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Sample records for cmv usando-se pcr

  1. Monitoramento de pacientes com AIDS para o desenvolvimento de doença por citomegalovirus (CMV usando-se PCR multiplex

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    Terra Ana Paula Sarreta

    2000-01-01

    Full Text Available O citomegalovirus é um patógeno importante em indivíduos infectados pelo vírus da imunodeficiência humana (HIV. A carga viral do CMV parece ser preditora da sintomatologia das infecções citomegálicas em pacientes com a síndrome da imunodeficiência adquirida (AIDS. Um protocolo de PCR multiplex que fornece informações de quantificação de DNA amplificando somente um ou dois genes alvo do CMV foi utilizado neste trabalho. Amostras mensais de sangue foram coletadas de 270 pacientes com AIDS. Vinte pacientes tiveram positividade para dois alvos por 3 ou mais consecutivas e apresentaram doença por CMV no decorrer do estudo. Os pacientes que não apresentaram positividade ou alternância de amostras positivas somente para um gene viral não desenvolveram doença ligada ao CMV. Os resultados sugerem que a PCR multiplex é um protocolo útil para a identificação dos pacientes com alta carga viral e maior risco de desenvolvimento de doença por CMV.

  2. Quantitation of cytomegalovirus (CMV) DNA in leukocytes of human immunodeficiency virus-infected subjects with and without CMV disease by using PCR and the SHARP Signal Detection System.

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    Boivin, G; Handfield, J; Murray, G; Toma, E; Lalonde, R; Lazar, J G; Bergeron, M G

    1997-02-01

    We report the development of a simple and rapid PCR assay for quantitation of the cytomegalovirus (CMV) DNA load in polymorphonuclear leukocytes. Using this system, a very good correlation was found between a high number of CMV copies in the blood and the presence of CMV disease in subjects with AIDS.

  3. Performance Evaluation of the Real-Q Cytomegalovirus (CMV) Quantification Kit Using Two Real-Time PCR Systems for Quantifying CMV DNA in Whole Blood.

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    Park, Jong Eun; Kim, Ji Youn; Yun, Sun Ae; Lee, Myoung Keun; Huh, Hee Jae; Kim, Jong Won; Ki, Chang Seok

    2016-11-01

    Standardized cytomegalovirus (CMV) DNA quantification is important for managing CMV disease. We evaluated the performance of the Real-Q CMV Quantification Kit (Real-Q assay; BioSewoom, Korea) using whole blood (WB), with nucleic acid extraction using MagNA Pure 96 (Roche Diagnostics, Germany). Real-time PCR was performed on two platforms: the 7500 Fast real-time PCR (7500 Fast; Applied Biosystems, USA) and CFX96 real-time PCR detection (CFX96; Bio-Rad, USA) systems. The WHO international standard, diluted with CMV-negative WB, was used to validate the analytical performance. We used 90 WB clinical samples for comparison with the artus CMV RG PCR kit (artus assay; Qiagen, Germany). Limits of detections (LODs) in 7500 Fast and CFX96 were 367 and 479 IU/mL, respectively. The assay was linear from the LOD to 10⁶ IU/mL (R² ≥0.9886). The conversion factors from copies to IU in 7500 Fast and CFX96 were 0.95 and 1.06, respectively. Compared with the artus assay, for values 1,000 copies/mL, 73.3% and 80.6% of samples in 7500 Fast and CFX96, respectively, had real-time PCR platforms.

  4. Evaluation of different cytomegalovirus (CMV) DNA PCR protocols for analysis of dried blood spots from consecutive cases of neonates with congenital CMV infections.

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    Soetens, Oriane; Vauloup-Fellous, Christelle; Foulon, Ina; Dubreuil, Pascal; De Saeger, Ben; Grangeot-Keros, Liliane; Naessens, Anne

    2008-03-01

    Two protocols for the extraction of cytomegalovirus (CMV) DNA and two methods for the amplification of CMV DNA in dried blood spots were evaluated for the retrospective diagnosis of congenital CMV infection. During the period from 1996 to 2006, a urine screening program detected 76 congenitally infected neonates. Stored Guthrie cards with blood from 55 cases and 12 controls were tested. Two spots of dried blood were cut from each card and evaluated in two centers. CMV DNA was extracted from a whole single spot. Center 1 used phenol-chloroform extraction and ethanol precipitation followed by a conventional PCR. Center 2 used the NucliSens easyMAG automated DNA/RNA extraction platform (bioMérieux) followed by a real-time PCR. For evaluation of the extraction method, DNA extracted from each blood spot was evaluated by the amplification method used by the collaborating center. The sensitivities were 66% for center 1 and 73% for center 2. None of the controls were positive. A sensitivity as high as 82% could be obtained by combining the most sensitive extraction method (the phenol-chloroform procedure) with the most sensitive PCR method (real-time PCR). The detection rate was not influenced by the duration of storage of the spots. The sensitivity was higher with blood from congenitally infected cases due to a primary maternal CMV infection, regardless of the protocol used. However, the difference reached significance only for the least-sensitive protocol (P = 0.036).

  5. Determination of CMV infection in CSF of children with meningoencephalitis: PCR method

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    Noorbakhsh S

    2009-01-01

    Full Text Available "nBackground: In recent years, many ill cases with cytomegalovirus reactivation in non-immuno compromised persons reported. Goal of study: to determine the CMV infection in cerebrospinal fluid of aseptic meningoencephalitis children hospitalized in Rasul & Mofid hospital (2005-2007. "nMethods: In a cross sectional study 132 cases selected with simple sampling. CMV-DNA in their Cerebro spinal fluids searched by qualitative PCR. "nResults: The age range of the study patients was 5 month- 13 years, median age= 2±3.7 years; 87(65.9% male and 45(34.1% was female. The presenting signs and symptoms were convulsion 77(69.4%; meningitis 25(18.8%, loss of consciousness 47(37%; neurologic defects 15.9%. DNA extrated in 11 cases. Mycoplasma- DNA in 2cases; DNA-CMV detected 2(1.5%. Positive DNA HSV found in 7(15.3% of patients. DNA- HSV type- 15.3% (7/132 cases. An infant 5 month age with developmental delay, microcephaly and recurrent convulsions. A 1 year girl with brain atrophy and progressive hydrocephaly with intracranial shunt "nConclusions: Differentiation between herpes meningoencephalitis and other encephalopathy based on clinical signs in children is too difficult. CMV (1.5% has lower rate than herpes simplex type-1 (5.7%. In addition to CMV and HSV1 all of herpes family viruses (varicella, herpes 6, 7, Epstein barr virus could have role in  children with meningoencephalitis. In recent years a sensitive, rapid, simple diagnostic  test "Single tube Multiplex PCR" in cerebro spinal fluid recommend. Rapid diagnosis and faster treatment is necessary for decreasing mortality and morbidity in all of herpes meningoencephalitis cases

  6. [Analytical performances of real-time PCR by Abbott RealTime CMV with m2000 for the detection of cytomegalovirus in urine].

