

    定量RT-PCR(Quantitative reverse transcriptase-PCR)是在反转录和定量PCR的基础上发展起来的一种特异性检测基因表达的技术.主要包括相对定量RT-PCR(Relative quantitative RT-PCR)、竞争性定量RT-PCR(Competitive quantitative RT-PCR)、比较定量RT-PCR(Comparative quantitative RT-PCR)和实时定量RT-PCR(Realtime quantitativeRT-PCR)四种.目前定量RT-PCR在植物学研究中的应用越来越广泛,如植物营养学研究、植物发育学研究、植物抗逆机理研究、转基因植物的检测、病原菌的检测、植物与微生物互作机理研究、植物抗病性检测等方面.本文综述了定量RT-PCR的原理及在植物学中的应用.

  1. One-step lowrank wave extrapolation

    KAUST Repository

    Sindi, Ghada Atif

    2014-01-01

    Wavefield extrapolation is at the heart of modeling, imaging, and Full waveform inversion. Spectral methods gained well deserved attention due to their dispersion free solutions and their natural handling of anisotropic media. We propose a scheme a modified one-step lowrank wave extrapolation using Shanks transform in isotropic, and anisotropic media. Specifically, we utilize a velocity gradient term to add to the accuracy of the phase approximation function in the spectral implementation. With the higher accuracy, we can utilize larger time steps and make the extrapolation more efficient. Applications to models with strong inhomogeneity and considerable anisotropy demonstrates the utility of the approach.

  2. Proteomic analysis and qRT-PCR verification of temperature response to Arthrospira (Spirulina platensis.

    Directory of Open Access Journals (Sweden)

    Wang Huili

    Full Text Available Arthrospira (Spirulina platensis (ASP is a representative filamentous, non-N2-fixing cyanobacterium that has great potential to enhance the food supply and possesses several valuable physiological features. ASP tolerates high and low temperatures along with highly alkaline and salty environments, and can strongly resist oxidation and irradiation. Based on genomic sequencing of ASP, we compared the protein expression profiles of this organism under different temperature conditions (15°C, 35°Cand 45°C using 2-DE and peptide mass fingerprinting techniques. A total of 122 proteins having a significant differential expression response to temperature were retrieved. Of the positively expressed proteins, the homologies of 116 ASP proteins were found in Arthrospira (81 proteins in Arthrospira platensis str. Paraca and 35 in Arthrospira maxima CS-328. The other 6 proteins have high homology with other microorganisms. We classified the 122 differentially expressed positive proteins into 14 functions using the COG database, and characterized their respective KEGG metabolism pathways. The results demonstrated that these differentially expressed proteins are mainly involved in post-translational modification (protein turnover, chaperones, energy metabolism (photosynthesis, respiratory electron transport, translation (ribosomal structure and biogenesis and carbohydrate transport and metabolism. Others proteins were related to amino acid transport and metabolism, cell envelope biogenesis, coenzyme metabolism and signal transduction mechanisms. Results implied that these proteins can perform predictable roles in rendering ASP resistance against low and high temperatures. Subsequently, we determined the transcription level of 38 genes in vivo in response to temperature and identified them by qRT-PCR. We found that the 26 differentially expressed proteins, representing 68.4% of the total target genes, maintained consistency between transcription and

  3. APPLICATION OF RT-PCR FOR ACETOBACTER SPECIES DETECTION IN RED WINE

    Directory of Open Access Journals (Sweden)

    Attila Kántor

    2014-02-01

    Full Text Available Acetic acid bacteria play a negative role in wine making because they increase the volatile acidity of wines. They can survive in the various phases of alcoholic fermentation and it is very important to control their presence and ulterior development. The main objective of the present work is to test fast, sensitive and reliable technique such as real-time PCR (rt-PCR and detecting the presence of Acetobacter aceti, Acetobacter pasteurianus, Gluconobacter oxydans, Gluconacetobacter liquefaciens and Gluconacetobacter hansenii in red wine. The aim of our study was the identification of some species of acetic acid bacteria in red wine during the fermentation process using a classical microbiological method. The changes in different groups of microorganisms were monitored in total counts of bacteria, and Acetobacter cells. Microbiological parameters were observed during the current collection and processing of wine in 2012. Samples (Modry Portugal, MP and Frankovka modra, FM were taken during the fermentation process in wine enterprises and were storage with different conditions. The total counts of bacteria ranged from 4.21 in the wine MP at 4°C of storage to 5.81 log CFU.mL-1 in the wine MP at 25°C of storage, but the total counts of bacteria ranged from 4.85 in the wine FM at 4°C of storage to 5.63 log CFU.mL-1 in the wine FM at 25°C of storage. The higher number of Acetobacter cells was found in wine MP at 25°C.

  4. Proteomic analysis and qRT-PCR verification of temperature response to Arthrospira (Spirulina) platensis.

    Science.gov (United States)

    Huili, Wang; Xiaokai, Zhao; Meili, Lin; Dahlgren, Randy A; Wei, Chen; Jaiopeng, Zhou; Chengyang, Xu; Chunlei, Jin; Yi, Xu; Xuedong, Wang; Li, Ding; Qiyu, Bao

    2013-01-01

    Arthrospira (Spirulina) platensis (ASP) is a representative filamentous, non-N2-fixing cyanobacterium that has great potential to enhance the food supply and possesses several valuable physiological features. ASP tolerates high and low temperatures along with highly alkaline and salty environments, and can strongly resist oxidation and irradiation. Based on genomic sequencing of ASP, we compared the protein expression profiles of this organism under different temperature conditions (15°C, 35°Cand 45°C) using 2-DE and peptide mass fingerprinting techniques. A total of 122 proteins having a significant differential expression response to temperature were retrieved. Of the positively expressed proteins, the homologies of 116 ASP proteins were found in Arthrospira (81 proteins in Arthrospira platensis str. Paraca and 35 in Arthrospira maxima CS-328). The other 6 proteins have high homology with other microorganisms. We classified the 122 differentially expressed positive proteins into 14 functions using the COG database, and characterized their respective KEGG metabolism pathways. The results demonstrated that these differentially expressed proteins are mainly involved in post-translational modification (protein turnover, chaperones), energy metabolism (photosynthesis, respiratory electron transport), translation (ribosomal structure and biogenesis) and carbohydrate transport and metabolism. Others proteins were related to amino acid transport and metabolism, cell envelope biogenesis, coenzyme metabolism and signal transduction mechanisms. Results implied that these proteins can perform predictable roles in rendering ASP resistance against low and high temperatures. Subsequently, we determined the transcription level of 38 genes in vivo in response to temperature and identified them by qRT-PCR. We found that the 26 differentially expressed proteins, representing 68.4% of the total target genes, maintained consistency between transcription and translation levels. The

  5. Identification of suitable grapevine reference genes for qRT-PCR derived from heterologous species.

    Science.gov (United States)

    Tashiro, Rebecca M; Philips, Joshua G; Winefield, Christopher S

    2016-02-01

    Identification and validation of suitable reference genes that exhibit robust transcriptional stability across many sample types is an absolute requirement of all qRT-PCR experiments. Often, however, only small numbers of reference genes, validated across limited sample types, are available for non-model species. This points to a clear need to assess and validate a wider range of potential reference genes than is currently available. We therefore looked to test and validate a large number of potential reference genes across a wide range of tissue types and treatments to determine the applicability of these reference genes for use in grapevine and other non-model plant species. Potential reference genes were selected based on stability of gene transcription in the model plant species Arabidopsis or due to their common use in the grapevine community. The selected reference genes were analyzed across two datasets consisting of a range of either 'Sauvignon blanc' or 'Pinot noir' tissues. A total of 11 potential reference genes were screened across the two datasets. Gene stability was analyzed by GeNorm, a widely used Excel application, or an ANOVA-based method developed in red clover. Both analysis methods showed that all 11 potential reference genes are stably expressed in the datasets tested, but the rankings of gene stability differed based on the datasets and analysis method used. Furthermore, the transcript stability of these genes, initially identified in Arabidopsis and now validated in grapevine, suggests applicability across a wide range of non-model plant species in addition to their utility in grapevine.

  6. Proteomic Analysis and qRT-PCR Verification of Temperature Response to Arthrospira (Spirulina) platensis

    Science.gov (United States)

    Huili, Wang; Xiaokai, Zhao; Meili, Lin; Dahlgren, Randy A.; Wei, Chen; Jaiopeng, Zhou; Chengyang, Xu; Chunlei, Jin; Yi, Xu; Xuedong, Wang; Li, Ding; Qiyu, Bao

    2013-01-01

    Arthrospira (Spirulina) platensis (ASP) is a representative filamentous, non-N2-fixing cyanobacterium that has great potential to enhance the food supply and possesses several valuable physiological features. ASP tolerates high and low temperatures along with highly alkaline and salty environments, and can strongly resist oxidation and irradiation. Based on genomic sequencing of ASP, we compared the protein expression profiles of this organism under different temperature conditions (15°C, 35°Cand 45°C) using 2-DE and peptide mass fingerprinting techniques. A total of 122 proteins having a significant differential expression response to temperature were retrieved. Of the positively expressed proteins, the homologies of 116 ASP proteins were found in Arthrospira (81 proteins in Arthrospira platensis str. Paraca and 35 in Arthrospira maxima CS-328). The other 6 proteins have high homology with other microorganisms. We classified the 122 differentially expressed positive proteins into 14 functions using the COG database, and characterized their respective KEGG metabolism pathways. The results demonstrated that these differentially expressed proteins are mainly involved in post-translational modification (protein turnover, chaperones), energy metabolism (photosynthesis, respiratory electron transport), translation (ribosomal structure and biogenesis) and carbohydrate transport and metabolism. Others proteins were related to amino acid transport and metabolism, cell envelope biogenesis, coenzyme metabolism and signal transduction mechanisms. Results implied that these proteins can perform predictable roles in rendering ASP resistance against low and high temperatures. Subsequently, we determined the transcription level of 38 genes in vivo in response to temperature and identified them by qRT-PCR. We found that the 26 differentially expressed proteins, representing 68.4% of the total target genes, maintained consistency between transcription and translation levels. The

  7. Real-time Quantitative RT-PCR for CT9 Level in Human Cancer

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    CT9 is a recently cloned cancer-testis antigen, which is a member of the bromodomain and extraterminal family.Each member of this protein family contains two N-terminal bromodomain motifs. We investigated the distribution of CT9 in different tissues and the possibility for it to be used as a potential therapeutic target in cancer treament. By using the real-time RT-PCR method and 18SrRNA as an internal standard, we analyzed the CT9 expression in several normal human tissues and in the tissues of patients suffering from cancer. The result of this study shows that the highest level of mRNA is only present in testis tissue because the CT9 expression has not been detected in other normal tissues. In 6 of 10 cases of gastric adenocarcinoma, in 3 of 10 cases of esophageal squamous cell carcinoma, in 2 of 9 cases of endometrial carcinoma and only in 1 of 12 cases of brain cancer, the low level expression of CT9 was detected. In none of the 12 cases of cervical squamous cell carcinoma, the expression of CT9 was detected. Since the high level expression of CT9 is only found in the normal testis tissue, but the low expression in cancer tissues, for example tissues of cervical squamous cell carcinoma, brain cancer, endometrial adenocarcinoma, esophageal squamous cell carcinoma, we conclude that CT9 cannot be used as a cancer therapeutic target molecule for cervical squamous cell carcinoma, brain cancer, endometrial adenocarcinoma, esophageal squamous cell carcinoma.

  8. Reference gene validation for quantitative RT-PCR during biotic and abiotic stresses in Vitis vinifera.

    Directory of Open Access Journals (Sweden)

    Alexandre Filipe Borges

    Full Text Available Grapevine is one of the most cultivated fruit crop worldwide with Vitis vinifera being the species with the highest economical importance. Being highly susceptible to fungal pathogens and increasingly affected by environmental factors, it has become an important agricultural research area, where gene expression analysis plays a fundamental role. Quantitative reverse transcription polymerase chain reaction (qRT-PCR is currently amongst the most powerful techniques to perform gene expression studies. Nevertheless, accurate gene expression quantification strongly relies on appropriate reference gene selection for sample normalization. Concerning V. vinifera, limited information still exists as for which genes are the most suitable to be used as reference under particular experimental conditions. In this work, seven candidate genes were investigated for their stability in grapevine samples referring to four distinct stresses (Erysiphe necator, wounding and UV-C irradiation in leaves and Phaeomoniella chlamydospora colonization in wood. The expression stability was evaluated using geNorm, NormFinder and BestKeeper. In all cases, full agreement was not observed for the three methods. To provide comprehensive rankings integrating the three different programs, for each treatment, a consensus ranking was created using a non-weighted unsupervised rank aggregation method. According to the last, the three most suitable reference genes to be used in grapevine leaves, regardless of the stress, are UBC, VAG and PEP. For the P. chlamydospora treatment, EF1, CYP and UBC were the best scoring genes. Acquaintance of the most suitable reference genes to be used in grapevine samples can contribute for accurate gene expression quantification in forthcoming studies.

  9. Reference gene validation for quantitative RT-PCR during biotic and abiotic stresses in Vitis vinifera.

    Science.gov (United States)

    Borges, Alexandre Filipe; Fonseca, Catarina; Ferreira, Ricardo Boavida; Lourenço, Ana Maria; Monteiro, Sara

    2014-01-01

    Grapevine is one of the most cultivated fruit crop worldwide with Vitis vinifera being the species with the highest economical importance. Being highly susceptible to fungal pathogens and increasingly affected by environmental factors, it has become an important agricultural research area, where gene expression analysis plays a fundamental role. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is currently amongst the most powerful techniques to perform gene expression studies. Nevertheless, accurate gene expression quantification strongly relies on appropriate reference gene selection for sample normalization. Concerning V. vinifera, limited information still exists as for which genes are the most suitable to be used as reference under particular experimental conditions. In this work, seven candidate genes were investigated for their stability in grapevine samples referring to four distinct stresses (Erysiphe necator, wounding and UV-C irradiation in leaves and Phaeomoniella chlamydospora colonization in wood). The expression stability was evaluated using geNorm, NormFinder and BestKeeper. In all cases, full agreement was not observed for the three methods. To provide comprehensive rankings integrating the three different programs, for each treatment, a consensus ranking was created using a non-weighted unsupervised rank aggregation method. According to the last, the three most suitable reference genes to be used in grapevine leaves, regardless of the stress, are UBC, VAG and PEP. For the P. chlamydospora treatment, EF1, CYP and UBC were the best scoring genes. Acquaintance of the most suitable reference genes to be used in grapevine samples can contribute for accurate gene expression quantification in forthcoming studies.

  10. Detection and confirmation of PPR virus antigen in sheep and goats by sandwich-ELISA and RT-PCR in Andhra Pradesh, India

    Directory of Open Access Journals (Sweden)

    G. Saritha

    2015-06-01

    Full Text Available Peste des petits ruminants (PPR is a highly contagious disease of domestic and wild small ruminants. Rapid and accurate laboratory assay are essential to enable the implementation of appropriate control strategies to restrict the spread of PPR. The present study was designed to detect the PPR virus (PPRV antigen (N-gene in nasal swabs and tissue samples. A total of 195 samples comprising of 138 nasal swabs from PPR suspected sheep (n=72 and goats (n=66, and 57 tissue samples comprising of lymph nodes from dead sheep (n=39 and goats (n=18 were collected from certain parts of Andhra Pradesh. The samples were subjected to sandwich-ELISA followed by RT-PCR for confirmatory diagnosis. In this study, PPRV could be detected in 27.53% (n=38/138 nasal swabs and 49.12% (n=28/57 tissue samples. Data showed that PPRV infection is widespread in the Andhra Pradesh, India.