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    De Monte, Anne; Cannavo, Isabelle; Caramella, Anne; Ollier, Laurence; Giordanengo, Valérie

    2016-01-01

    Congenital cytomegalovirus (CMV) infection is the leading cause of sensoneurinal disability due to infectious congenital disease. The diagnosis of congenital CMV infection is based on the search of CMV in the urine within the first two weeks of life. Viral culture of urine is the gold standard. However, the PCR is highly sensitive and faster. It is becoming an alternative choice. The objective of this study is the validation of real-time PCR by Abbott RealTime CMV with m2000 for the detection of cytomegalovirus in urine. Repeatability, reproducibility, detection limit and inter-sample contamination were evaluated. Urine samples from patients (n=141) were collected and analyzed simultaneously in culture and PCR in order to assess the correlation of these two methods. The sensitivity and specificity of PCR were also calculated. The Abbott RealTime CMV PCR in urine is an automated and sensitive method (detection limit 200 UI/mL). Fidelity is very good (standard deviation of repeatability: 0.08 to 0.15 LogUI/mL and reproducibility 0.18 LogUI/mL). We can note a good correlation between culture and Abbott RealTime CMV PCR (kappa 96%). When considering rapid culture as reference, real-time PCR was highly sensitive (100%) and specific (98.2%). The real-time PCR by Abbott RealTime CMV with m2000 is optimal for CMV detection in urine.

  7. A comparison of CMV detection in gastrointestinal mucosal biopsies using immunohistochemistry and PCR performed on formalin-fixed, paraffin-embedded tissue.

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    Mills, Anne M; Guo, Frances P; Copland, Andrew P; Pai, Reetesh K; Pinsky, Benjamin A

    2013-07-01

    Cytomegalovirus (CMV) can precipitate and exacerbate gastrointestinal (GI) mucosal injury. The gold standard for CMV detection in formalin-fixed, paraffin-embedded (FFPE) tissue is immunohistochemistry (IHC). Although CMV polymerase chain reaction (PCR) on fresh tissue may be a valuable adjunct to IHC, its utility is unknown for FFPE tissues. We therefore evaluated quantitative, real-time CMV PCR in a total of 102 FFPE GI biopsy specimens from 74 patients with a history of hematopoietic stem cell or solid organ transplant, inflammatory bowel disease, human immunodeficiency virus infection, or unspecified colitis. CMV DNA was detected by PCR in 90.9% (30/33) of IHC-positive, 14.5% (8/55) of IHC-negative, and 20.0% (1/5) of IHC-equivocal FFPE tissues. Quantitation of CMV DNA copies normalized to β-globin demonstrated a wide range of values (median 0.276; range, 0.0004 to 144.50). Importantly, 93.3% (14/15) of patients with IHC-positive, active colitis showed no evidence of CMV in matched concurrent, histologically normal biopsies tested by PCR. These results suggest that CMV PCR on FFPE GI biopsies complements IHC and has the potential to identify additional patients who may benefit from anti-CMV therapy.

  8. Diagnosis of cytomegalovirus infections by qualitative and quantitative PCR in HIV infected patients Diagnóstico de infecção por CMV em pacientes infectados pelo HIV utilizando PCR qualitativa e quantitativa

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    Aldo de Albuquerque CUNHA

    2002-01-01

    Full Text Available A high incidence of cytomegalovirus (CMV infections is observed in Brazil. These viruses are causatives of significant morbidity and mortality among patients with advanced human immunodeficiency virus (HIV infection. This work, shows the application of a PCR on determination of CMV load in the buffy coat and plasma. We analyzed the samples of 247 HIV infected patients in order to diagnose CMV infection and disease. We developed a semi-quantitative PCR that amplifies part of the glycoprotein B (gB gene of CMV. The semi-quantitative PCR was carried out only in positive clinical samples in a qualitative PCR confirmed by a nested-PCR. CD4 lymphocyte count, HIV viral load and CMV disease symptom were correlated with CMV load. CMV genome was detected in the buffy coat of 82 of 237 (34.6% patients, in 10 of these the CMV load was determined varying between 928 and 332 880 viral copies/mug DNA. None of these 237 patients developed any suggestive manifestation of CMV disease. For the other 10 HIV infected patients selected based on the suspicion of CMV disease, CMV genome was detected in only one case. This patient presented a high CMV load, 8 000 000 copies/mug DNA, and developed a disseminated form of CMV disease including hepatitis and retinitis. Our results were greatly influenced by the impact of the highly active antiretroviral therapy that reduced incidence of CMV viremia and occurrence of CMV disease in the HIV infected patients.Uma alta incidência de infecção pelo citomegalovirus (CMV é observada no Brasil. Este vírus é responsável por significante morbi-mortalidade entre pacientes infectados pelo vírus da imunodeficiência humana (HIV. Neste estudo, mostramos a aplicação de uma PCR quantitativa para determinar a carga de CMV nos leucócitos do sangue periférico e no plasma de 247 pacientes infectados pelo HIV. As amostras clínicas foram previamente testadas por uma PCR qualitativa e confirmadas por uma nested-PCR para posteriormente

  9. Improved quantitative PCR protocols for adenovirus and CMV with an internal inhibition control system and automated nucleic acid isolation.

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    Henke-Gendo, C; Ganzenmueller, T; Kluba, J; Harste, G; Raggub, L; Heim, A

    2012-06-01

    With the establishment of routine virus load (DNAemia) screening for Human adenovirus (HAdV) and Cytomegalovirus (CMV) in post-transplant care quality standards for quantitative PCR-assays are increasing. Established real-time PCR assays were improved with a fully automated DNA-extraction and with a competitive internal control DNA packaged into a lambda phage which serves as an extraction and amplification control in each sample. HAdV and CMV DNA were detected and quantified simultaneously in various types of diagnostic samples like blood, feces or respiratory tract materials. Inhibition was observed in 0.33-0.66% of over 14,000 diagnostic samples, an infrequent but nevertheless not negligible event, which is observed mainly in stool samples. CMV viral load in broncho-alveolar lavage fluid (BALF) ranged between positive but below the quantitation limit of 1,000 copies/ml up to 1.8 × 10(7) copies/ml with a median of 6.0 × 10(3) copies/ml. Forty-one (4.7%) BALF samples had a viral load above 5.0 × 10(5) copies/ml, which was proposed as a threshold for the diagnosis of pneumonia. HAdV viral loads ranged between positive but below the quantitation limit of 1,000 copies/ml to a very high concentration of 1.3 × 10(11) copies/ml in stool and BALF samples. A HAdV-DNAemia of >10(4) copies/ml was found only in patients with stool viral load of above 10(5) copies/ml. These data support the hypothesis that quantitation in diagnostic materials other than blood may give valuable diagnostic information and that further evaluation of this approach is reasonable.

  10. Evaluation of a standardised real-time PCR based DNA-detection method (Realstar®) in whole blood for the diagnosis of primary human cytomegalovirus (CMV) infections in immunocompetent patients.

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    Berth, M; Benoy, I; Christensen, N

    2016-02-01

    Cytomegalovirus (CMV) DNA detection in blood could, as a supplementary test to serology, improve the accuracy and speed of diagnosis of an acute CMV infection. In this study we evaluated the performance of a commercially available and standardised CMV PCR assay in whole blood for the diagnosis of a primary infection in immunocompetent adults. Moreover, the kinetics of viral DNA was evaluated in order to provide a time frame in which viral DNA could be detected during an acute primary infection. Whole blood samples were collected from 66 patients with an acute CMV infection, 65 patients with an acute Epstein-Barr virus infection, 27 patients with various other acute infections (parvovirus B19, HIV, Toxoplasma gondii), 20 patients with past CMV infections (>1 year) and 20 apparently healthy persons. For CMV DNA detection and quantification a commercially available real-time PCR was applied (RealStar®, altona Diagnostics). The clinical sensitivity of CMV PCR in whole blood for the diagnosis of a recent primary CMV infection was 93.9 % and the diagnostic specificity 99.2 %. In the majority of the patients CMV DNA was not detectable anymore approximately within 4 weeks after the first blood sample was taken. From these data we concluded that, together with a suggestive serological profile, a positive CMV PCR result in whole blood can be regarded as a diagnostic confirmation of a recent CMV infection on a single blood sample in an immunocompetent patient. However, a negative CMV PCR result does not exclude a recent CMV infection.

  11. PCR multiplex para a detecção de BSV e CMV em bananeiras micropropagadas Multiplex PCR for detection of Banana streak virus and Cucumber mosaic virus from micropropagated banana

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    Daniel Vasquez Figueiredo

    2007-09-01

    Full Text Available Um protocolo de PCR multiplex foi estabelecido para a detecção do Banana streak virus (BSV e do Cucumber mosaic virus (CMV em bananeiras micropropagadas. Estes vírus são responsáveis por perdas na produção de bananas em todo o mundo. Alguns trabalhos descrevem a integração do BSV no genoma B da bananeira. Contudo, a existência de bananeiras híbridas livres do BSV tem sido demonstrada. Ademais, determinadas estirpes do CMV não são transmitidas mecanicamente sob condições de laboratório, nem tampouco detectadas por testes sorológicos. Como conseqüência, a indexação de matrizes para cultura de tecido algumas vezes se mostra ineficiente. A metodologia apresentada neste trabalho sobrepõe esta dificuldade, pois se baseia na detecção do ácido nucléico viral presente em amostras foliares de bananeira. Na reação, foram usados os oligonucleotídeos BADNA 1A e BADNA 4, para a detecção do BSV, e "CMV senso" e "CMV antisenso" para a detecção do CMV. Após a eletroforese foi verificada a presença de dois fragmentos de DNA amplificados simultaneamente, um dos quais com 597 pb correspondente ao BSV e o outro, com 488 pb, correspondente ao CMV. Este resultado indica que o PCR multiplex pode ser utilizado como uma ferramenta adicional na indexação do BSV e do CMV em bananeiras propagadas por cultura de tecido.A protocol for multiplex PCR assay was established to detect both Banana streak virus (BSV and Cucumber mosaic virus (CMV in micropropagated banana cultivars. These viruses are responsible for losses in banana production worldwide. Previous reports have described the integration of BSV in banana genome B. However, the existence of hybrid BSV-free banana cultivars has been reported. Moreover, there have been reports that some CMV strains cannot be mechanically transmitted under laboratory conditions and are not always detected by serological assays. Therefore, the selection of virus-free mother plants for tissue culture is

  12. 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.

  13. Comparative study of RT-PCR, pp-65 Test and histopathology for detection of CMV infection after kidney transplantation%RT-PCR,pp65抗原及组织病理学测定肾移植术后巨细胞病毒感染的比较研究

    Institute of Scientific and Technical Information of China (English)

    韩永; 黄海燕; 许晓光; 肖漓; 周文强; 冯凯; 蔡明; 石炳毅

    2011-01-01

    目的 比较巨细胞病毒(CMV)定量PCR检测,CMV-pp65抗原检测及组织病理学在肾移植术后CMV感染中的诊断价值.方法 回顾性分析28例肾移植患者临床资料,并结合文献进行讨论.结果 共有11例患者发生无临床症状的巨细胞病毒感染,经静脉注射更昔洛韦,11例全部治愈.结论 CMV定量PCR,CMV-pp65抗原检测及组织病理学均能作为肾移植术后CMV感染早期诊断的有效手段,三者比较,CMV定量PCR更敏感,CMV-pp65抗原检测更特异,组织病理学则具有创伤性,因此三者共同应用对CMV感染早期诊断更有意义.%Objective To compare the efficiency of cytomegalovirus (CMV) quantitative PCR, CMV pp65 antigen test and histo-pathology for detection of CMV infection and their clinical significance in the recipients of renal transplantation. Methods The clinical data of 28 patients who received kidney splantation were retrospectively analyzed along with literature review. Results CMV infection occurred asymptomatically in total 11 patients, all of them were cured through intravenous ganciclovir. Conclusion It is concluded that CMV quantitative PCR, CM V-pp65 antigen test and histopathology can detect the infection of CMV early and effectively in the recipients of renal transplantation. CMV quantitative PCR is more sensitive, and CMV-pp65 is more specific. His-topathology as golden standard is an invasive examination. The detection of CMV quantitative PCR, CMV-pp65 and histopathology are the same necessary for early diagnosis and guiding treatment of CMV infection more meaningful.