  11. Methods for Preparation of MS2 Phage-Like Particles and Their Utilization as Process Control Viruses in RT-PCR and qRT-PCR Detection of RNA Viruses From Food Matrices and Clinical Specimens.

    Science.gov (United States)

    Mikel, P; Vasickova, P; Kralik, P

    2015-02-25

    RNA viruses are pathogenic agents of many serious infectious diseases affecting humans and animals. The detection of pathogenic RNA viruses is based on modern molecular methods, of which the most widely used methods are the reverse transcription polymerase chain reaction (RT-PCR) and the real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). All steps of RT-PCR and qRT-PCR should be strictly controlled to ensure the validity of obtained results. False-negative results may be caused not only by inhibition of RT or/and PCR steps but also by failure of the nucleic acid extraction step, particularly in the case of viral RNA extraction. The control of nucleic acid extraction generally involves the utilization of a non-pathogenic virus (process control virus) of similar structural properties to those of the target virus. Although in clinical samples the use of such process control virus is only recommended, in other kinds of settings such as food matrices its use is necessary. Currently, several different process control viruses are used for these purposes. Process control viruses can also be constructed artificially using technology for production of MS2 phage-like particles, which have many advantages in comparison with other used controls and are especially suited for controlling the detection and quantification of certain types of RNA viruses. The technology for production of MS2 phage-like particles is theoretically well established, uses the knowledge gained from the study of the familiar bacteriophage MS2 and utilizes many different approaches for the construction of the various process control viruses. Nevertheless, the practical use of MS2 phage-like particles in routine diagnostics is relatively uncommon. The current situation with regard to the use of MS2 phage-like particles as process control viruses in detection of RNA viruses and different methods of their construction, purification and use are summarized and discussed in this

  12. Diagnosis of Cetacean morbillivirus: A sensitive one step real time RT fast-PCR method based on SYBR(®) Green.

    Science.gov (United States)

    Sacristán, Carlos; Carballo, Matilde; Muñoz, María Jesús; Bellière, Edwige Nina; Neves, Elena; Nogal, Verónica; Esperón, Fernando

    2015-12-15

    Cetacean morbillivirus (CeMV) (family Paramyxoviridae, genus Morbillivirus) is considered the most pathogenic virus of cetaceans. It was first implicated in the bottlenose dolphin (Tursiops truncatus) mass stranding episode along the Northwestern Atlantic coast in the late 1980s, and in several more recent worldwide epizootics in different Odontoceti species. This study describes a new one step real-time reverse transcription fast polymerase chain reaction (real-time RT-fast PCR) method based on SYBR(®) Green to detect a fragment of the CeMV fusion protein gene. This primer set also works for conventional RT-PCR diagnosis. This method detected and identified all three well-characterized strains of CeMV: porpoise morbillivirus (PMV), dolphin morbillivirus (DMV) and pilot whale morbillivirus (PWMV). Relative sensitivity was measured by comparing the results obtained from 10-fold dilution series of PMV and DMV positive controls and a PWMV field sample, to those obtained by the previously described conventional phosphoprotein gene based RT-PCR method. Both the conventional and real-time RT-PCR methods involving the fusion protein gene were 100- to 1000-fold more sensitive than the previously described conventional RT-PCR method.

  13. Detection of Strawberry mottle virus and Strawberry mild yellow edge virus with multiplex RT-PCR based on internal control%利用内标为基础的多重 RT-PCR 技术检测草莓斑驳病毒和草莓轻型黄边病毒

    Institute of Scientific and Technical Information of China (English)

    王红; 王菁菁; 张科立; 成卓敏

    2014-01-01

    草莓斑驳病毒(SMoV)和草莓轻型黄边病毒(SMYEV)分布较广,危害较重。应用CTAB法提取草莓总RNA,以优质的草莓总RNA为模板,结合扩增内标的检测体系,建立了SMoV和SMYEV的多重RT-PCR检测体系,可以对草莓田间植株和试管苗进行快速稳定的病毒检测。%Strawberry mottle virus(SMoV) and Strawberry mild yellow edge virus(SMYEV), which are found world-wide, can cause serious crop losses .Total RNA for reverse transcriptase-polymerase chain reaction ( RT-PCR) was extracted from strawberry with CTAB method .A multiplex RT-PCR assay was used for simultaneous detecting SMoV and SMYEV based on ndh B gene as the internal control .Both field plants and test-tube plantlets of strawberries were detected effectively .

  14. Use of automated real-time reverse transcription-polymerase chain reaction (RT-PCR) to monitor experimental swine vesicular disease virus infection in pigs

    DEFF Research Database (Denmark)

    Reid, S.M.; Paton, D.J.; Wilsden, G.;

    2004-01-01

    of the template extraction method was required to counteract the effects of RT-PCR inhibitors in faeces. It was concluded that the automated real-time RT-PCR is a useful diagnostic method for SVD in clinically or subclinically affected pigs and contributed to the study of the pathogenesis of SVD in the pigs....

  15. Destruction of Cucumber green mottle mosaic virus by heat treatment and rapid detection of virus inactivation by RT-PCR.

    Science.gov (United States)

    Kim, Sang-Min; Nam, Sang-Hyun; Lee, Jung-Myung; Yim, Kyu-Ock; Kim, Kook-Hyung

    2003-12-31

    Heat treatment is commonly used to control viral contamination of seeds. To study virus inactivation, virus was purified from seeds contaminated with Cucumber green mottle mosaic virus (CGMMV) after various heat treatments. CGMMV particles were observed to be physically disrupted by high temperature. Analysis of viral RNA revealed that the 5' and 3' termini of the genome were protected whereas regions between 2-2.5, 3.2-3.7 and 4-4.8 kb from the 5' terminus were not. Heat inactivation of virus on seeds was confirmed by RT-PCR using primers for a heat-sensitive region. The RT-PCR approach developed here may prove preferable to time- and labor-intensive bioassays for assessing virus heat inactivation.

  16. Validation of reference genes in Penicillium echinulatum to enable gene expression study using real-time quantitative RT-PCR.

    Science.gov (United States)

    Zampieri, Denise; Nora, Luísa C; Basso, Vanessa; Camassola, Marli; Dillon, Aldo J P

    2014-08-01

    Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a methodology that facilitates the quantification of mRNA expression in a given sample. Analysis of relative gene expression by qRT-PCR requires normalization of the data using a reference gene that is expressed at a similar level in all evaluated conditions. Determining an internal control gene is essential for gene expression studies. Gene expression studies in filamentous fungi frequently use the β-actin gene (actb), β-tubulin, and glyceraldehyde-3-phosphate dehydrogenase as reference genes because they are known to have consistent expression levels. Until now, no study has been performed to select an internal control gene for the filamentous fungal species Penicillium echinulatum. The aim of this study was to evaluate and validate internal control genes to enable the study of gene expression in P. echinulatum using qRT-PCR. P. echinulatum strain S1M29 was grown in conditions to either induce (cellulose and sugar cane bagasse) or repress (glucose) gene expression to analyze 23 candidate normalization genes for stable expression. Two software programs, BestKeeper and geNorm, were used to assess the expression of the candidate normalization genes. The results indicate that the actb reference gene is more stably expressed in P. echinulatum. This is the first report in the literature that determines a normalization gene for this fungus. From the results obtained, we recommend the use of the P. echinulatum actb gene as an endogenous control for gene expression studies of cellulases and hemicellulases by qRT-PCR.

  17. Molecular staging of pathologically negative sentinel lymph nodes from melanoma patients using multimarker, quantitative real-time rt-PCR.

    Science.gov (United States)

    Hilari, Josep M; Mangas, Cristina; Xi, Liqiang; Paradelo, Cristina; Ferrándiz, Carlos; Hughes, Steven J; Yueh, Cindy; Altomare, Ivy; Gooding, William E; Godfrey, Tony E

    2009-01-01

    The aim of this study was to evaluate the prognostic potential of quantitative reverse-transcription, polymerase chain reaction (qRT-PCR) in melanoma patients with pathologically negative sentinel lymph nodes (SLN). Our study included 195 node-negative melanoma patients with a Breslow thickness greater than 0.76 mm (n = 158), or less than 0.76 mm but who had Clark level IV-V, microscopic ulceration, or pathological signs of regression (n = 32), and five patients with melanoma of unknown thickness. SLNs were examined by serial-section histopathology. A portion of each SLN was frozen for qRT-PCR analysis using markers Tyrosinase, MART1, SSX2, MAGEA3, PAX3, and GalNAc-T. In addition, two other markers (PLAB and L1CAM) were evaluated for melanoma specificity but not for SLN analysis. Median follow-up was 64 months, during which time there were 15 (7.7%) recurrences. A total of 370 lymph nodes were analyzed by qRT-PCR. No association was found between quantitative expression level of any marker and disease recurrence. Previously published primer designs were tested for PAX3 and GalNAc-T and revealed that alternative PAX3 transcripts are differentially expressed in melanoma and benign lymph nodes. No associations with recurrence were found regardless of the transcripts amplified by different primer sets. PLAB and L1CAM did not appear to differentiate between malignant melanoma and benign melanocytes or lymph nodes in our analysis. We conclude that, in this large cohort of patients, multimarker qRT-PCR analysis of SLNs did not correlate with disease recurrence. Our data support specific PAX3 splice variants but not GalNAc-T, PLAB or L1CAM as possible markers for melanoma metastasis to SLNs.

  18. Two metallothionein gene family members in buckwheat: Expression analysis in flooding stress using Real Time RT-PCR technology

    Directory of Open Access Journals (Sweden)

    Majić Dragana B.

    2008-01-01

    Full Text Available Metallothioneins (MTs are an extensive and diverse family of small cysteine-rich proteins with metal-binding ability that are involved in metal homeostasis and detoxification. Two cDNA clones of the MT3 type, differing in 3’ UTRs, were isolated from the developing buckwheat seed cDNA library. Following sequence analyses, expression profiles during flooding stress were monitored by Real Time RT PCR technology.

  19. Isolation of bovine coronavirus (bcoV) in vero cell line and its confirmation by direct FAT and RT-PCR.

    Science.gov (United States)

    Hansa, A; Rai, R B; Dhama, K; Wani, M Y; Saminathan, M; Ranganath, G J

    2013-11-01

    Bovine Coronavirus (BCoV) is widespread both in dairy and beef cattle throughout the world. The virus is one of the largest RNA virus and has specific tropism for intestinal and pulmonary epithelial cells. It is responsible for huge economic losses by causing winter dysentery in adult dairy cattle and respiratory and intestinal tract infections leading to pneumo-enteritis in young calves. Isolation of BCoV has been reported to be difficult. Studies regarding epidemiology, virus isolation and molecular detection from India are very few. In the present study Vero cell line was used for isolation of the BCoV from Enzyme Linked Immunosorbent Assay (ELISA) positive samples. Direct florescent antibody technique (dFAT) and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to confirm the isolated virus strains at antigenic and genomic levels, respectively. Out of the 15 positive fecal samples, virus from only seven was able to infect vero cell line. Subsequently BCoV got adapted to the vero cell line upto three passages, which was confirmed both at genomic and antigenic levels by dFAT and RT-PCR testing. It can be concluded that vero cell line can be used for isolation of BCoV, however due to the enormous stain diversity of the virus it is possible that many stains can't grow and get adapt in this cell line. Further studies are required for isolation of different viral strains, finding the susceptible cell lines and also to confirm the variations among the BCoV isolates at antigenic/genomic levels.

  20. Early Detection and Serotyping of Dengue Viruses Clinical Isolates Using Reverse Transcription Polymerase Chain Reaction (RT-PCR 2 Primers

    Directory of Open Access Journals (Sweden)

    Abdul Rahman Siregar

    2015-11-01

    Full Text Available Recently several methods for confirming Dengue Virus have been developed involve virus isolation, detection of virus antigen, and nucleic acid using PCR. It has been reported that rapid detection method for confirming DHF by Multiplex RT-PCR had been successfully developed. It was more effective than the other methods with a high sensitivity and specivicity were 100% at the early phase (day 1-3. This study was designed to develop rapid detection and serotyping methods for Dengue Virus using RT-PCR 2 primers (Dcon and preM with annealing temperature was 57oC. The whole blood samples were collected from suspected dengue fever patients that had been confirmed with NS1 kit from R.S. Persahabatan DKI Jakarta and R.S. Prof. Dr. Sardjito DI Yogyakarta during Februari-August 2009. The PCR products showed that in 12 samples, 100 % were postitive with different pattern among the serotypes especially for DEN1 and DEN2, but not for DEN3 and Den4.  This method was also able to confirm the double infection DEN2-DEN3, but not for the other ones because of the unspecific pattern. From the results, it indicated that the 2 primers can be a promising early detection and serotyping method of Dengue Virus which infected the DHF patients. Key words: Dengue Virus, DHF, early detection, serotyping, RT-PCR 2 primers.

  1. Development and application of a SYBR green RT-PCR for first line screening and quantification of porcine sapovirus infection.

    Science.gov (United States)

    Mauroy, Axel; Van der Poel, Wim H M; der Honing, Renate Hakze-Van; Thys, Christine; Thiry, Etienne

    2012-10-17

    Sapoviruses are single stranded positive sense RNA viruses belonging to the family Caliciviridae. The virus is detected in different species including the human and the porcine species as an enteric pathogen causing asymptomatic to symptomatic enteritis. In this study, we report the development of a rapid real time qRT-PCR based on SYBR Green chemistry for the diagnosis of porcine sapovirus infection in swine. The method allows the detection of porcine sapoviruses and the quantification of the genomic copies present in stool samples. During its development, the diagnostic tool showed good correlation compared with the gold standard conventional RT-PCR and was ten-fold more sensitive. When the method was applied to field samples, porcine noroviruses from genogroup 2 genotype 11b were also detected. The method was also applied to swine samples from the Netherlands that were positive for PoSaV infection. Phylogenetic results obtained from the samples showed that PoSaV sequences were genetically related to the currently described genogroup III, to the proposed genogroup VII and also to the MI-QW19 sequence (close to the human SaV sequences). A rapid, sensitive, and reliable diagnosis method was developed for porcine sapovirus diagnosis. It correlated with the gold standard conventional RT-PCR. Specificity was good apart for genogroup 2 genotype 11b porcine noroviruses. As a first line screening diagnosis method, it allows a quicker and easier decision on doubtful samples.