  14. CMV - gastroenteritis/colitis

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    Colitis - cytomegalovirus; Gastroenteritis - cytomegalovirus; Gastrointestinal CMV disease ... or after bone marrow or organ transplant Ulcerative colitis or Crohn disease Rarely, serious CMV infection involving ...

  15. Development of cytomegalovirus (CMV) disease may be predicted in HIV-infected patients by CMV polymerase chain reaction and the antigenemia test

    DEFF Research Database (Denmark)

    Dodt, K K; Jacobsen, P H; Hofmann, B;

    1997-01-01

    evaluated PCR and the antigenemia tests as methods for early detection of CMV disease. METHODS: Two-hundred HIV-seropositive subjects with CD4 T-cell counts below 100 x 10(6)/l were monitored with CMV polymerase chain reaction (PCR), the antigenemia test, blood cultures and CMV immunoglobulin (Ig) G and Ig...

  16. Cytomegalovirus (CMV) and Pregnancy

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    ... Close contact includes activities like changing diapers and kissing. What is congenital cytomegalovirus (congenital CMV)? Pregnant women ... or saliva. Try to avoid mouth-to-mouth kissing with children in day-care. Do not share ...

  17. Late-onset CMV disease following CMV prophylaxis.

    LENUS (Irish Health Repository)

    Donnelly, C

    2012-02-01

    BACKGROUND: Cytomegalovirus (CMV) is the most common opportunistic infection after solid-organ transplantation, increasing morbidity and mortality. Three months of oral valganciclovir have been shown to provide effective prophylaxis. Late-onset CMV disease, occurring after the discontinuation of prophylaxis, is now increasingly recognised. AIMS: To investigate the incidence and the time of detection of CMV infections in liver transplant recipients who received CMV prophylaxis. METHODS: Retrospective review of 64 high- and moderate-risk patients with 1 year of follow-up. RESULTS: The incidence of CMV infection was 12.5%, with 4.7% disease. All cases of symptomatic CMV disease were of late-onset. CONCLUSIONS: The incidence of CMV infections in this study was low compared with literature reports; however, the late-onset disease is an emerging problem. Detection of late-onset disease may be delayed because of less frequent clinic follow-up visits. Increased regular laboratory monitoring may allow earlier detection at the asymptomatic infection stage.

  18. A case of ganciclovir-resistant cytomegalovirus (CMV) retinitis in a patient with AIDS: longitudinal molecular analysis of the CMV viral load and viral mutations in blood compartments.

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    Boivin, G; Gilbert, C; Morissette, M; Handfield, J; Goyette, N; Bergeron, M G

    1997-06-01

    To study the temporal relationships between cytomegalovirus (CMV) viral load and specific UL97 mutations in polymorphonuclear leukocytes (PMNL) and plasma samples from a patient with AIDS who developed ganciclovir-resistant CMV retinitis. Sequential PMNL and plasma samples were analysed for determination of the CMV viral load using non-molecular methods and a quantitative polymerase chain reaction (PCR) assay. Screening of the same samples for the most common mutations conferring ganciclovir resistance was performed using nested PCR and restriction enzyme analysis. At the time of progression of CMV retinitis (after 6 months of ganciclovir), a rapid increase in the CMV DNA load was found in both PMNL and plasma samples. This increase paralleled the emergence of a specific mutation (V594) in the same samples and recovery of ganciclovir-resistant blood isolates. In this patient, however, the only tests that substantially predicted the progression of CMV disease were the quantitative PCR assay using PMNL and to a lesser extent the pp65 antigenemia assay. Quantitative evaluation of the CMV viral load in PMNL using sensitive assays such as PCR appears to be a promising approach for monitoring antiviral therapy in subjects with AIDS. In addition, common mutations conferring ganciclovir resistance can be detected directly in PMNL and plasma samples.

  19. Detection of donor-derived CMV-specific T cells in cerebrospinal fluid in a case of CMV meningoencephalitis after cord blood stem cell transplantation.

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    Ikegame, Kazuhiro; Kato, Ruri; Fujioka, Tatsuya; Okada, Masaya; Kaida, Katsuji; Ishii, Shinichi; Yoshihara, Satoshi; Inoue, Takayuki; Taniguchi, Kyoko; Tamaki, Hiroya; Soma, Toshihiro; Ogawa, Hiroyasu

    2013-02-01

    Cytomegalovirus (CMV) meningoencephalitis is a rather rare complication after allogeneic stem cell transplantation. We describe here the case of a 59-year-old man with acute myeloid leukemia who developed CMV meningoencephalitis after cord blood transplantation. The patient presented with a sudden onset of neurological symptoms, such as convulsion, on day 37. The analysis of cerebrospinal fluid (CSF) sample revealed an increase in the number of cells, which were of donor (cord blood) origin, consisting mainly of T cells. No bacteria were detected in the CSF sample. Real-time PCR analysis revealed that the CSF sample was positive for CMV, but was negative for HHV-6, adenovirus, or BK virus. The patient was diagnosed with CMV meningoencephalitis and received cidofovir. His neurological symptoms were gradually improved and completely disappeared by day 60. CMV-specific dextramer-positive CD8(+) T cells were detected in the peripheral blood and CSF samples, with the frequency being much higher in the CSF. To our knowledge, this is the first report on the appearance of CMV-specific T cells in CSF samples from a patient with CMV meningoencephalitis. Cord blood-derived CMV-specific T cells may develop early after transplantation, enter the intrathecal compartment, and likely contribute to the regulation of CMV-meningoencephalitis.

  20. Diagnostic moléculaire du Cytomégalovirus (CMV), de l’herpès virus humain de type 6 (HHV6) et d’Epstein-Barr virus (EBV) par PCR en temps réel chez les femmes enceintes VIH séropositives et séronégatives à Ouagadougou, Burkina Faso

    Science.gov (United States)

    Ouedraogo, Alice Rogomenoma; Kabre, Madeleine; Bisseye, Cyrille; Zohoncon, Théodora Mahoukèdè; Asshi, Maleki; Soubeiga, Serge Théophile; Diarra, Birama; Traore, Lassina; Djigma, Florencia Wendkuuni; Ouermi, Djénéba; Pietra, Virginio; Barro, Nicolas; Simpore, Jacques

    2016-01-01

    Introduction Les herpès virus EBV, CMV et HHV-6 sont des virus qui évoluent sous le modèle pandémique et sont responsables d’infections congénitales pouvant provoquer des séquelles graves chez les nouveau-nés. L’objectif de cette étude était de déterminer les prévalences de CMV, EBV et HHV-6 chez les femmes enceintes VIH(+) et VIH(-) à Ouagadougou. Méthodes Dans cette étude 200 échantillons de plasma sanguin de femmes enceintes dont 100 femmes VIH(+) et 100 femmes VIH(-) ont été diagnostiqués par PCR multiplex en temps réel pour les trois infections (EBV, CMV et HHV-6). Résultats Sur l’ensemble des 200 échantillons analysés, 18 (9,0%) étaient positifs à au moins un des trois virus, 12 (6,0%) étaient positifs au EBV, 13 (6,5%) au CMV et 12 (6,0%) positifs au HHV-6. Parmi les 18 cas d’infections, nous avons trouvé 10 cas (55,6%) de coïnfections dont 90,0% (9/10) d’infection multiple EBV/CMV/HHV6 et 10,0% de coinfection EBV/HHV6. Le taux d’infection HHVs était plus élevé chez les femmes VIH(-) que celles VIH(+) (12,0% versus 6,0%). Parmi les VIH(+), la PCR a révélé 7,1% (soit 6/85) d’infection HHVs chez celles qui n’étaient pas sous ARV contre 0% chez celles sous ARV. Conclusion Les herpès virus sont fréquents chez les femmes enceintes au Burkina Faso et pourraient constituer une menace chez ces dernières à cause des complications et des risques d’infection pour le nouveau-né. PMID:27800078

  1. CMV and HSV-2 myeloradiculitis in an HIV infected patient

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    Adriana Pinto

    2011-06-01

    Full Text Available While CMV myeloradiculitis is a known complication in AIDS patients with severe immunosuppression, HSV-2 necrotizing myeloradiculitis is rare and often associated with disabling a fatal outcome. We hereby describe a 46 year-old HIV infected patient with profound and sustained immunosuppression who presented with an acute ascending paraparesis and urinary retention. Lumbar spine MRI showed contrast enhancement at the conus medullaris and cauda equine, and both CMV and HSV-2 CSF PCR were positive. Despite treatment, the patient died 20 days later. We review the main diagnostic and therapeutic aspects of herpes virus associated myeloradiculitis and discuss the approach in similar cases.

  2. Expression of the late cytomegalovirus (CMV) pp150 transcript in leukocytes of AIDS patients is associated with a high viral DNA load in leukocytes and presence of CMV DNA in plasma.

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    Boivin, G; Handfield, J; Toma, E; Lalonde, R; Bergeron, M G

    1999-05-01

    The expression of a late cytomegalovirus (CMV) transcript (pp150) was sought in peripheral blood leukocytes (PBL) of subjects with AIDS and correlated with the amounts of CMV DNA in PBL and plasma, by means of quantitative polymerase chain reaction (PCR). The detection of the late CMV transcript was associated with a high number of CMV DNA copies in PBL (P=.0015) and with a positive CMV PCR assay in plasma (P<.001). Expression of CMV pp150 mRNA was best predicted by viral DNA thresholds corresponding to 7058 and 30 copies in PBL and plasma, respectively. The detection of CMV pp150 mRNA was associated with the presence of CMV disease in a univariate analysis but not in a multivariate analysis after controlling for the viral DNA load in PBL. Thus, active viral replication as determined by a high CMV DNA load in PBL is reflected by expression of the late CMV transcript in the same cells and by the presence of CMV DNA in plasma.

  3. CMV IN HEMATOPOIETIC STEM CELL TRANSPLANTATION

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    Rafael de la Camara

    2016-06-01

    Full Text Available Due to its negative impact in the outcome of stem cell transplant (SCT and solid organ transplant patients (SOT CMV has been called “the troll of transplantation”. One of the greatest advances in the management of SCT has been the introduction of the preemptive strategy. Since its introduction, the incidence of the viremia, as expected, remains unchanged but there has been a marked decline in the incidence of early CMV disease. But in spite of the advances in prevention of CMV disease, CMV is still today an important cause of morbidity and mortality. Late CMV disease is still occurring in a significant proportion of patients and the so-called indirect effects of CMV are causing significant morbidity and mortality. Fortunately, there have been several advances in the development of new antivirals, adoptive immunotherapy and DNA-CMV vaccines that might transform the management of CMV in the near future.

  4. CMV and Immunosenescence: from basics to clinics

    Directory of Open Access Journals (Sweden)

    Solana Rafael

    2012-10-01

    Full Text Available Abstract Alone among herpesviruses, persistent Cytomegalovirus (CMV markedly alters the numbers and proportions of peripheral immune cells in infected-vs-uninfected people. Because the rate of CMV infection increases with age in most countries, it has been suggested that it drives or at least exacerbates “immunosenescence”. This contention remains controversial and was the primary subject of the Third International Workshop on CMV & Immunosenescence which was held in Cordoba, Spain, 15-16th March, 2012. Discussions focused on several main themes including the effects of CMV on adaptive immunity and immunosenescence, characterization of CMV-specific T cells, impact of CMV infection and ageing on innate immunity, and finally, most important, the clinical implications of immunosenescence and CMV infection. Here we summarize the major findings of this workshop.