  2. Validation of reference genes for quantitative gene expression studies in Volvox carteri using real-time RT-PCR.

    Science.gov (United States)

    Kianianmomeni, Arash; Hallmann, Armin

    2013-12-01

    Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) is a sensitive technique for analysis of gene expression under a wide diversity of biological conditions. However, the identification of suitable reference genes is a critical factor for analysis of gene expression data. To determine potential reference genes for normalization of qRT-PCR data in the green alga Volvox carteri, the transcript levels of ten candidate reference genes were measured by qRT-PCR in three experimental sample pools containing different developmental stages, cell types and stress treatments. The expression stability of the candidate reference genes was then calculated using the algorithms geNorm, NormFinder and BestKeeper. The genes for 18S ribosomal RNA (18S) and eukaryotic translation elongation factor 1α2 (eef1) turned out to have the most stable expression levels among the samples both from different developmental stages and different stress treatments. The genes for the ribosomal protein L23 (rpl23) and the TATA-box binding protein (tbpA) showed equivalent transcript levels in the comparison of different cell types, and therefore, can be used as reference genes for cell-type specific gene expression analysis. Our results indicate that more than one reference gene is required for accurate normalization of qRT-PCRs in V. carteri. The reference genes in our study show a much better performance than the housekeeping genes used as a reference in previous studies.

  3. Development and application of a SYBR green RT-PCR for first line screening and quantification of porcine sapovirus infection

    Directory of Open Access Journals (Sweden)

    Mauroy Axel

    2012-10-01

    Full Text Available Abstract Background Sapoviruses are single stranded positive sense RNA viruses belonging to the family Caliciviridae. The virus is detected in different species including the human and the porcine species as an enteric pathogen causing asymptomatic to symptomatic enteritis. In this study, we report the development of a rapid real time qRT-PCR based on SYBR Green chemistry for the diagnosis of porcine sapovirus infection in swine. Results The method allows the detection of porcine sapoviruses and the quantification of the genomic copies present in stool samples. During its development, the diagnostic tool showed good correlation compared with the gold standard conventional RT-PCR and was ten-fold more sensitive. When the method was applied to field samples, porcine noroviruses from genogroup 2 genotype 11b were also detected. The method was also applied to swine samples from the Netherlands that were positive for PoSaV infection. Phylogenetic results obtained from the samples showed that PoSaV sequences were genetically related to the currently described genogroup III, to the proposed genogroup VII and also to the MI-QW19 sequence (close to the human SaV sequences. Conclusions A rapid, sensitive, and reliable diagnosis method was developed for porcine sapovirus diagnosis. It correlated with the gold standard conventional RT-PCR. Specificity was good apart for genogroup 2 genotype 11b porcine noroviruses. As a first line screening diagnosis method, it allows a quicker and easier decision on doubtful samples.

  4. Identification of reliable reference genes for qRT-PCR studies of the developing mouse mammary gland

    Science.gov (United States)

    van de Moosdijk, Anoeska Agatha Alida; van Amerongen, Renée

    2016-01-01

    Cell growth and differentiation are often driven by subtle changes in gene expression. Many challenges still exist in detecting these changes, particularly in the context of a complex, developing tissue. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) allows relatively high-throughput evaluation of multiple genes and developmental time points. Proper quantification of gene expression levels by qRT-PCR requires normalization to one or more reference genes. Traditionally, these genes have been selected based on their presumed “housekeeping” function, with the implicit assumption that they are stably expressed over the entire experimental set. However, this is rarely tested empirically. Here we describe the identification of novel reference genes for the mouse mammary gland based on their stable expression in published microarray datasets. We compared eight novel candidate reference genes (Arpc3, Clock, Ctbp1, Phf7, Prdx1, Sugp2, Taf11 and Usp7) to eight traditional ones (18S, Actb, Gapdh, Hmbs, Hprt, Rpl13a, Sdha and Tbp) and analysed all genes for stable expression in the mouse mammary gland from pre-puberty to adulthood using four different algorithms (GeNorm, DeltaCt, BestKeeper and NormFinder). Prdx1, Phf7 and Ctbp1 were validated as novel and reliable, tissue-specific reference genes that outperform traditional reference genes in qRT-PCR studies of postnatal mammary gland development. PMID:27752147

  5. Molecular Characterization and Clinical Impact of TMPRSS2-ERG Rearrangement on Prostate Cancer: Comparison between FISH and RT-PCR

    Directory of Open Access Journals (Sweden)

    A. Fernández-Serra

    2013-01-01

    Full Text Available Prostate cancer (PCa is a very heterogeneous disease, and there are constraints in its current diagnosis. Serum PSA levels, digital rectal examination (DRE, and histopathologic analysis often drive to overdiagnosis and overtreatment. Since 2005, the presence of the genetic rearrangement between transmembrane-serine protease gene (TMPRSS2 and the erythroblast transformation-specific (ETS member ERG (v-ets erythroblastosis virus E26 oncogene homolog avian has been demonstrated in almost half of PCa cases. Both FISH and RT-PCR are useful tools for detecting these rearrangements, but very few comparatives between both techniques have been published. In this study, we included FFPE tumors from 294 PCa patients treated with radical prostatectomy with more than 5 years of followup. We constructed a total of 20 tissue microarrays in order to perform break-apart and tricolor probe FISH approaches that were compared with RT-PCR, showing a concordance of 80.6% (P<0.001. The presence of TMPRSS2-ERG rearrangement was observed in 56.6% of cases. No association between TMPRSS2-ERG status and clinicopathological parameters nor biochemical progression and clinical progression free survival was found. In conclusion, this study demonstrates that both FISH and RT-PCR are useful tools in the assessment of the TMPRSS2-ERG fusion gene status in PCa patients and that this genetic feature per se lacks prognostic value.

  6. Detection of GLV, OYDV and LYSV in potato onion (Allium cepa L.,Aggregatum group) by RT-PCR

    Institute of Scientific and Technical Information of China (English)

    XU Qijiang; CHEN Dian; TONG Youli; LI Yuhua

    2007-01-01

    Three pairs of primers were designed and synthesized from nucleotide sequences of garlic latent virus (GLV), onion yellow dwarf virus (OYDV), and leek yellow stripe virus (LYSV) by using PCR primer design software. The expected fragments about 170 bp,287 bp, and 191 bp were amplified by RT-PCR for GLV, OYDV, and LYSV, respectively in disease-infected plants of potato onion(Allium cepa L., Aggregatum group), but such fragments were not obtained from healthy-looking plants and virus-free seedlings of shoot-tips. The amplified products ofGLV, OYDV and LYSV were cloned into pGEM-T vectors, and transformed into Escherichia coli.JM109. The recombinant plasmids were obtained and sequenced. The nucleotide sequences were compared with corresponding viral nucleotide sequences reported in GenBank by performing a NCBI BLAST. The analysis showed that their homology attained 75% toplants was diluted to a series of 10-1 to 10-5 and the detection sensitivity of RT-PCR was 10-4 (about 4 ng). Thus, a method of identification and detection by RT-PCR of GLV, OYDV, and SLYV was established.

  7. Rapid detection of peste des petits ruminants virus by a reverse transcription loop-mediated isothermal amplification assay.

    Science.gov (United States)

    Li, Lin; Bao, Jingyue; Wu, Xiaodong; Wang, Zhiliang; Wang, Junwei; Gong, Mingxia; Liu, Chunju; Li, Jinming

    2010-12-01

    Peste des petits ruminants virus (PPRV) is the causative agent of peste des petits ruminants (PPR), an economically important viral disease of small ruminants. In this report, a one-step, single-tube, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of PPRV. A set of six LAMP primers were designed based on the matrix gene sequence of PPRV to amplify the target RNA by incubation at 63°C for 60min with Bst DNA polymerase and reverse transcriptase. The amplified products could be observed by the naked eye. The specificity of the RT-LAMP assay was validated by amplifying eight strains of PPRV isolated in different geographical areas. No cross-reactivity with other related viruses, including rinderpest virus, canine distemper virus and measles virus, was detected. The sensitivity of the assay was similar to that of real-time reverse transcription polymerase chain reaction (RT-PCR) and 10-fold higher than that of conventional RT-PCR. Twenty clinical samples were evaluated by the RT-LAMP assay, and the results were consistent with those of real-time RT-PCR. As a simple, rapid and accurate detection method, this RT-LAMP assay has important potential applications in the clinical diagnosis of PPR and the surveillance of PPRV. Copyright © 2010 Elsevier B.V. All rights reserved.

  8. Rapid detection of foot-and-mouth disease virus, influenza A virus and classical swine fever virus by high-speed real-time RT-PCR.

    Science.gov (United States)

    Wernike, Kerstin; Beer, Martin; Hoffmann, Bernd

    2013-10-01

    High sensitivity, minor risk of cross-contamination and in particular the rapid reaction time make quantitative real-time polymerase chain reaction (qPCR) assays well suited for outbreak investigations as well as for monitoring epidemics of pathogens. In this study qPCR assays for three highly contagious animal diseases, namely foot-and-mouth-disease (FMD), influenza A (IA) and classical swine fever (CSF) have been developed. Furthermore, an amplification control targeting 18S ribosomal RNA was included. Each assay was validated with samples from infected animals using three different standard qPCR-machines in two thermal profiles: one standard and one high-speed approach, respectively. The high-speed PCR assays allowed the reliable diagnosis of FMD, influenza A and CSF in less than 28 min with an analytical sensitivity of at least 200 genome copies/μl in every case, with slight differences regarding reaction time and sensitivity for the individual PCR-cycler instruments. Therefore, the newly established rapid RT-PCR systems will be a valuable method for the monitoring and control of these three important viruses and will be a robust option for the development of novel molecular pen-side tests. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

  9. A one step real time PCR method for the quantification of hepatitis delta virus RNA using an external armored RNA standard and intrinsic internal control.

    Science.gov (United States)

    Karataylı, Ersin; Altunoğlu, Yasemin Çelik; Karataylı, Senem Ceren; Alagöz, S Gökçe K; Cınar, Kubilay; Yalçın, Kendal; Idilman, Ramazan; Yurdaydın, Cihan; Bozdayı, A Mithat

    2014-05-01

    Hepatitis delta virus (HDV) RNA viral load measurement is critical in diagnosis and monitoring the response to antiviral treatment. Our aim is to design a real time PCR method for accurate quantitation of HDV RNA in clinical specimens using an armored RNA as external standard, and an intrinsic internal control. A plasmid bearing delta antigen region of genotype I HDV genome was used to develop an armored RNA. Serial dilutions of the armored HDV RNA standard with 10(12)copy/mL were used as standards for quantitation. A primer-probe set derived from HDAg region was used in one step EZ RT PCR kit chemistry which uses rTth enzyme allowing reverse transcription and polymerization in the same tube. The kit also uses the advantage of uracil-N-glycosylase (UNG) enzyme treatment to prevent PCR contamination. The established assay has a dynamic range of 10(2)-10(11)copy/mL with a PCR efficiency of 96.9%. Detection limit was 858±32copy/mL with 95% confidence interval. Intra- and inter-assay variabilities were low for high, medium and low levels of viremia. Incorporation of freely circulating GAPDH in serum into the assay as an intrinsic internal control prevented false negative results and failures in PCR amplifications due to inhibitors, inefficient extraction procedures or enzymatic reactions. In conclusion, this study defines a novel assay for sensitive and reliable quantification of HDV RNA using an armored HDV RNA as a standard and GAPDH in plasma or serum as an intrinsic internal control in a single tube. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Evaluation of ALK gene rearrangement in central nervous system metastases of non-small-cell lung cancer using two-step RT-PCR technique.

    Science.gov (United States)

    Nicoś, M; Krawczyk, P; Wojas-Krawczyk, K; Bożyk, A; Jarosz, B; Sawicki, M; Trojanowski, T; Milanowski, J

    2017-05-22

    RT-PCR technique has showed a promising value as pre-screening method for detection of mRNA containing abnormal ALK sequences, but its sensitivity and specificity is still discussable. Previously, we determined the incidence of ALK rearrangement in CNS metastases of NSCLC using IHC and FISH methods. We evaluated ALK gene rearrangement using two-step RT-PCR method with EML4-ALK Fusion Gene Detection Kit (Entrogen, USA). The studied group included 145 patients (45 females, 100 males) with CNS metastases of NSCLC and was heterogeneous in terms of histology and smoking status. 21% of CNS metastases of NSCLC (30/145) showed presence of mRNA containing abnormal ALK sequences. FISH and IHC tests confirmed the presence of ALK gene rearrangement and expression of ALK abnormal protein in seven patients with positive result of RT-PCR analysis (4.8% of all patients, 20% of RT-PCR positive patients). RT-PCR method compared to FISH analysis achieved 100% of sensitivity and only 82.7% of specificity. IHC method compared to FISH method indicated 100% of sensitivity and 97.8% of specificity. In comparison to IHC, RT-PCR showed identical sensitivity with high number of false positive results. Utility of RT-PCR technique in screening of ALK abnormalities and in qualification patients for molecularly targeted therapies needs further validation.

  11. Comparative Study on Real-time PCR and RT-PCR Testing Methods for Detecting human Metapneumo Virus%Real-time PCR与RT-PCR检测儿童人偏肺病毒

    Institute of Scientific and Technical Information of China (English)

    郭鑫; 崔玉霞; 诸葛姝芮; 刘兴梅

    2015-01-01

    目的:比较荧光定量PCR( real-time PCR)与普通反转录酶-聚合酶链锁反应( RT-PCR)对人偏肺病毒( hMPV)的检测价值。方法:Real-time PCR和RT-PCR同时对500例急性下呼吸道感染( ALRTI)患儿鼻咽部分泌物进行hMPV检测,分析两种方法的特异性和灵敏性。结果:Real-time PCR和RT-PCR的检出率分别为16%及9.8%,RT-PCR与Real-time PCR比较,灵敏度为31.3%(25/80),特异度为94.3%(396/420)。结论:Real-time PCR检测hMPV敏感性高于RT-PCR,是临床检测儿童鼻咽部分泌物hMPV感染的有效的方法。%Objective:To investigate the effect of real-time quantitative PCR and RT-PCR in detec-tion of human metapneumo virus. Methods:Real-time PCR and RT-PCR are adopted to test hMPV on throat exudate of 500 acute lower respiratory tract infection( ALRTI)child patients,analyzing the spe-cificity and sensitivity of both assays. Results:Comparing Real-time PCR and RT-PCR,the detection rates were 16 % to 9. 8%. Comparing Real-time PCR and RT-PCR,the sensitivity and specificity were 31. 3 %(25/80)and 94. 3%(396/420)respectively. Conclusion:The sensitivity of Real-time PCR is significantly higher than RT-PCR in detection of hMPV,which provid an effective method for detection of hMPV.

  12. Penerapan Metode Diagnosis Cepat Virus Avian Influenza H5N1 dengan Metode Single Step Multiplex RT-PCR

    Directory of Open Access Journals (Sweden)

    Aris Haryanto

    2010-12-01

    Full Text Available Avian influenza (AI virus is a segmented single stranded (ss RNA virus with negative polarity andbelong to the Orthomyxoviridae family. Diagnose of AI virus can be performed using conventional methodsbut it has low sensitivity and specificity. The objective of the research was to apply rapid, precise, andaccurate diagnostic method for AI virus and also to determine its type and subtype based on the SingleStep Multiplex Reverse Transcriptase-Polymerase Chain Reaction targeting M, H5, and N1 genes. In thismethod M, H5 and NI genes were simultaneously amplified in one PCR tube. The steps of this researchconsist of collecting viral RNAs from 10 different AI samples originated from Maros Disease InvestigationCenter during 2007. DNA Amplification was conducted by Simplex RT-PCR using M primer set. Then, bysingle step multiplex RT-PCR were conducted simultaneously using M, H5 and N1 primers set. The RTPCRproducts were then separated on 1.5% agarose gel, stained by ethidum bromide and visualized underUV transilluminator. Results showed that 8 of 10 RNA virus samples could be amplified by Simplex RTPCRfor M gene which generating a DNA fragment of 276 bp. Amplification using multiplex RT-PCRmethod showed two of 10 samples were AI positive using multiplex RT-PCR, three DNA fragments weregenerated consisting of 276 bp for M gene, 189 bp for H5 gene, and 131 bp for N1. In this study, rapid andeffective diagnosis method for AI virus can be conducted by using simultaneous Single Step Multiplex RTPCR.By this technique type and subtype of AI virus, can also be determined, especially H5N1.