  5. Congenital CMV infection: prevalence in newborns and the impact on hearing deficit.

    Science.gov (United States)

    Engman, Mona-Lisa; Malm, Gunilla; Engstrom, Lotta; Petersson, Karin; Karltorp, Eva; Tear Fahnehjelm, Kristina; Uhlen, Inger; Guthenberg, Claes; Lewensohn-Fuchs, Ilona

    2008-01-01

    Congenital cytomegalovirus (CMV) infection is asymptomatic in 90% of infected newborns but approximately 10-20% of these infants are at risk of developing sequelae later, mostly hearing deficit. The aims of the study were to investigate the prevalence of congenital CMV infection in a Swedish population of newborns and investigate the relative risk of hearing deficit in newborns with congenital CMV infection. The dried blood spot (DBS) samples of 6060 newborns in southern Stockholm during 12 months (October 2003-June 2004; August 2004-October 2004) were analysed for CMV DNA by TaqMan based real-time PCR. Hearing deficit was assessed by otoacoustic emission (OAE) within a newborn screening programme. 12 infants out of 6060 or 0.2% (95% CI 0.1-0.3%) had congenital CMV infection. One boy among the 12 infected infants had unilateral hearing loss, indicating that the risk of hearing loss is greatly increased (about 20 times) in CMV infected infants. No child developed ocular complications such as chorioretinopathy during 3 y of follow-up. Congenital CMV has an impact on child health but can easily be overlooked due to lack of signs in the neonatal period. Surveillance for congenital CMV is important in addition to programmes for prevention and treatment.

  6. Examining the species-specificity of rhesus macaque cytomegalovirus (RhCMV in cynomolgus macaques.

    Directory of Open Access Journals (Sweden)

    Angie K Marsh

    Full Text Available Cytomegalovirus (CMV is a highly species-specific virus that has co-evolved with its host over millions of years and thus restricting cross-species infection. To examine the extent to which host restriction may prevent cross-species research between closely related non-human primates, we evaluated experimental infection of cynomolgus macaques with a recombinant rhesus macaque-derived CMV (RhCMV-eGFP. Twelve cynomolgus macaques were randomly allocated to three groups: one experimental group (RhCMV-eGFP and two control groups (UV-inactivated RhCMV-eGFP or media alone. The animals were given two subcutaneous inoculations at week 0 and week 8, and a subset of animals received an intravenous inoculation at week 23. No overt clinical or haematological changes were observed and PBMCs isolated from RhCMV-eGFP inoculated animals had comparable eGFP- and IE-1-specific cellular responses to the control animals. Following inoculation with RhCMV-eGFP, we were unable to detect evidence of infection in any blood or tissue samples up to 4 years post-inoculation, using sensitive viral co-culture, qPCR, and Western blot assays. Co-culture of urine and saliva samples demonstrated the presence of endogenous cynomolgus CMV (CyCMV cytopathic effect, however no concomitant eGFP expression was observed. The absence of detectable RhCMV-eGFP suggests that the CyCMV-seropositive cynomolgus macaques were not productively infected with RhCMV-eGFP under these inoculation conditions. In a continued effort to develop CMV as a viral vector for an HIV/SIV vaccine, these studies demonstrate that CMV is highly restricted to its host species and can be highly affected by laboratory cell culture. Consideration of the differences between lab-adapted and primary viruses with respect to species range and cell tropism should be a priority in evaluating CMV as vaccine vector for HIV or other pathogens at the preclinical development stage.

  7. Refractory acute respiratory failure due to Pneumocystis jiroveci (PCP) and Cytomegalovirus (CMV) pneumonitis: A case report and review of literature.

    Science.gov (United States)

    Shah, Kairav; Cherabuddi, Kartikeya; Beal, Stacy G; Kalyatanda, Gautam

    2017-01-01

    Opportunistic infections with Pneumocystis jiroveci pneumonia (PCP) are common in patients with HIV (human immunodeficiency virus) and are encountered once the CD4 count decreases below 200 cells/mm3. Cytomegalovirus (CMV) tends to cause disease once the CD4 count drops below 50 cells/mm3. CMV pneumonitis is not common in this population. However, detecting its presence in broncho-alveolar lavage (BAL) fluid has been associated with increased morbidity and mortality. The role of antiviral therapy against CMV remains unclear. We report a newly diagnosed HIV patient with a CD4 count of 44 cells/mm3 presenting with acute respiratory failure secondary to PCP that failed to respond to 3 weeks of standard therapy with trimethoprim-sulfamethoxazole and corticosteroids. He was later diagnosed to have a CMV co-infection causing pneumonitis with BAL cytology findings showing CMV cytopathic effects and PCP. Plasma CMV DNA PCR was 17,424 copies/mL. He responded well after introduction of intravenous ganciclovir. The presence of histopathologic changes demonstrating viral cytopathic effects on BAL cytology along with a high plasma CMV DNA PCR should raise the specificity for diagnosing CMV pneumonitis. True PCP and CMV pneumonitis can occur, and the addition of antiviral therapy with ganciclovir may benefit such patients in the right clinical scenario.

  8. Evaluation of cytomegalovirus (CMV)-specific T-cell immunity for the assessment of the risk of active CMV infection in non-immunosuppressed surgical and trauma intensive care unit patients.

    Science.gov (United States)

    Clari, María A; Aguilar, Gerardo; Benet, Isabel; Belda, Javier; Giménez, Estela; Bravo, Dayana; Carbonell, José A; Henao, Liliana; Navarro, David

    2013-10-01

    The current study was designed to assess the predictive value of the evaluation of cytomegalovirus (CMV)-specific T-cell immunity early following admission to the intensive care unit for inferring the risk of active CMV infection in non-immunosuppressed surgical and trauma patients. A total of 31 CMV-seropositive patients were included. Patients were screened for the presence of CMV DNA in plasma and in tracheal aspirates by real-time PCR. Enumeration of CMV pp65 and IE-1-specific IFN-γ CD8(+) and CD4(+) T cells was performed by flow cytometry for intracellular cytokine staining. Virological and immunological monitoring was conducted once or twice a week. Active CMV infection occurred in 17 out of 31 patients. Undetectable levels of pp65 and IE-1-specific IFN-γ CD8(+) and CD4(+) T-cell subsets cells were observed in 10 patients who developed active CMV infection and in one who did not (at a median of 2 days following ICU admission). Peak CMV DNA loads in both tracheal aspirates and plasma were substantially higher (P = 0.018 and P = 0.091, respectively) in patients with undetectable IFN-γ T-cell responses than in patients with detectable responses. The expansion of both CMV-specific T-cell subsets following detection of active CMV infection was demonstrated in 9 out of 14 patients with active CMV infection. In conclusion, the evaluation of CMV pp65 and IE-1-specific IFN-γ-producing CD8(+) and CD4(+) T cells early following ICU admission may allow the identification of patients most at risk of either having or developing an episode of active CMV infection, particularly those associated with high-level virus replication.

  9. CMV and immunosenescence: from basics to clinics

    NARCIS (Netherlands)

    Solana, R.; Tarazona, R.; Aiello, A.E.; Akbar, A.N.; Appay, V.; Beswick, M.; Bosch, J.A.; Campos, C.; Cantisán, S.; Cicin-Sain, L.; Derhovanessian, E.; Ferrando-Martínez, S.; Frasca, D.; Fulöp, T.; Govind, S.; Gruben-Loebenstein, B.; Hill, A.; Hurme, M.; Kern, F.; Larbi, A.; López-Botet, M.; Maier, A.B.; McElhaney, J.E.; Moss, P.; Naumova, E.; Nikolich-Zugich, J.; Pera, A.; Rector, J.L.; Riddell, N.; Sanchez-Correa, B.; Sansoni, P.; Sauce, D.; van Lier, R.; Wang, G.C.; Wills, M.R.; Zieliński, M.; Pawelec, G.

    2013-01-01

    Alone among herpesviruses, persistent Cytomegalovirus (CMV) markedly alters the numbers and proportions of peripheral immune cells in infected-vs-uninfected people. Because the rate of CMV infection increases with age in most countries, it has been suggested that it drives or at least exacerbates

  10. Isolation and Characterization of Pepper Genes Interacting with the CMV-P1 Helicase Domain.

    Directory of Open Access Journals (Sweden)

    Yoomi Choi

    Full Text Available Cucumber mosaic virus (CMV is a destructive pathogen affecting Capsicum annuum (pepper production. The pepper Cmr1 gene confers resistance to most CMV strains, but is overcome by CMV-P1 in a process dependent on the CMV-P1 RNA1 helicase domain (P1 helicase. Here, to identify host factors involved in CMV-P1 infection in pepper, a yeast two-hybrid library derived from a C. annuum 'Bukang' cDNA library was screened, producing a total of 76 potential clones interacting with the P1 helicase. Beta-galactosidase filter lift assay, PCR screening, and sequencing analysis narrowed the candidates to 10 genes putatively involved in virus infection. The candidate host genes were silenced in Nicotiana benthamiana plants that were then inoculated with CMV-P1 tagged with the green fluorescent protein (GFP. Plants silenced for seven of the genes showed development comparable to N. benthamiana wild type, whereas plants silenced for the other three genes showed developmental defects including stunting and severe distortion. Silencing formate dehydrogenase and calreticulin-3 precursor led to reduced virus accumulation. Formate dehydrogenase-silenced plants showed local infection in inoculated leaves, but not in upper (systemic leaves. In the calreticulin-3 precursor-silenced plants, infection was not observed in either the inoculated or the upper leaves. Our results demonstrate that formate dehydrogenase and calreticulin-3 precursor are required for CMV-P1 infection.

  11. Evaluation of glycoprotein B genotypes and load of CMV infecting blood leukocytes on prognosis of AIDS patients

    Directory of Open Access Journals (Sweden)

    Aldo Albuquerque Cunha

    2011-04-01

    Full Text Available BACKGROUND: Cytomegalovirus (CMV remains an important pathogen to immunocompromised patients even in the era of HAART. The present study aimed at evaluating the influence of CMV viral load and its gB genotypes on AIDS patients' outcome. METHODS: Blood samples of 101 AIDS patients were collected and tested for HIV load, CD4 - cell count and opportunistic pathogens, including CMV. Semi-nested PCRs were run to detect CMV genome and in the positive samples, gB genotyping and CMV load were established using enzymatic restriction and real time PCR, respectively. All patients were clinically followed for four years. RESULTS: In thirty patients (31% CMV was detected and all fatal cases (n = 5 occurred in this group of patients (p = 0.007, but only two patients had CMV disease (1.9%. However, viral load was not statistically associated with any analyzed parameter. The most frequently observed CMV genotype was gB2 (45.16% followed by gB3 (35.48%. gB2 genotype was more frequently found in patients with CD4-cell counts under 200 cells/mm³ (p = 0.0017, and almost all fatal cases (80% had gB2 genotype. CONCLUSIONS: Our study suggests that CMV and its polymorphisms in biologically relevant genes, such as the gB encoding ORF, may still influence the prognosis and outcome of AIDS patients. The gB2 genotype was associated to patient's bad outcome.

  12. cmv treatment in south africa review

    African Journals Online (AJOL)

    THE SOUTHERN AFRICAN JOURNAL OF HIV MEDICINE APRIL 2010. Almost all humans ... was noted to increase with time.10 A cross-sectional study screening all ... VALGANCICLOVIR PRICE REDUCTION IN SA FOR. CMV TREATMENT ...

  13. Reactivation of intestinal CMV in a renal transplant patient after 10 years from the transplant

    Directory of Open Access Journals (Sweden)

    Maria Landi

    2013-04-01

    Full Text Available Introduction.We analyzed the clinical case of a 51 years old man, kidney transplanted on December 2002. On April 2011, he had acute rectal bleeding, renal chronic rejection (creatinine 2.9 mg/dl, Hgb 8.7 g/dl, positive anti-CMV antibodies (IgG. A colonoscopy showed diverticulosis of the rectum associated with deepithelialisation. The patient was treated with maintenance immunosuppressive post-transplant therapy. On June 2011, the colonoscopy showed a stenosing lesion of the sigmoid colon, and blood sampling and intestinal biopsy were performed to search Cytomegalovirus (CMV DNA by PCR. Methods. The presence of CMV-DNA was sought by automatic extractor QIACUBE, using QIAamp DNA BLOOD Mini Kit (Qiagen for whole blood and QIAamp DNA Mini Kit (Qiagen for biopsy.The extracted DNA was then amplified by Real Time PCR using Q-CMV RealTime Complete Kit (Nanogen, on instrument Applied Biosystems 7300. Results. At disease onset the viral load in whole blood was 208000 Geq/ml, and biopsy was positive. Antiviral therapy with Ganciclovir led to the negativity of the viral load and remission of symptoms. Conclusions. The clinical case described presented a reactivation of CMV infection in the intestine after more than 10 years from kidney transplantation, while the highest incidence of CMV reactivation usually occurs during the first year. In our opinion, the reactivation can be traced to long-term immunosuppressive therapy (maintenance posttransplant therapy in combination with a state of inflammation of the intestinal mucosa. In fact, patients with IBD treated with steroid drugs, in particular the group of refractory to therapy and thus have a recovery of the inflammatory process, are exposed to reactivation of CMV with intestinal localization.