  13. Microarray and quantitative RT-PCR analyses in calcium-channel blockers induced gingival overgrowth tissues of periodontitis patients.

    Science.gov (United States)

    Shimizu, Taro; Kubota, Takehiko; Nakasone, Naohiro; Abe, Daisuke; Morozumi, Toshiya; Yoshie, Hiromasa

    2011-03-01

    The purpose of the present study was to analyse transcriptomes and mRNA expression levels for specific genes in calcium-channel blocker-induced gingival overgrowth (GO) tissues. Eight gingival tissues samples (from both GO negative and positive sites) were harvested from four GO patients for microarray analyses. Twelve candidate genes were selected for further quantitative real time reverse transcription-polymerase chain reaction (qRT-PCR) analyses. Ten GO tissues from periodontitis patients and ten control gingival tissues from healthy subjects were compared by qRT-PCR. Mann-Whitney U-test was used for statistical evaluation. In GO positive tissues, 163-1631 up-regulated and 100-695 down-regulated genes were identified with more than two-fold changes compared with GO negative tissues amongst patients by microarray experiments. No commonly expressed genes amongst the eight sets of microarray data were found. The clustering analysis confirmed that the entire transcriptome patterns showed similarities in individuals, but differences amongst the four patients. The qRT-PCR and statistical analyses for the candidate genes, though, revealed differential gene expressions between GO-positive and negative tissues. We found that matrix metalloproteinase (MMP)-1 and MMP-12 as well as cathepsin-L were significantly up-regulated whilst keratin-10 and transforming growth factor-β1 were significantly down-regulated in GO tissues of periodontitis patients compared with the control gingival tissues of healthy subjects. The microarray analyses revealed that GO pathogenesis was complex and individually varied, though GO-affected gingival tissues were controlled at least by genes related to collagen metabolisms including regulated MMPs, cathepsin-L, growth factors, and keratins to maintain tissue homeostasis in vivo. Copyright © 2010 Elsevier Ltd. All rights reserved.

  14. Detection of avian leukosis virus subgroups in albumen of commercial and native fowl eggs using RT-PCR in Iran.

    Science.gov (United States)

    Rajabzadeh, Mostafa; Dadras, Habibollah; Mohammadi, Ali

    2010-12-01

    Avian leukosis viruses (ALVs) belong to Alpharetrovirus genus of the family Retroviridae that are widespread in nature. Different subgroups of ALV commonly infect egg-laying hens. They are responsible for economic losses due to both mortality and depressed performance in chickens. To investigate the presence of these viruses in chickens in Iran, 560 egg albumens were selected from different farms of Fars province, Iran. These eggs were obtained from flocks of two research centers of native fowl production (60 eggs), a broiler grandparent farm (100 eggs), three broiler breeder farms (300 eggs), and a commercial layer flock (100 eggs). Firstly, for primary screening a degenerative primer set (PU1 and PU2) were used in reverse transcriptase-polymerase chain reaction (RT-PCR). Positive cases were detected in 47 of 300 (15.7%) samples from three broiler breeders, 40 of 100 (40%) samples from commercial layer, 53 of 60 (88.3%) samples from flocks of two research centers of native fowl production, and none from the samples of broiler grandparent. Then RT-PCR was undertaken with primers PA1 and PA2 on the positive samples. RT-PCR analysis detected ALVs in two of 47 (4.3%) samples from three broiler breeders, 13 of 40 (32.5%) samples from commercial layer, and 19 of 53 (35.8%) samples from flocks of two research centers of native fowl production. The sequencing results showed that subgroup E of ALV was the most detected virus among chicken eggs and subgroup B was more prevalent in the eggs of native fowls. This is the first report of the ALV subgroup B and E in egg albumen in Iran.

  15. Identification of qRT-PCR reference genes for analysis of opioid gene expression in a hibernator.

    Science.gov (United States)

    Otis, Jessica P; Ackermann, Laynez W; Denning, Gerene M; Carey, Hannah V

    2010-04-01

    Previous work has suggested that central and peripheral opioid signaling are involved in regulating torpor behavior and tissue protection associated with the hibernation phenotype. We used quantitative real-time PCR (qRT-PCR) to measure mRNA levels of opioid peptide precursors and receptors in the brain and heart of summer ground squirrels (Ictidomys tridecemlineatus) and winter hibernating squirrels in the torpid or interbout arousal states. The use of appropriate reference genes for normalization of qRT-PCR gene expression data can have profound effects on the analysis and interpretation of results. This may be particularly important when experimental subjects, such as hibernating animals, undergo significant morphological and/or functional changes during the study. Therefore, an additional goal of this study was to identify stable reference genes for use in qRT-PCR studies of the 13-lined ground squirrel. Expression levels of 10 potential reference genes were measured in the small intestine, liver, brain, and heart, and the optimal combinations of the most stable reference genes were identified by the GeNorm Excel applet. Based on this analysis, we provide recommendations for reference genes to use in each tissue that would be suitable for comparative studies among different activity states. When appropriate normalization of mRNA levels was used, there were no changes in opioid-related genes in heart among the three activity states; in brain, DOR expression was highest during torpor, lowest in interbout arousal and intermediate in summer. The results support the idea that changes in DOR expression may regulate the level of neuronal activity in brain during the annual hibernation cycle and may contribute to hibernation-associated tissue protection.

  16. Development of an in situ magnetic beads based RT-PCR method for electrochemiluminescent detection of rotavirus

    Science.gov (United States)

    Zhan, Fangfang; Zhou, Xiaoming

    2012-12-01

    Rotaviruses are double-stranded RNA viruses belonging to the family of enteric pathogens. It is a major cause of diarrhoeal disease in infants and young children worldwide. Consequently, rapid and accurate detection of rotaviruses is of great importance in controlling and preventing food- and waterborne diseases and outbreaks. Reverse transcription-polymerase chain reaction (RT-PCR) is a reliable method that possesses high specificity and sensitivity. It has been widely used to detection of viruses. Electrochemiluminescence (ECL) can be considered as an important and powerful tool in analytical and clinical application with high sensitivity, excellent specificity, and low cost. Here we have developed a method for the detection of rotavirus by combining in situ magnetic beads (MBs) based RT-PCR with ECL. RT of rotavirus RNA was carried out in a traditional way and the resulting cDNA was directly amplified on MBs. Forward primers were covalently bounded to MBs and reverse primers were labeled with tris-(2, 2'-bipyridyl) ruthenium (TBR). During the PCR cycling, the TBR labeled products were directly loaded and enriched on the surface of MBs. Then the MBs-TBR complexes could be analyzed by a magnetic ECL platform without any post-modification or post-incubation which avoid some laborious manual operations and achieve rapid yet sensitive detection. In this study, rotavirus from fecal specimens was successfully detected within 2 h, and the limit of detection was estimated to be 104copies/μL. This novel in situ MBs based RT-PCR with ECL detection method can be used for pathogen detection in food safety field and clinical diagnosis.

  17. Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process

    Directory of Open Access Journals (Sweden)

    Borges-Pérez Andrés

    2008-12-01

    Full Text Available Abstract Background The elucidation of gene expression patterns leads to a better understanding of biological processes. Real-time quantitative RT-PCR has become the standard method for in-depth studies of gene expression. A biologically meaningful reporting of target mRNA quantities requires accurate and reliable normalization in order to identify real gene-specific variation. The purpose of normalization is to control several variables such as different amounts and quality of starting material, variable enzymatic efficiencies of retrotranscription from RNA to cDNA, or differences between tissues or cells in overall transcriptional activity. The validity of a housekeeping gene as endogenous control relies on the stability of its expression level across the sample panel being analysed. In the present report we describe the first systematic evaluation of potential internal controls during tomato development process to identify which are the most reliable for transcript quantification by real-time RT-PCR. Results In this study, we assess the expression stability of 7 traditional and 4 novel housekeeping genes in a set of 27 samples representing different tissues and organs of tomato plants at different developmental stages. First, we designed, tested and optimized amplification primers for real-time RT-PCR. Then, expression data from each candidate gene were evaluated with three complementary approaches based on different statistical procedures. Our analysis suggests that SGN-U314153 (CAC, SGN-U321250 (TIP41, SGN-U346908 ("Expressed" and SGN-U316474 (SAND genes provide superior transcript normalization in tomato development studies. We recommend different combinations of these exceptionally stable housekeeping genes for suited normalization of different developmental series, including the complete tomato development process. Conclusion This work constitutes the first effort for the selection of optimal endogenous controls for quantitative real

  18. Validation of artificial microRNA expression by poly(A) tailing-based RT-PCR

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Rui Shi, Chenmin Yang, Ronald Sederoff & Vincent Chiang ### Abstract Here we describe a protocol for validating expression of artificial microRNAs (amiRNAs) by poly(A) tailing-based RT-PCR. Total RNAs, including amiRNA, are poly(A) tailed using E.coli. poly(A) polymerase. Poly(A) tailed amiRNA can be converted into cDNA along with mRNAs in a reverse transcription reaction primed by a standard poly(T) anchor adaptor. AmiRNA can then be amplified and quantitated by real-tim...

  19. 马尔堡病毒的实时荧光RT-PCR检测方法研究%Study on real time RT - PCR detection method for Marburg virus

    Institute of Scientific and Technical Information of China (English)

    洪烨; 邓燕凤; 郑夔; 相大鹏; 黄吉城; 戴俊; 师永霞; 李小波; 幸芦琴; 郭波旋

    2011-01-01

    Objective To set up real time RT - PCR detection method for Marburg virus. Methods Some representative nucleic acid segment of Marburg virus as positive control were synth esized, and several primers, probes and reaction system of real time RT - PCR were designed to explore the best detection condition. The PCR condition was optimized to improve the sensitivity and specificity of the assay. Results The specificity of the assay for real time RT - PCR for Marburg virus was high and there was no cross reactions with Dengue virus, Japanese encephalitis virus and Chikungunya virus. The sensitivity of the assay was 100 gene copies per test. Conclusion This method is suitable for laboratory detection of Marburg virus because of its high sensitivity and specificity.%目的 建立马尔堡病毒的实时荧光RT-PCR检测方法.方法 人工合成马尔堡病毒特异性核酸序列作为阳性对照模板,设计实时荧光RT-PCR引物、探针并构建反应体系,对反应条件进行优化,验证该方法的特异性、灵敏度.结果 建立的马尔堡病毒实时荧光RT-PCR检测方法对马尔堡病毒核酸检测有高度特异性,与1型~4型登革病毒、日本脑炎病毒和基孔肯雅病毒均无交叉反应,检测灵敏度为102拷贝/反应.结论 该方法灵敏度高、特异性强,适用于对马尔堡病毒的快速检验.

  20. The quantification of tomato microRNAs response to viral infection by stem-loop real-time RT-PCR.

    Science.gov (United States)

    Feng, Junli; Wang, Kai; Liu, Xin; Chen, Shaoning; Chen, Jishuang

    2009-05-15

    MicroRNAs (miRNAs) are RNA molecules consisting of 20-24 nucleotides that play important roles in regulating plant's gene expression for growth and development, cell viability and stress responses. Viral infection often has a noticeable influence on host gene expression, which may result in a range of developmental abnormalities. To investigate the molecular mechanisms underlying viral infection, miRNA pathway and host gene expression, we report herein the application of the novel miRNAs quantification method in tomato, using a stem-loop reverse transcription followed by SYBR Green PCR assay. For the seven tested miRNAs of Solanum lycopersicum, which are related to the regulation of plant development, hormone response, and their own biogenesis, this quantification method showed high sensitivity, specificity, and wide dynamic range. Precise quantification could be achieved with as little as 0.01 ng of total RNAs for most cases. Additionally, their target mRNAs could be quantified from the same RNA sample simultaneously, by the conventional real-time RT-PCR assay. In comparison with mock inoculation, accumulation levels of the tested miRNAs and target mRNAs were found obviously altered in tomato seedlings, indicating that the miRNA pathway was interrupted by Cucumber mosaic virus and Tomato aspermy virus infection.

  1. Use of automated real-time reverse transcription-polymerase chain reaction (RT-PCR) to monitor experimental swine vesicular disease virus infection in pigs

    DEFF Research Database (Denmark)

    Reid, S.M.; Paton, D.J.; Wilsden, G.

    2004-01-01

    Automated real-time RT-PCR was evaluated as a diagnostic tool for swine vesicular disease virus (SVDV) infection on a range of samples (vesicular epithelium, serum, nasal swabs, faeces) from four inoculated and three in-contact pigs over a period of 28 days. Traditional diagnostic procedures (virus....... The RT-PCR and virus isolation were generally comparable in detecting SVDV in the serum and nasal swabs from inoculated and in-contact pigs up to day 6 after infection; it was possible, however, to isolate virus for a longer period from the faeces of a few pigs. This suggested that further optimization...... of the template extraction method was required to counteract the effects of RT-PCR inhibitors in faeces. It was concluded that the automated real-time RT-PCR is a useful diagnostic method for SVD in clinically or subclinically affected pigs and contributed to the study of the pathogenesis of SVD in the pigs....

  2. Standardization of Gene Expression Quantification by Absolute Real-Time qRT-PCR System Using a Single Standard for Marker and Reference Genes

    Directory of Open Access Journals (Sweden)

    Yi-Hong Zhou

    2010-08-01

    Full Text Available In the last decade, genome-wide gene expression data has been collected from a large number of cancer specimens. In many studies utilizing either microarray-based or knowledge-based gene expression profiling, both the validation of candidate genes and the identification and inclusion of biomarkers in prognosis-modeling has employed real-time quantitative PCR on reverse transcribed mRNA (qRT-PCR because of its inherent sensitivity and quantitative nature. In qRT-PCR data analysis, an internal reference gene is used to normalize the variation in input sample quantity. The relative quantification method used in current real-time qRT-PCR analysis fails to ensure data comparability pivotal in identification of prognostic biomarkers. By employing an absolute qRT-PCR system that uses a single standard for marker and reference genes (SSMR to achieve absolute quantification, we showed that the normalized gene expression data is comparable and independent of variations in the quantities of sample as well as the standard used for generating standard curves. We compared two sets of normalized gene expression data with same histological diagnosis of brain tumor from two labs using relative and absolute real-time qRT-PCR. Base-10 logarithms of the gene expression ratio relative to ACTB were evaluated for statistical equivalence between tumors processed by two different labs. The results showed an approximate comparability for normalized gene expression quantified using a SSMR-based qRT-PCR. Incomparable results were seen for the gene expression data using relative real-time qRT-PCR, due to inequality in molar concentration of two standards for marker and reference genes. Overall results show that SSMR-based real-time qRT-PCR ensures comparability of gene expression data much needed in establishment of prognostic/predictive models for cancer patients—a process that requires large sample sizes by combining independent sets of data.

  3. Expression patterns of prion protein gene in differential genotypes sheep: quantification using molecular beacon real-time RT-PCR.

    Science.gov (United States)

    Wang, Chuan; Wu, Run; Li, Fa-Di; Liu, Lei; Zhang, Xiao-Li; Zhao, Chun-Lin; Diao, Xiao-Long; Guan, Hong-Wei

    2011-06-01

    Determination of the transcription level of cellular prion protein (PrP(C)) is essential for understanding its role in organisms and revealing mechanism of susceptibility and resistance to scrapie. However, the expression of prion protein (PrP) mRNA in sheep has not been quantified in great detail in digestive tract which is important during scrapie spread through oral route. Herein, we report on measurement of sheep PrP mRNA using absolute quantitative real-time RT-PCR. Total RNA was isolated from five different regions of the central nervous system (CNS), four regions of lymphoid system, eleven regions of digestive tract, and two reproductive organ tissues of eight sheep of two different genotypes (ARR/ARQ and ARH/ARQ) and PrP mRNA was quantified by real-time RT-PCR using molecular beacon. The results showed that highest levels of PrP mRNA were expressed in thalamus and cerebrum (P mRNA expression in sheep for further studies of pathogenesis of prion diseases.

  4. Real time polymerase chain reaction (rt-pcr for mycobacterium tuberculosis in serpiginous choroiditis- A study of 29 cases

    Directory of Open Access Journals (Sweden)

    RadhaAnnamalai, Jyotirmay Biswas, Sudharshan S, Gayathri R, Lily Therese, Viswanathan S, NamithaBhuvaneswari

    2014-07-01

    Full Text Available Purpose: A study of real time Polymerase Chain Reaction for Mycobacterium tuberculosis (M. tuberculosis DNA in 29 cases of active serpiginous choroiditis. Design: Case control study. Methods: DNA extraction from the aqueous humor was carried out using QIAMP DNA extraction kit. Real- time Polymerase Chain reaction (RT-PCR for MTB was carried out using Genosen’s Mtb complex quantitative Real time PCR kit. All patients were also subjected to complete blood count, venereal disease research laboratory test, chest radiograph, QuantiFERON TB Gold test on the blood and polymerase chain reaction on a sample of aqueous humor. Results: Aqueous aspirate showed copies of mycobacterium tuberculosis DNA in one out of twenty nine cases of serpiginous choroiditis. Direct smear and culture for mycobacteria was negative in all cases. Conclusion: RT-PCR identifies MTB DNA in suspected latent tuberculosis in serpiginous choroiditis with high specificity. Serpiginous choroiditis and multifocal choroiditis due to tuberculosis may resemble each other clinically but have distinct clinical features which can be confirmed by real time polymerase chain reaction performed on the aqueous humor The association between serpiginous choroiditis and tuberculosis would be a chance association or if present a rare association.

  5. PolyA RT-PCR-based quantification of microRNA by using universal TaqMan probe.

    Science.gov (United States)

    Luo, Xin; Zhang, Jin; Wang, Huijun; Du, Yingying; Yang, Lu; Zheng, Fengyun; Ma, Duan

    2012-04-01

    Quantification of microRNAs (miRNAs) in tissues under normal and pathological conditions is important for elucidating miRNA functions. Based on a PolyA RT-PCR method we have described (J Zhang et al. Biochem Biophys Res Commun 2008 377:136-140), a modified miRNA quantification method was developed and validated using a universal TaqMan probe complementary to the reverse transcript primer. This method effectively detects miRNA expression in cell lines and tissues. The TaqMan probe is more accurate and reliable than the SYBR Green method since it was free from primer dimers. A series of miRNAs were tested in five different mouse tissues: the method differentiated different miRNAs of the same family. This universal TaqMan probe-based PolyA RT-PCR method showed its advantages in precision, simplicity and high-throughput capability compared with other miRNA-detecting methods.

  6. A competitive RT-PCR method for the quantitative analysis of cytokine mRNAs in mouse tissues.

    Science.gov (United States)

    Zhou, N M; Matthys, P; Polacek, C; Fiten, P; Sato, A; Billiau, A; Froyen, G

    1997-03-01

    The authors describe the design and validation of a competitive RT-PCR method for the efficient and reproducible quantitation of mRNA molecules of IFN-gamma, TNF-alpha, IL-4 and IL-10 in mouse spleen RNA extracts. Before being subjected to RT-PCR, the RNA extracts were supplemented with internal control RNAs (IC-RNAs), which were constructed by inserting DNA fragments in the cDNA of the respective cytokines. The efficiency of amplification of the target and the IC-RNA was shown to remain equal over a wide range of cycle numbers. Reproducibility was such that differences in mRNA contents that were greater than 17% could be detected between two RNA samples run in parallel. Normal mouse spleen tissue was found to contain 10(7)-10(8) molecules of TNF-alpha, IFN-gamma, IL-4 and IL-10 mRNA per micrograms total RNA extracted. Injection of animals with anti-CD3 antibody, a well-known cytokine inducer, resulted in a moderate increase in TNF-alpha and IL-10 mRNA levels (14- and 24-fold, respectively), and in a substantially greater increase in the levels of mRNA for IL-4 and IFN-gamma (199- and 851-fold, respectively). These results demonstrate an accurate and reliable quantitation of cytokine mRNA levels in animal tissues.

  7. Evaluation of reference genes for real-time RT-PCR expression studies in the plant pathogen Pectobacterium atrosepticum

    Directory of Open Access Journals (Sweden)

    Toth Ian K

    2007-09-01

    Full Text Available Abstract Background Real-time RT-PCR has become a powerful technique to monitor low-abundance mRNA expression and is a useful tool when examining bacterial gene expression inside infected host tissues. However, correct evaluation of data requires accurate and reliable normalisation against internal standards. Thus, the identification of reference genes whose expression does not change during the course of the experiment is of paramount importance. Here, we present a study where manipulation of cultural growth conditions and in planta experiments have been used to validate the expression stability of reference gene candidates for the plant pathogen Pectobacterium atrosepticum, belonging to the family Enterobacteriaceae. Results Of twelve reference gene candidates tested, four proved to be stably expressed both in six different cultural growth conditions and in planta. Two of these genes (recA and ffh, encoding recombinase A and signal recognition particle protein, respectively, proved to be the most stable set of reference genes under the experimental conditions used. In addition, genes proC and gyrA, encoding pyrroline-5-carboxylate reductase and DNA gyrase, respectively, also displayed relatively stable mRNA expression levels. Conclusion Based on these results, we suggest recA and ffh as suitable candidates for accurate normalisation of real-time RT-PCR data for experiments investigating the plant pathogen P. atrosepticum and potentially other related pathogens.

  8. Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress.

    Science.gov (United States)

    Nicot, Nathalie; Hausman, Jean-François; Hoffmann, Lucien; Evers, Danièle

    2005-11-01

    Plant stress studies are more and more based on gene expression. The analysis of gene expression requires sensitive, precise, and reproducible measurements for specific mRNA sequences. Real-time RT-PCR is at present the most sensitive method for the detection of low abundance mRNA. To avoid bias, real-time RT-PCR is referred to one or several internal control genes, which should not fluctuate during treatments. Here, the non-regulation of seven housekeeping genes (beta-tubulin, cyclophilin, actin, elongation factor 1-alpha (ef1alpha), 18S rRNA, adenine phosphoribosyl transferase (aprt), and cytoplasmic ribosomal protein L2) during biotic (late blight) and abiotic stresses (cold and salt stress) was tested on potato plants using geNorm software. Results from the three experimental conditions indicated that ef1alpha was the most stable among the seven tested. The expression of the other housekeeping genes tested varied upon stress. In parallel, a study of the variability of expression of hsp20.2, shown to be implicated in late blight stress, was realized. The relative quantification of the hsp20.2 gene varied according to the internal control and the number of internal controls used, thus highlighting the importance of the choice of internal controls in such experiments.

  9. Development of 2, 7-Diamino-1, 8-Naphthyridine (DANP) Anchored Hairpin Primers for RT-PCR Detection of Chikungunya Virus Infection.

    Science.gov (United States)

    Chen, Huixin; Parimelalagan, Mariya; Takei, Fumie; Hapuarachchi, Hapuarachchige Chanditha; Koay, Evelyn Siew-Chuan; Ng, Lee Ching; Ho, Phui San; Nakatani, Kazuhiko; Chu, Justin Jang Hann

    2016-08-01

    A molecular diagnostic platform with DANP-anchored hairpin primer was developed and evaluated for the rapid and cost-effective detection of Chikungunya virus (CHIKV) with high sensitivity and specificity. The molecule 2, 7-diamino-1, 8-naphthyridine (DANP) binds to a cytosine-bulge and emits fluorescence at 450 nm when it is excited by 400 nm light. Thus, by measuring the decline in fluorescence emitted from DANP-primer complexes after PCR reaction, we could monitor the PCR progress. By adapting this property of DANP, we have previously developed the first generation DANP-coupled hairpin RT-PCR assay. In the current study, we improved the assay performance by conjugating the DANP molecule covalently onto the hairpin primer to fix the DANP/primer ratio at 1:1; and adjusting the excitation emission wavelength to 365/430 nm to minimize the background signal and a 'turn-on' system is achieved. After optimizing the PCR cycle number to 30, we not only shortened the total assay turnaround time to 60 minutes, but also further reduced the background fluorescence. The detection limit of our assay was 0.001 PFU per reaction. The DANP-anchored hairpin primer, targeting nsP2 gene of CHIKV genome, is highly specific to CHIKV, having no cross-reactivity to a panel of other RNA viruses tested. In conclusion, we report here a molecular diagnostic assay that is sensitive, specific, rapid and cost effective for CHIKV detection and can be performed where no real time PCR instrumentation is required. Our results from patient samples indicated 93.62% sensitivity and 100% specificity of this method, ensuring that it can be a useful tool for rapid detection of CHIKV for outbreaks in many parts of the world.

  10. Development of 2, 7-Diamino-1, 8-Naphthyridine (DANP) Anchored Hairpin Primers for RT-PCR Detection of Chikungunya Virus Infection

    Science.gov (United States)

    Chen, Huixin; Parimelalagan, Mariya; Takei, Fumie; Hapuarachchi, Hapuarachchige Chanditha; Koay, Evelyn Siew-Chuan; Ng, Lee Ching; Ho, Phui San; Nakatani, Kazuhiko; Chu, Justin Jang Hann

    2016-01-01

    A molecular diagnostic platform with DANP-anchored hairpin primer was developed and evaluated for the rapid and cost-effective detection of Chikungunya virus (CHIKV) with high sensitivity and specificity. The molecule 2, 7-diamino-1, 8-naphthyridine (DANP) binds to a cytosine-bulge and emits fluorescence at 450 nm when it is excited by 400 nm light. Thus, by measuring the decline in fluorescence emitted from DANP—primer complexes after PCR reaction, we could monitor the PCR progress. By adapting this property of DANP, we have previously developed the first generation DANP-coupled hairpin RT-PCR assay. In the current study, we improved the assay performance by conjugating the DANP molecule covalently onto the hairpin primer to fix the DANP/primer ratio at 1:1; and adjusting the excitation emission wavelength to 365/430 nm to minimize the background signal and a ‘turn-on’ system is achieved. After optimizing the PCR cycle number to 30, we not only shortened the total assay turnaround time to 60 minutes, but also further reduced the background fluorescence. The detection limit of our assay was 0.001 PFU per reaction. The DANP-anchored hairpin primer, targeting nsP2 gene of CHIKV genome, is highly specific to CHIKV, having no cross-reactivity to a panel of other RNA viruses tested. In conclusion, we report here a molecular diagnostic assay that is sensitive, specific, rapid and cost effective for CHIKV detection and can be performed where no real time PCR instrumentation is required. Our results from patient samples indicated 93.62% sensitivity and 100% specificity of this method, ensuring that it can be a useful tool for rapid detection of CHIKV for outbreaks in many parts of the world. PMID:27571201

  11. Detection of rabies in camel, goat and cattle in Sudan using Fluorescent antibody test (FAT and hemi nested Polymerase Chain Reaction (hnRT-PCR

    Directory of Open Access Journals (Sweden)

    Baraa Abdalaziz Ahmed

    2016-09-01

    Full Text Available Objective: The objective of this study was to identify rabies virus in camels and other animals in Sudan. Materials and methods: Four camel samples were collected from Garraht Elzawia, Kab-kabia and North Darfur areas in Sudan. The samples were collected based on clinical signs. In addition, two camel samples were obtained from Khartoum and Tambool, one goat sample was collected from El-Fashir, and one cattle sample was obtained from Atbara. The samples were transported to the Veterinary Research Institute (VRI at Khartoum, Sudan for further studies. The samples were subjected for nested and hemi nested RT-PCR (hnRT-PCR along with the gold standard Fluorescent antibody test (FAT to diagnose rabies. Results: Out of eight samples, seven were found to be positive by both FAT and RT-PCR methods. The remaining one sample was positive by FAT but negative by hnRT-PCR indicating the suitablity of hnRT-PCR along with FAT for accurate diagnosis of rabies in animals. Conclusion: The study concluded that FAT and RT-PCR are useful tools for research and diagnosis of rabies. [J Adv Vet Anim Res 2016; 3(3.000: 274-277

  12. A review of RT-PCR technologies used in veterinary virology and disease control: sensitive and specific diagnosis of five livestock diseases notifiable to the World Organisation for Animal Health.

    Science.gov (United States)

    Hoffmann, Bernd; Beer, Martin; Reid, Scott M; Mertens, Peter; Oura, Chris A L; van Rijn, Piet A; Slomka, Marek J; Banks, Jill; Brown, Ian H; Alexander, Dennis J; King, Donald P

    2009-10-20

    Real-time, reverse transcription polymerase chain reaction (rRT-PCR) has become one of the most widely used methods in the field of molecular diagnostics and research. The potential of this format to provide sensitive, specific and swift detection and quantification of viral RNAs has made it an indispensable tool for state-of-the-art diagnostics of important human and animal viral pathogens. Integration of these assays into automated liquid handling platforms for nucleic acid extraction increases the rate and standardisation of sample throughput and decreases the potential for cross-contamination. The reliability of these assays can be further enhanced by using internal controls to validate test results. Based on these advantageous characteristics, numerous robust rRT-PCRs systems have been developed and validated for important epizootic diseases of livestock. Here, we review the rRT-PCR assays that have been developed for the detection of five RNA viruses that cause diseases that are notifiable to the World Organisation for Animal Health (OIE), namely: foot-and-mouth disease, classical swine fever, bluetongue disease, avian influenza and Newcastle disease. The performance of these tests for viral diagnostics and disease control and prospects for improved strategies in the future are discussed.

  13. Analysis of Porcine Reproductive and Respiratory Syndrome Virus Isolates from Eastern China with RT-PCR-RFLP%猪繁殖与呼吸综合征病毒华东地区分离株RT-PCR-RFLP分析

    Institute of Scientific and Technical Information of China (English)

    邓雨修; 姜平; 李玉峰; 王先炜; 蒋文明; 汤景元

    2004-01-01

    Porcine reproductive and respiratory syndrome virus(PRRSV) isolates identified in samples from 50 field cases originated from eastern China herds were studied. ORF5 gene of PRRSV isolates was amplified by reverse transcript polymerase chain reaction(RT-PCR) and restriction fragment length polymorphism (RFLP) patterns for enzymes Mlu Ⅰ, Hinc Ⅱ and Sac Ⅱ were determined on these ORF5 genes. The RFLP pattern obtained from 39 isolates was 2-2-1(78%), 3 isolates was 1-1-1(6%) and 8 isolates identified in 2003 was 1-3-1 (16%). The results showed PRRSV strains have variations and existed no less than 3 gene subtypes in the area.

  14. RT-PCR detection of Grapevine fleck virus%葡萄斑点病毒的RT-PCR检测

    Institute of Scientific and Technical Information of China (English)

    卓娜; 王国平; 邓丛良; 洪霓

    2011-01-01

    Grapevine fleck is a common grapevine virus disease in the world,which is caused by Grapevine fleck virus(GFkV).A pair of primers F1/R1 were synthesized based on the reported GFkV sequence,and the products with expected 179 bp were amplified by RT-PCR from the leaves of grapevine samples Centennial seedless,Victoria and Shengbao which showed fleck symptoms.These products were cloned and sequenced.Sequence comparison showed that the similarities among sequences from the three samples were 97.2% to 98.9% and shared 96.6% to 97.8% similarities with the reported sequence(GeneBank accession no.AJ309022).The virus RNAs were extracted from buds and phloem tissues of the dormant grapevine canes by magnetic nano-particles(MNP) method,and the TaqMan probe and primers were designed based on the above sequences.The MNP RT-PCR method was primarily established for the detection of GFkV.The results showed that the GFkV titers were relatively higher in Centennial seedless than in the other two samples,and it was 10 times higher in dormant buds than in phloem.The detection sensitivity for GFkV by MNP-Real time-RT-PCR was about 100 μg grapevine tissues.%由葡萄斑点病毒(Grapevine fleck virus,GFkV)引起的葡萄斑点病是世界上普遍发生的葡萄病毒病害。采用RT-PCR从表现明显斑点症状的葡萄样品维多利亚、无核白鸡心和胜宝叶片中检测到预期大小为179 bp的片段。对这些扩增片段进行克隆、序列测定及比对分析,结果表明,来源于这3个样品的GFkV序列间相似性为97.2%~98.9%,与NCBI已登录GFkV序列AJ309022的相似性为96.6%~97.8%。在此基础上,采用纳米磁珠法从以上3个葡萄品种的休眠枝条韧皮部与休眠芽中提取病毒RNA

  15. Diagnosis of intestinal parasites in a rural community of Venezuela: Advantages and disadvantages of using microscopy or RT-PCR.

    Science.gov (United States)

    Incani, Renzo Nino; Ferrer, Elizabeth; Hoek, Denise; Ramak, Robbert; Roelfsema, Jeroen; Mughini-Gras, Lapo; Kortbeek, Titia; Pinelli, Elena

    2017-03-01

    A cross-sectional study was carried out to determine the prevalence and diagnostic performance of microscopy and real time PCR (RT-PCR) for 14 intestinal parasites in a Venezuelan rural community with a long history of persistent intestinal parasitic infections despite the implementation of regular anthelminthic treatments. A total of 228 participants were included in this study. A multiplex RT-PCR was used for the detection of Dientamoeba fragilis, Giardia intestinalis, Cryptosporidium sp. and a monoplex RT-PCR for Entamoeba histolytica. Furthermore, a multiplex PCR was performed for detection of Ascaris lumbricoides, Strongyloides stercoralis, Necator americanus and Ancylostoma duodenale. Combined microscopy-PCR revealed prevalences of 49.3% for A. lumbricoides, 10.1% for N. americanus (no A. duodenale was detected), 2.0% for S. stercoralis, 40.4% for D. fragilis, 35.1% for G. intestinalis, and 7.9% for E. histolytica/dispar. Significant increases in prevalence at PCR vs. microscopy were found for A. lumbricoides, G. intestinalis and D. fragilis. Other parasites detected by microscopy alone were Trichuris trichiura (25.7%), Enterobius vermicularis (3.4%), Blastocystis sp. (65.8%), and the non-pathogenic Entamoeba coli (28.9%), Entamoeba hartmanni (12.3%), Endolimax nana (19.7%) and Iodamoeba bütschlii (7.5%). Age- but no gender-related differences in prevalences were found for A. lumbricoides, T. trichiura, G. intestinalis, and E. histolytica/dispar. The persistently high prevalences of intestinal helminths are probably related to the high faecal pollution as also evidenced by the high prevalences of non-pathogenic intestinal protozoans. These results highlight the importance of using sensitive diagnostic techniques in combination with microscopy to better estimate the prevalence of intestinal parasites, especially in the case of D. fragilis trophozoites, which deteriorate very rapidly and would be missed by microscopy. In addition, the differentiation between

  16. 新型鸭呼肠孤病毒RT-PCR方法的建立与应用%Establishment and Its Application of RT-PCR for Detection of Novel duck reovirus

    Institute of Scientific and Technical Information of China (English)

    王劭; 陈少莺; 陈仕龙; 程晓霞; 林锋强; 朱小丽; 江斌; 李兆龙

    2011-01-01

    研究建立检测新型鸭呼肠孤病毒(novel duck reovirus,NDRV)的RT-PCR方法,并运用建立的检测方法对分离毒与人工感染样品进行应用检测.根据NDRV-NP03株S3基因全序列(NDRV-NP03,GenBank登录号:GQ888710),设计合成了一对引物,以NDRV分离株为模板,建立了检测NDRV的RT-PCR方法.结果显示:该方法仅能从NDRV分离毒中扩增到与预期大小相符长度为586 bp的特异性目的片段,检测灵敏度达到2 pg病毒RNA,而其它病毒,番鸭呼肠孤病毒(Muscovy duck reovirus,MDRV)、禽呼肠孤病毒(Avian reovirus,ARV)、鸡传染性法氏囊病病毒(Infectious bursal disease virus,IBDV)、番鸭细小病毒(Muscovy duck parvovirus,MDPV)、鹅细小病毒(Goose parvovirus,GPV)、鸭副粘病毒(Duck paramyxovirus,DPMV)、鸭病毒性肝炎病毒(Duck hepatitis virus,DHV)等样品的扩增结果均为阴性.应用该方法对8株NDRV分离毒和3份人工感染鸭肝脾组织进行检测均为阳性.表明建立的RT-PCR方法特异性强、敏感度高,可用于NDRV的临床诊断和流行病学调查.%To develop a RT-PCR assay for the detection of novel duck reovirus (NDRV) and apply the developed assay to isolated strains and artificial infected samples, according to NDRV-NP03 S3 gene in the GenBank(GenBank:GQ888710), a pair of primers for amplying NDRV specifical fragment were designed and synthesized.RT-PCR technique detecting the RNA of NDRV was constructed.RT-PCR results showed that a 586 bp specifical fragment could be isolated only from the NDRV-NP03 strain RNA, and the sensitivity of RT-PCR reached to 2 pg NDRV-RNA.And the negative results were achieved from the other viruses, Muscovy duck reovirus(MDRV), Avian reovirus(ARV), Infectious bursal disease virus(IBDV), Muscovy duck parvovirus(MDPV), Goose parvovirus(GPV),Duck paramyxovirus(DPMV) and Duck hepatitis virus(DHV).The positive rate of RT-PCR method for detecting viral pathogens in 8 field NDRV isolates and 3 samples of the liver and

  17. Expression patterns of Doppel in differential ovine PRNP genotypes: quantification using real-time RT-PCR.

    Science.gov (United States)

    Wang, C; Zhao, C-L; Liu, L; Wu, R; Zhang, X-L

    2015-10-09

    Doppel is a homologue of cellular prion protein (PrP)-like protein (PrPC). Different tissue samples were collected from the central nervous system plus four regions of lymphoid system, eleven regions of digestive tract and two reproductive organs from four ARR/ARQ and four ARH/ARQ sheep, genotypes of the PrP gene. Total RNA was isolated from these samples, and Doppel mRNA was quantified by real-time RT-PCR using SYBR Green. Doppel mRNA expression was higher in the ovary, hypothalamus and brain than in other tissues, and it significantly differed between the two genotypes in brain, ileum, cecum, rectum, colon, and uterus. This study demonstrated that Doppel mRNA expression in sheep with ARR/ARQ or ARH/ARQ genotypes was very different. These findings could be helpful in future studies of the relationship between PrP and Doppel.

  18. Cloning and detection of metallothionein mRNA by RT-PCR in mangrove oysters (Crassostrea rhizophorae).

    Science.gov (United States)

    Rebelo, Mauro F; Pfeiffer, Wolfgang C; da Silva, Hamilton; Moraes, Milton O

    2003-08-20

    A semi-quantitative RT-PCR protocol was developed to directly evaluate metallothionein (MT) mRNA expression in different tissues of mangrove oysters (Crassostrea rhizophorae), using beta-Actin (ACT) as a normalizing gene. Clones with high degree of identity from partial coding sequences were obtained for both MT and ACT. Although not statistically significant, high relative accumulation of MT mRNA was observed in the digestive gland (DGG), but not in the gills, from samples collected from both control and contaminated sites. Nevertheless, MT expression was not comparable to the high levels of metal in the contaminated oysters. Results indicate that the variation in relative MT mRNA levels from different samples of the same site could be due to multiple gene copies or different MT isoform induction.

  19. Detection of HSP mRNA Transcription in Transport Stressed Pigs by Fluorescence Quantitative RT-PCR

    Institute of Scientific and Technical Information of China (English)

    LI Yu-bao; BAO En-dong; WANG Zhi-liang; ZHAO Ru-qian

    2007-01-01

    The RNA transcripted in vitro was used as the standard quantitative template to make the standard curve and establish the fluorescence quantitative RT-PCR (FQ-PCR) method. By means of FQ-PCR, the transcription changes of HSP70 and HSPg0 mRNA in the livers and hearts of transport stressed pigs were studied. The level of HSP70 mRNA transcription increased continuously from the beginning of transportation. The inductions of HSP70 mRNA transcription in the livers and hearts of 10 h transport stressed pigs were 2.5 and 4.1 times higher than that of the un-transport stressed pigs (P<0.01).However, the transcription levels of HSPg0 mRNA in the livers and hearts decreased with the transport stress.

  20. Quantification of dissimilatory (bi)sulphite reductase gene expression in Desulfobacterium autotrophicum using real-time RT-PCR

    DEFF Research Database (Denmark)

    Neretin, LN; Schippers, A.; Pernthaler, A.

    2003-01-01

    We developed a real-time RT-PCR method for the quantification of dissimilatory (bi)sulphite reductase (DSR) mRNA in Desulfobacterium autotrophicum cells. The amount of DSR mRNA was determined relative to the amount of 16S rRNA at different growth conditions during transition from exponential...... changed significantly during growth (up to 310-fold from the early to the late exponential phase during respiration with thiosulphate). The maximum DSR mRNA per-cell contents correlated with cell-specific sulphate reduction rates for all experiments. Environmental applications for the quantification...... to stationary phase: sulphate respiration with lactate, thiosulphate respiration with lactate, sulphate respiration with H-2 and pyruvate fermentation. The dsr gene was expressed constitutively, although DSR mRNA content per-cell varied under different growth conditions. The maximum DSR mRNA per-cell content...

  1. Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample

    Directory of Open Access Journals (Sweden)

    Marone Maria

    2001-01-01

    Full Text Available We describe a semiquantitative RT-PCR protocol optimized in our laboratory to extract RNA from as little as 10,000 cells and to measure the expression levels of several target mRNAs from each sample. This procedure was optimized on the human erythroleukemia cell line TF-1 but was successfully used on primary cells and on different cell lines. We describe the detailed procedure for the analysis of Bcl-2 levels. Aldolase A was used as an internal control to normalize for sample to sample variations in total RNA amounts and for reaction efficiency. As for all quantitative techniques, great care must be taken in all optimization steps: the necessary controls to ensure a rough quantitative (semi-quantitative analysis are described here, together with an example from a study on the effects of TGF-&bgr;1 in TF-1 cells.

  2. Microarray-based identification and RT-PCR test screening for epithelial-specific mRNAs in peripheral blood of patients with colon cancer

    Directory of Open Access Journals (Sweden)

    Coppola Domenico

    2006-10-01

    Full Text Available Abstract Background The efficacy of screening for colorectal cancer using a simple blood-based assay for the detection of tumor cells disseminated in the circulation at an early stage of the disease is gaining positive feedback from several lines of research. This method seems able to reduce colorectal cancer mortality and may replace colonoscopy as the most effective means of detecting colonic lesions. Methods In this work, we present a new microarray-based high-throughput screening method to identifying candidate marker mRNAs for the early detection of epithelial cells diluted in peripheral blood cells. This method includes 1. direct comparison of different samples of colonic mucosa and of blood cells to identify consistent epithelial-specific mRNAs from among 20,000 cDNA assayed by microarray slides; 2. identification of candidate marker mRNAs by data analysis, which allowed selection of only 10 putative differentially expressed genes; 3. Selection of some of the most suitable mRNAs (TMEM69, RANBP3 and PRSS22 that were assayed in blood samples from normal subjects and patients with colon cancer as possible markers for the presence of epithelial cells in the blood, using reverse transcription – polymerase chain reaction (RT-PCR. Results Our present results seem to provide an indication, for the first time obtained by genome-scale screening, that a suitable and consistent colon epithelium mRNA marker may be difficult to identify. Conclusion The design of new approaches to identify such markers is warranted.

  3. Molecular epidemiological study of feline coronavirus strains in Japan using RT-PCR targeting nsp14 gene.

    Science.gov (United States)

    Tanaka, Yoshikazu; Sasaki, Takashi; Matsuda, Ryo; Uematsu, Yosuke; Yamaguchi, Tomohiro

    2015-03-11

    Feline infectious peritonitis is a fatal disease of cats caused by infection with feline coronavirus (FCoV). For detecting or genotyping of FCoV, some RT-PCR plus nested PCR techniques have been reported previously. However, referring to the whole genome sequences (WGSs) registered at NCBI, there are no detection methods that can tolerate the genetic diversity among FCoV population. In addition, the quasispecies nature of FCoV, which consists of heterogeneous variants, has been also demonstrated; thus, a universal method for heteropopulations of FCoV variants in clinical specimens is desirable. To develop an RT-PCR method for detection and genotyping of FCoV, we performed comparative genome analysis using WGSs of 32 FCoV, 7 CCoV and 5 TGEV strains obtained from NCBI. As the PCR target, we focused on the nsp14 coding region, which is highly conserved and phylogenetically informative, and developed a PCR method targeting nsp14 partial sequences. Among 103 ascites, 45 pleural effusion and 214 blood specimens from clinically ill cats, we could detect FCoV in 55 (53.4%), 14 (31.1%) and 19 (8.9%) specimens using the present method. Direct sequencing of PCR products and phylogenetic analysis allowed discrimination between type I- and II-FCoV serotypes. Our nsp14 amino acid sequence typing (nsp14 aa ST) showed that the FCoV clone with sequence type (ST) 42, which was the most predominant genotype of WGS strains, was prevalent in domestic cats in Japan. Our nsp14 PCR scheme will contribute to virus detection, epidemiology and ecology of FCoV strains.

  4. Methods for detection and differentiation of existing and new crinivirus species through multiplex and degenerate primer RT-PCR.

    Science.gov (United States)

    Wintermantel, William M; Hladky, Laura L

    2010-12-01

    A method was developed for rapid identification and differentiation of both known and novel crinivirus species involving both multiplex and degenerate reverse transcription-polymerase chain reaction (RT-PCR). The multiplex method can discriminate among known criniviruses infecting vegetable and small fruit crops, and rapidly identify viruses associated with disease symptoms, as well as identification of mixed crinivirus infections. Four host groups for multiplex detection of criniviruses were selected based on the types of crops where specific criniviruses would be expected to occur. Each detection group contained three to four crop-specific primers designed to the same region of the gene encoding the highly conserved RNA-dependent RNA polymerase gene (RdRp) of criniviruses for rapid, single-reaction determination of which crinivirus(es) may be infecting a plant. Degenerate reverse primers used for RT and in PCR were designed to amplify all members of each host group, and were coupled with species-specific forward primers resulting in four separate single-reaction cocktails for detection of most criniviruses sequenced to date, whether present in single or mixed virus infections. Additional viruses can be added to multiplex detection by adjustment of primer concentration for balanced detection of target viruses. In order to identify unknown putative criniviruses or those for which sequence information is not yet available, a genus-wide, universal degenerate primer set was developed. These primers also targeted the crinivirus RdRp gene, and amplify a wide range of crinivirus sequences. Both detection systems can be used with most RNA extraction methods, and with RT-PCR reagents common in most laboratories.

  5. RT-PCR standardization and bone mineralization after low-level laser therapy on adult osteoblast cells

    Science.gov (United States)

    do Bomfim, Fernando R. C.; Sella, Valéria R. G.; Zanaga, Jéssica Q.; Pereira, Nayara S.; Nouailhetas, Viviane L. A.; Plapler, Hélio

    2014-03-01

    Purpose: Osteoblasts are capable to produce different compounds directly connected to bone mineralization process. This study aims to standardize the reverse transcriptase polymerase chain reaction (RT-PCR) for adult osteoblasts to observe the effect of low level laser therapy on bone mineralization. Methods: Five-millimeter long fragments obtained from the mead femoral region of male Wistar rats were assigned into group A (n=10, laser) and group B (n=10, no laser), submitted to mechanic and enzymatic digestion. After 7 days, cultures of group A were irradiated daily on a single spot with a GaInAs laser, λ=808nm, 200mW/cm2, 2J/cm2, bean diameter of 0,02mm, 5 seconds for 6 days. Group B was manipulated but received no laser irradiation. After 13 days the cells were trypsinized for 15 minute and stabilized with RNA later® for RNA extraction with Trizol®. cDNA synthesis used 10μg of RNA and M-MLV® enzyme. PCR was accomplished using the β-actin gene as a control. Another aliquot was fixed for Hematoxylin-Eosin and Von Kossa staining to visualize bone mineralization areas. Results: Under UV light we observed clearly the amplification of β-actin gene around 400bp. HE and Von Kossa staining showed osteoblast clusters, a higher number of bone cells and well defined mineralization areas in group A. Conclusion: The cell culture, RNA extraction and RT-PCR method for adult osteoblasts was effective, allowing to use these methods for bone mineralization studies. Laser improved bone mineralization and further studies are needed involving osteogenesis, calcium release mechanisms and calcium related channels.

  6. Evaluation and selection of candidate reference genes for normalization of quantitative RT-PCR in Withania somnifera (L. Dunal.

    Directory of Open Access Journals (Sweden)

    Varinder Singh

    Full Text Available Quantitative real-time PCR (qRT-PCR is now globally used for accurate analysis of transcripts levels in plants. For reliable quantification of transcripts, identification of the best reference genes is a prerequisite in qRT-PCR analysis. Recently, Withania somnifera has attracted lot of attention due to its immense therapeutic potential. At present, biotechnological intervention for the improvement of this plant is being seriously pursued. In this background, it is important to have comprehensive studies on finding suitable reference genes for this high valued medicinal plant. In the present study, 11 candidate genes were evaluated for their expression stability under biotic (fungal disease, abiotic (wounding, salt, drought, heat and cold stresses, in different plant tissues and in response to various plant growth regulators (methyl jasmonate, salicylic acid, abscisic acid. The data as analyzed by various software packages (geNorm, NormFinder, Bestkeeper and ΔCt method suggested that cyclophilin (CYP is a most stable gene under wounding, heat, methyl jasmonate, different tissues and all stress conditions. T-SAND was found to be a best reference gene for salt and salicylic acid (SA treated samples, while 26S ribosomal RNA (26S, ubiquitin (UBQ and beta-tubulin (TUB were the most stably expressed genes under drought, biotic and cold treatment respectively. For abscisic acid (ABA treated samples 18S-rRNA was found to stably expressed gene. Finally, the relative expression level of the three genes involved in the withanolide biosynthetic pathway was detected to validate the selection of reliable reference genes. The present work will significantly contribute to gene analysis studies in W. somnifera and facilitate in improving the quality of gene expression data in this plant as well as and other related plant species.

  7. Identification of suitable reference genes for quantitative RT-PCR during 3T3-L1 adipocyte differentiation.

    Science.gov (United States)

    Zhang, Juan; Tang, Hongju; Zhang, Yuqing; Deng, Ruyuan; Shao, Li; Liu, Yun; Li, Fengying; Wang, Xiao; Zhou, Libin

    2014-05-01

    Quantitative reverse transcription PCR (qRT-PCR) is becoming increasingly important in the effort to gain insight into the molecular mechanisms underlying adipogenesis. However, the expression profile of a target gene may be misinterpreted due to the unstable expression of the reference genes under different experimental conditions. Therefore, in this study, we investigated the expression stability of 10 commonly used reference genes during 3T3-L1 adipocyte differentiation. The mRNA expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and transferrin receptor (TFRC) significantly increased during the course of 3T3-L1 adipocyte differentiation, which was decreased by berberine, an inhibitor of adipogenesis. Three popular algorithms, GeNorm, NormFinder and BestKeeper, identified 18 ribosomal RNA and hydroxymethylbilane synthase (HMBS) as the most stable reference genes, while GAPDH and TFRC were the least stable ones. Peptidylprolyl isomerase A [PIPA (cyclophilin A)], ribosomal protein, large, P0 (36-B4), beta-2-microglobulin (B2M), α1-tubulin, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and β-actin showed relatively stable expression levels. The choice of reference genes with various expression stabilities exerted a profound influence on the expression profiles of 2 target genes, peroxisome proliferator-activated receptor (PPAR)γ2 and C/EBPα. In addition, western blot analysis revealed that the increased protein expression of GAPDH was markedly inhibited by berberine during adipocyte differentiation. This study highlights the importance of selecting suitable reference genes for qRT-PCR studies of gene expression during the process of adipogenesis.

  8. Evaluation of candidate reference genes for gene expression normalization in Brassica juncea using real time quantitative RT-PCR.

    Directory of Open Access Journals (Sweden)

    Ruby Chandna

    Full Text Available The real time quantitative reverse transcription PCR (qRT-PCR is becoming increasingly important to gain insight into function of genes. Given the increased sensitivity, ease and reproducibility of qRT-PCR, the requirement of suitable reference genes for normalization has become important and stringent. It is now known that the expression of internal control genes in living organism vary considerably during developmental stages and under different experimental conditions. For economically important Brassica crops, only a couple of reference genes are reported till date. In this study, expression stability of 12 candidate reference genes including ACT2, ELFA, GAPDH, TUA, UBQ9 (traditional housekeeping genes, ACP, CAC, SNF, TIPS-41, TMD, TSB and ZNF (new candidate reference genes, in a diverse set of 49 tissue samples representing different developmental stages, stress and hormone treated conditions and cultivars of Brassica juncea has been validated. For the normalization of vegetative stages the ELFA, ACT2, CAC and TIPS-41 combination would be appropriate whereas TIPS-41 along with CAC would be suitable for normalization of reproductive stages. A combination of GAPDH, TUA, TIPS-41 and CAC were identified as the most suitable reference genes for total developmental stages. In various stress and hormone treated samples, UBQ9 and TIPS-41 had the most stable expression. Across five cultivars of B. juncea, the expression of CAC and TIPS-41 did not vary significantly and were identified as the most stably expressed reference genes. This study provides comprehensive information that the new reference genes selected herein performed better than the traditional housekeeping genes. The selection of most suitable reference genes depends on the experimental conditions, and is tissue and cultivar-specific. Further, to attain accuracy in the results more than one reference genes are necessary for normalization.

  9. Evaluation and selection of candidate reference genes for normalization of quantitative RT-PCR in Withania somnifera (L.) Dunal.

    Science.gov (United States)

    Singh, Varinder; Kaul, Sunil C; Wadhwa, Renu; Pati, Pratap Kumar

    2015-01-01

    Quantitative real-time PCR (qRT-PCR) is now globally used for accurate analysis of transcripts levels in plants. For reliable quantification of transcripts, identification of the best reference genes is a prerequisite in qRT-PCR analysis. Recently, Withania somnifera has attracted lot of attention due to its immense therapeutic potential. At present, biotechnological intervention for the improvement of this plant is being seriously pursued. In this background, it is important to have comprehensive studies on finding suitable reference genes for this high valued medicinal plant. In the present study, 11 candidate genes were evaluated for their expression stability under biotic (fungal disease), abiotic (wounding, salt, drought, heat and cold) stresses, in different plant tissues and in response to various plant growth regulators (methyl jasmonate, salicylic acid, abscisic acid). The data as analyzed by various software packages (geNorm, NormFinder, Bestkeeper and ΔCt method) suggested that cyclophilin (CYP) is a most stable gene under wounding, heat, methyl jasmonate, different tissues and all stress conditions. T-SAND was found to be a best reference gene for salt and salicylic acid (SA) treated samples, while 26S ribosomal RNA (26S), ubiquitin (UBQ) and beta-tubulin (TUB) were the most stably expressed genes under drought, biotic and cold treatment respectively. For abscisic acid (ABA) treated samples 18S-rRNA was found to stably expressed gene. Finally, the relative expression level of the three genes involved in the withanolide biosynthetic pathway was detected to validate the selection of reliable reference genes. The present work will significantly contribute to gene analysis studies in W. somnifera and facilitate in improving the quality of gene expression data in this plant as well as and other related plant species.

  10. A general method for nested RT-PCR amplification and sequencing the complete HCV genotype 1 open reading frame

    Directory of Open Access Journals (Sweden)

    Tavis John E

    2005-12-01

    Full Text Available Abstract Background Hepatitis C virus (HCV is a pathogenic hepatic flavivirus with a single stranded RNA genome. It has a high genetic variability and is classified into six major genotypes. Genotype 1a and 1b cause the majority of infections in the USA. Viral genomic sequence information is needed to correlate viral variation with pathology or response to therapy. However, reverse transcription-polymerase chain reaction (RT-PCR of the HCV genome must overcome low template concentration and high target sequence diversity. Amplification conditions must hence have both high sensitivity and specificity yet recognize a heterogeneous target population to permit general amplification with minimal bias. This places divergent demands of the amplification conditions that can be very difficult to reconcile. Results RT and nested PCR conditions were optimized independently and systematically for amplifying the complete open reading frame (ORF from HCV genotype 1a and 1b using several overlapping amplicons. For each amplicon, multiple pairs of nested PCR primers were optimized. Using these primers, the success rate (defined as the rate of production of sufficient DNA for sequencing with any one of the primer pairs for a given amplicon for amplification of 72 genotype 1a and 1b patient plasma samples averaged over 95% for all amplicons. In addition, two sets of sequencing primers were optimized for each genotype 1a and 1b. Viral consensus sequences were determined by directly sequencing the amplicons. HCV ORFs from 72 patients have been sequenced using these primers. Sequencing errors were negligible because sequencing depth was over 4-fold and both strands were sequenced. Primer bias was controlled and monitored through careful primer design and control experiments. Conclusion Optimized RT-PCR and sequencing conditions are useful for rapid and reliable amplification and sequencing of HCV genotype 1a and 1b ORFs.

  11. Quantification of GPCR mRNA using real-time RT-PCR.

    Science.gov (United States)

    Brattelid, Trond; Levy, Finn Olav

    2011-01-01

    Characterisation of G-protein-coupled receptor (GPCR) mRNA expression under normal, different pharmacological and pathological conditions in experimental animal models and human tissue biopsies by quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is a valuable approach to understand the regulation of GPCR expression. RT-qPCR is specific and sensitive with a broad dynamic range, which allows precise quantification of mRNA species of interest. In addition to measuring the relative levels of mRNA in a tissue or changes in expression levels between groups of genes of interest, RT-qPCR is also used to identify splice variants and single nucleotide polymorphisms (SNPs) of GPCRs. Even though RT-qPCR has become the standard method for quantification of gene expression, RT-qPCR is sensitive to RNA quality, assay design, normalisation approach and data analysis. This protocol is meant as a guide to RT-qPCR methodology with references to the best standard methods available at present.

  12. Use of Real-time RT-PCR Analysis for mRNA Expression of Tobacco Ferritin Gene (NtFer1)

    Institute of Scientific and Technical Information of China (English)

    JIANG Tingbo; LI Fengjuan; YANG Chuanping

    2006-01-01

    To understand the use of real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) for detecting the relative abundance of mRNA, the expression of a tobacco ferritin gene (NtFer1) was detected by Northern blot and real-time RT-PCR. The results indicated that both of the two methods were able to detect mRNA expression of NtFer1 clearly and similarly, namely NtFer1 expression was responsive to iron-overload, and the abundance of NtFer1 mRNA was greatly increased after iron loaded for 6 h. To compare the effect and sensitivity of two methods, results revealed that Northern blot need 30 μg of total RNA and at least 3 days for the total protocol performance, whereas real-time RT-PCR only need 2 μg of total RNA and 1.5 h. The real-time RT-PCR is rather sensitive and effective than Northern blot. Real-time RT-PCR analysis can be used to rapidly detect the relative abundance of mRNA expression instead of Northern blot analysis.

  13. 套式逆转录PCR检测戊型肝炎病毒RNA的临床应用%Detection of HEV RNA by Nested RT-PCR and its clinical significance

    Institute of Scientific and Technical Information of China (English)

    徐鸿绪; 刘敏; 沈振宇; 陈雯; 胡旭初

    2012-01-01

    OBJECTIVE To investigate the clinical significance of detection of hepatitis E virus (HEV) RNA in serum from patients with acute hepatitis E using nRT-PCR. METHODS The conserved region at ORF2 was used for the design of primers based on the alignment result of HEV registered sequences and other related sequences in GenBank. Two pairs of primers were designed by bioinformatics software. Then nested RT-PCR (nRT-PCR) technology was wsed to amplify Hepatitis E virus RNA of 126 patients. The nested RT-PCR technology was performed to detect HEV RNA in all these sera in comparison to anti-HEV IgM detected by ELISA assay. RESULTS A total of 67 of sera from 126 cases were positive for HEV RNA measured by nRT-PCR. All of the controls were negative. The results of nRT-PCR and anti-HEV IgM (ELISA) were concordant in 80. 91% samples. The two methods appeared a good concordance. Moreover, there was significant difference between the two methods, so they had complementarity and could not be substituted with each other. The first serum samples from three patients were positive for HEV RNA and negative for anti-HEV IgM. Following studies showed all the three sera samples were positive for anti-HEV IgM. HEV RNA in serum of the patients with acute HEV infection could be detected mostly from the first to the twelfth day. CONCLUSION The nRT-PCR method had high specificity and can be applied to the qualitative detection of the serum with hepatitis E virus. nRT-PCR is more sensitive than the assay of ELISA (detected anti-HEV IgM) in this study, and it may improve the early diagnosis of HEV for clinical diagnosis.%目的 评价套式逆转录PCR检测血清戊肝病毒(HEV) RNA的临床意义.方法 对GenBank数据库中的戊型肝炎病毒全基因组序列进行分析比对,并针对国内流行的HEV株序列,选择位于ORF2的保守区域设计引物,建立套式逆转录聚合酶链式反应(nRT-PCR)方法,并检测126例急性戊型肝炎患者,并与检测抗-HEV Ig

  14. 苦瓜植株不同组织中降糖血糖多肽活性成分的RT-PCR分析%RT-PCR Analysis of Hypoglycemic Polypeptide Components from Different Tissues of Momordica charantia

    Institute of Scientific and Technical Information of China (English)

    宋还雷; 谢先文; 顾红艳; 任大云; 王富军; 赵健

    2011-01-01

    Objective To investigate the distribution of hypoglycemic polypeptide P in different tissues of Momordica chamntia (MC). Methods The improved one-step Trizol extraction method was used for the isolation of total RNA from different tissues of MC. A RT-PCR method was used for analyzing the expression patterns of the hypoglycemic polypeptide P gene in MC. Results The RT-PCR analysis results showed that the expression of hypoglycemic polypeptide P gene could only be detected in seeds and fruit pulp of mature MC. Conclusion The hypoglycemic polypeptide P mainly expresses in seeds and fruit pulp of MC. These two tissues are the main medicinal parts for hypoglycemic therapy of MC.%目的 研究苦瓜降糖活性多肽P在苦瓜不同组织器官中的分布.方法 用改良Trizol一步法提取苦瓜不同组织的总RNA,采用RT-PCR方法扩增降糖多肽P基因,分析其表达.结果 成熟果实的种子和果肉中均可明显检测到降糖多肽P基因的表达,其他组织中则未能检测出.结论 苦瓜降糖多肽P活性成分主要在苦瓜果肉和种子中表达,这2种组织是苦瓜降血糖作用的主要药用部位.

  15. Detection of colon flora in peritoneal drain fluid after colorectal surgery: can RT-PCR play a role in diagnosing anastomotic leakage?

    Directory of Open Access Journals (Sweden)

    P Willemsen

    2009-12-01

    Full Text Available Background and objectives: A semi-quantitative Real-Time PCR strategy was developed to identify indicator organisms for an indication on anastomotic leakage in peritoneal drainage fluid, Escherichia coli and Enterococcus faecalis."nMaterials and Methods: The analytical performance of the amplification method was validated with 10 culture-positive and 7 culture negative peritoneal drain fluid samples, obtained from 9 different patients with a colorectal anastomosis."nResults: Real-Time PCR results were fully concordant with the microbiological culture results. However, among the culture negative samples, four false-positive RT-PCR results were found. All false-positives originated from a single patient with a surgical site infection. This may indicate an elevated sensitivity of the RT-PCR method."nConclusion: The results showed that the semi-quantitative RT-PCR method has the clear potential to be useful as a powerful tool in early detection of anastomotic leakage.

  16. Virus isolation vs RT-PCR: which method is more successful in detecting VHSV and IHNV in fish tissue sampled under field conditions?

    DEFF Research Database (Denmark)

    Knüsel, R.; Bergmann, S. M.; Einer-Jensen, Katja;

    2007-01-01

    (total of 859 fish) originating from a field survey on the occurrence of VHSV and IHNV in farmed and wild salmonids in Switzerland. These samples represented all sites with fish that were either identified as virus-positive by means of virus isolation (three sites, four positive tissue sample pools) and......This study compared the results of reverse transcription-polymerase chain reaction (RT-PCR) and traditional virus isolation on cell culture in detection of viral haemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV). RT-PCR was used for 172 tissue sample pools....../or demonstrated positive anti-VHSV-antibody titres (83 sites, 121 positive blood samples) in a serum plaque neutralization test (SPNT). The RT-PCR technique confirmed the four VHSV-positive tissue sample pools detected by virus isolation and additionally identified one VHSV-positive sample that showed positive...

  17. Clinical characteristics and outcomes of patients with primary lung adenocarcinoma harboring ALK rearrangements detected by FISH, IHC, and RT-PCR.

    Science.gov (United States)

    Wang, Jinghui; Cai, Yiran; Dong, Yujie; Nong, Jingying; Zhou, Lijuan; Liu, Guimei; Su, Dan; Li, Xi; Wu, Shafei; Chen, Xuejing; Qin, Na; Zeng, Xuan; Zhang, Haiqing; Zhang, Zongde; Zhang, Shucai

    2014-01-01

    EML4-ALK is a new driver gene of non-small cell lung cancer and a target of crizotinib. The objectives of this study were to determine the frequency of ALK rearrangements in a large cohort of patients with primary lung adenocarcinoma and to analyze the association of ALK rearrangements with clinicopathological characteristics and clinical outcomes. The roles of fluorescence in situ hybridization (FISH), Ventana immunohistochemistry (IHC), and reverse transcriptase polymerase chain reaction (RT-PCR) in the detection of ALK rearrangements were evaluated. The ALK rearrangement was detected in 430 specimens from individual patients with primary lung adenocarcinoma using FISH and Ventana IHC based on tissue microarrays. The EGFR status was detected in all of the specimens through DNA sequencing. An RT-PCR was performed on 200 of the specimens and confirmed by sequencing. Of the 430 patients, 46 (10.7%) harbored ALK rearrangements. The ALK rearrangements were associated with a younger age and the EGFR wild type in comparison with ALK-negative patients. The sensitivity and specificity of the Ventana IHC were 100% and 98.2%, respectively, and the concordance rate between the FISH and the Ventana IHC was 98.4%. The sensitivity and specificity of RT-PCR were 95.5% and 87.0%, respectively, and the concordance rate between the FISH and the RT-PCR was 89.0%. The Cox analysis indicated that an early stage and EGFR-activating mutations were independently associated with a longer OS. This study demonstrated that ALK rearrangements are associated with a younger age and the EGFR wild type rather than with other clinicopathological factors. Although the FISH and Ventana IHC have better concordance, and RT-PCR is a more sensitive method and can identify different variants or partners, the IHC and RT-PCR need to be further evaluated in clinical trials to identify their roles in guiding patients' targeted therapy using crizotinib.

  18. Clinical characteristics and outcomes of patients with primary lung adenocarcinoma harboring ALK rearrangements detected by FISH, IHC, and RT-PCR.

    Directory of Open Access Journals (Sweden)

    Jinghui Wang

    Full Text Available EML4-ALK is a new driver gene of non-small cell lung cancer and a target of crizotinib. The objectives of this study were to determine the frequency of ALK rearrangements in a large cohort of patients with primary lung adenocarcinoma and to analyze the association of ALK rearrangements with clinicopathological characteristics and clinical outcomes. The roles of fluorescence in situ hybridization (FISH, Ventana immunohistochemistry (IHC, and reverse transcriptase polymerase chain reaction (RT-PCR in the detection of ALK rearrangements were evaluated. The ALK rearrangement was detected in 430 specimens from individual patients with primary lung adenocarcinoma using FISH and Ventana IHC based on tissue microarrays. The EGFR status was detected in all of the specimens through DNA sequencing. An RT-PCR was performed on 200 of the specimens and confirmed by sequencing. Of the 430 patients, 46 (10.7% harbored ALK rearrangements. The ALK rearrangements were associated with a younger age and the EGFR wild type in comparison with ALK-negative patients. The sensitivity and specificity of the Ventana IHC were 100% and 98.2%, respectively, and the concordance rate between the FISH and the Ventana IHC was 98.4%. The sensitivity and specificity of RT-PCR were 95.5% and 87.0%, respectively, and the concordance rate between the FISH and the RT-PCR was 89.0%. The Cox analysis indicated that an early stage and EGFR-activating mutations were independently associated with a longer OS. This study demonstrated that ALK rearrangements are associated with a younger age and the EGFR wild type rather than with other clinicopathological factors. Although the FISH and Ventana IHC have better concordance, and RT-PCR is a more sensitive method and can identify different variants or partners, the IHC and RT-PCR need to be further evaluated in clinical trials to identify their roles in guiding patients' targeted therapy using crizotinib.

  19. A sensitive one-step real-time PCR for detection of avian influenza viruses using a MGB probe and an internal positive control

    Directory of Open Access Journals (Sweden)

    Delogu Mauro

    2006-05-01

    Full Text Available Abstract Background Avian influenza viruses (AIVs are endemic in wild birds and their introduction and conversion to highly pathogenic avian influenza virus in domestic poultry is a cause of serious economic losses as well as a risk for potential transmission to humans. The ability to rapidly recognise AIVs in biological specimens is critical for limiting further spread of the disease in poultry. The advent of molecular methods such as real time polymerase chain reaction has allowed improvement of detection methods currently used in laboratories, although not all of these methods include an Internal Positive Control (IPC to monitor for false negative results. Therefore we developed a one-step reverse transcription real time PCR (RRT-PCR with a Minor Groove Binder (MGB probe for the detection of different subtypes of AIVs. This technique also includes an IPC. Methods RRT-PCR was developed using an improved TaqMan technology with a MGB probe to detect AI from reference viruses. Primers and probe were designed based on the matrix gene sequences from most animal and human A influenza virus subtypes. The specificity of RRT-PCR was assessed by detecting influenza A virus isolates belonging to subtypes from H1–H13 isolated in avian, human, swine and equine hosts. The analytical sensitivity of the RRT-PCR assay was determined using serial dilutions of in vitro transcribed matrix gene RNA. The use of a rodent RNA as an IPC in order not to reduce the efficiency of the assay was adopted. Results The RRT-PCR assay is capable to detect all tested influenza A viruses. The detection limit of the assay was shown to be between 5 and 50 RNA copies per reaction and the standard curve demonstrated a linear range from 5 to 5 × 108 copies as well as excellent reproducibility. The analytical sensitivity of the assay is 10–100 times higher than conventional RT-PCR. Conclusion The high sensitivity, rapidity, reproducibility and specificity of the AIV RRT-PCR with

  20. Development of a real-time SYBR Green PCR assay for the rapid detection of Dermatophilus congolensis.

    Science.gov (United States)

    García, Alfredo; Martínez, Remigio; Benitez-Medina, José Manuel; Risco, David; Garcia, Waldo Luis; Rey, Joaquín; Alonso, Juan Manuel; Hermoso de Mendoza, Javier

    2013-01-01

    Methods such as real time (RT)-PCR have not been developed for the rapid detection and diagnosis of Dermatophilus (D.) congolensis infection. In the present study, a D. congolensis-specific SYBR Green RT-PCR assay was evaluated. The detection limit of the RT-PCR assay was 1 pg of DNA per PCR reaction. No cross-reaction with nucleic acids extracted from Pseudomonas aeruginosa, Mycobacterium tuberculosis, Staphylococcus aureus, or Austwickia chelonae was observed. Finally, the RT-PCR assay was used to evaluate clinical samples collected from naturally infected animals with D. congolensis. The results showed that this assay is a fast and reliable method for diagnosing dermatophilosis.

  1. Comparative Study on Fluorescence RT-PCR and RT-PCR Testing Methods for Detecting Avian Influenza Virus in Chickens%荧光RT-PCR与常规RT-PCR检测禽流感病毒比较研究

    Institute of Scientific and Technical Information of China (English)

    宫爱艳; 吴胜昔; 胡仁健; 王健

    2006-01-01

    采用目前公认的2种敏感特异的检测方法--荧光RT-PCR和常规RT-PCR--检测标准毒株禽流感病毒H9亚型(鸡胚尿囊液)及部分待检样品,进行灵敏度、特异性、安全性等方面的比较.结果表明:荧光RT-PCR方法在4 h内就能得出结果,检测尿囊液的禽流感病毒灵敏性达到10-7; RT-PCR方法 6 h出结果,其灵敏度为10-5,且荧光RT-PCR不需电泳,不接触EB,更安全.因此,荧光RT-PCR比常规RT-PCR更灵敏、快速和安全.

  2. Validation of reference genes for quantitative RT-PCR studies of gene expression in perennial ryegrass (Lolium perenne L.

    Directory of Open Access Journals (Sweden)

    Thrush Anthony

    2010-01-01

    Full Text Available Abstract Background Perennial ryegrass (Lolium perenne L. is an important pasture and turf crop. Biotechniques such as gene expression studies are being employed to improve traits in this temperate grass. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR is among the best methods available for determining changes in gene expression. Before analysis of target gene expression, it is essential to select an appropriate normalisation strategy to control for non-specific variation between samples. Reference genes that have stable expression at different biological and physiological states can be effectively used for normalisation; however, their expression stability must be validated before use. Results Existing Serial Analysis of Gene Expression data were queried to identify six moderately expressed genes that had relatively stable gene expression throughout the year. These six candidate reference genes (eukaryotic elongation factor 1 alpha, eEF1A; TAT-binding protein homolog 1, TBP-1; eukaryotic translation initiation factor 4 alpha, eIF4A; YT521-B-like protein family protein, YT521-B; histone 3, H3; ubiquitin-conjugating enzyme, E2 were validated for qRT-PCR normalisation in 442 diverse perennial ryegrass (Lolium perenne L. samples sourced from field- and laboratory-grown plants under a wide range of experimental conditions. Eukaryotic EF1A is encoded by members of a multigene family exhibiting differential expression and necessitated the expression analysis of different eEF1A encoding genes; a highly expressed eEF1A (h, a moderately, but stably expressed eEF1A (s, and combined expression of multigene eEF1A (m. NormFinder identified eEF1A (s and YT521-B as the best combination of two genes for normalisation of gene expression data in perennial ryegrass following different defoliation management in the field. Conclusions This study is unique in the magnitude of samples tested with the inclusion of numerous field-grown samples

  3. Quantification of human telomerase reverse transcriptase mRNA in testicular germ cell tumors by quantitative fluorescence real-time RT-PCR.

    Science.gov (United States)

    Schrader, Mark; Burger, Angelika M; Müller, Markus; Krause, Hans; Straub, Bernd; Smith, Gilian L; Newlands, Eward S; Miller, Kurt

    2002-01-01

    Telomerase is a ribonucleoprotein enzyme which is endogenously expressed in germ, stem and tumor cells, but absent in benign somatic cells. The two major telomerase components are human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT). It has been shown that hTERT is rate-limiting for telomerase activity and that it plays a central role in human carcinogenesis. Here, we investigated the potential of hTERT and hTR gene expression as diagnostic markers in testicular germ cell tumors (TGCT). hTERT mRNA and hTR expression were quantified in 55 te