  14. Construction and Quality Analysis of Transgenic Rehmannia glutinosa Containing TMV and CMV Coat Protein

    Directory of Open Access Journals (Sweden)

    Zhongqiu Teng

    2016-08-01

    Full Text Available Plant viruses, especially tobacco mosaic virus (TMV and cucumber mosaic virus (CMV are serious threats to Rehmannia glutinosa which is a “top grade” herb in China. In the present study, TMV- and CMV-resistant Rehmannia glutinosa Libosch. plants were constructed by transforming the protein (CP genes of TMV and CMV into Rehmannia glutinosa via a modified procedure of Agrobacterium tumefaciens-mediated transformation. Integration and expression of TMV CP and CMV CP transgenes in 2 lines, LBA-1 and LBA-2, were confirmed by PCR, Southern blot and RT-PCR. Both LBA-1 and LBA-2 were resistant to infection of homologous TMV and CMV strains. The quality of transgenic Rehmanniae Radix was evaluated based on fingerprint analysis and components quantitative analysis comparing with control root tubes. These results showed that chemical composition of transgenic Rehmanniae Radix were similar to non-transgenic ones, which demonstrated that the medical quality and biosafety of transgenic Rehmanniae Radix were equivalent to non-transgenic material when consumed as traditional Chinese medicinal (TCM.

  15. Construction and Quality Analysis of Transgenic Rehmannia glutinosa Containing TMV and CMV Coat Protein.

    Science.gov (United States)

    Teng, Zhongqiu; Shen, Ye; Li, Jing; Lin, Zhongping; Chen, Min; Wang, Min; Li, Man; Dong, Hongran; Huang, Luqi

    2016-08-27

    Plant viruses, especially tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) are serious threats to Rehmannia glutinosa which is a "top grade" herb in China. In the present study, TMV- and CMV-resistant Rehmannia glutinosa Libosch. plants were constructed by transforming the protein (CP) genes of TMV and CMV into Rehmannia glutinosa via a modified procedure of Agrobacterium tumefaciens-mediated transformation. Integration and expression of TMV CP and CMV CP transgenes in 2 lines, LBA-1 and LBA-2, were confirmed by PCR, Southern blot and RT-PCR. Both LBA-1 and LBA-2 were resistant to infection of homologous TMV and CMV strains. The quality of transgenic Rehmanniae Radix was evaluated based on fingerprint analysis and components quantitative analysis comparing with control root tubes. These results showed that chemical composition of transgenic Rehmanniae Radix were similar to non-transgenic ones, which demonstrated that the medical quality and biosafety of transgenic Rehmanniae Radix were equivalent to non-transgenic material when consumed as traditional Chinese medicinal (TCM).

  16. Circulating cytomegalovirus (cmv)-infected endothelial-cells in patients with an active cmv infection

    NARCIS (Netherlands)

    Grefte, A.; van der Giessen, M.; van Son, W.; The, T. Hauw

    1993-01-01

    In 10 of 14 patients with an active cytomegalovirus (CMV) infection, distinctive large cells (35-45 mum in diameter) were present in the peripheral blood. Morphologically these cells closely resembled the classic cytomegalic inclusion cells, generally regarded as a diagnostic hallmark of CMV infecti

  17. CMV and the art of memory maintenance.

    Science.gov (United States)

    Klenerman, Paul; Dunbar, P Rod

    2008-10-17

    The CD8(+) T cell responses to CMV gradually increase in magnitude over time-so-called memory "inflation." In this issue of Immunity, Snyder et al. (2008) examine the dynamics of memory inflation and demonstrate continuous turnover of inflating T cells, drawing on both memory cells and naive cells to replace them.

  18. Prevalence and associated characteristics of cytomegalovirus (CMV ...

    African Journals Online (AJOL)

    Objective: To determine the prevalence of IgG and IgM specific antibodies and ... for human immunodeficiency virus, hepatitis B and C viruses and syphilis were ... the immuno-compromised and adoption of a national policy on CMV infected ...

  19. Molecular cloning and expression of a bush related CmV1 gene in tropical pumpkin.

    Science.gov (United States)

    Wu, Tao; Cao, Jiashu

    2010-02-01

    A bush-type plant was selected from tropical pumpkin 'cga' (Cucurbita moschata Duchesne) in order to study the vine development in C. moschata. In this study, a novel gene encoding NADH dehydrogenase was isolated from the vine line (cgaV) of C. moschata, that was not expressed in the near isogenic bush line (cgaBu). This gene, designated as CmV1 (C. moschata vine 1), was 545 bp in length and was composed of a 477 bp open reading frame, which had 99% nucleotide similarity to the chloroplast ndhJ gene for NADH dehydrogenase subunit J from Brassica oleracea. The deduced amino acid sequence of CmV1 had 99% similarity to NADH dehydrogenase subunit J from Arabidopsis and had 98% similarity to NADH dehydrogenase subunit from Barbarea verna. Analysis of the basic characteristics of the CmV1 protein revealed that it has one Respiratory chain NADH dehydrogenase 30 kD subunit signature, three N-myristoylation sites, one Casein kinase II phosphorylation site, and one Protein kinase C phosphorylation site. Reverse transcriptase-PCR analysis showed that CmV1 was expressed at a high level in the internodes and hypocotyls and was expressed stronger in elongating internodes than in fully expanded internodes. In conclusion, results obtained in the present study suggest that CmV1 gene might play important roles in vine elongation of tropical pumpkin.

  20. [Detection of DNA human cytomegalovirus of a molecular methods: hybrid capture DNA CMV by immunocompromised].

    Science.gov (United States)

    Mhiri, Leila; Arrouji, Zakia; Slim, Amine; Ben Redjeb, Saida

    2006-10-01

    Human cytomegalovirus (HCMV), a member of the beta-virus herpes family, is a ubiquitous human pathogen. After a primary infection, HCMV establishes life latency. HCMV rarely causes symptomatic disease in an immunocompetent host, however, it is a major cause of infectious morbidity and mortality in immunocompromised individuals and developing fetuses. The HCMV genome consists of 240 kbp of double stranded DNA. Early diagnosis molecular of CMV infection is important. The objective of this study was to develop a molecular methods: Quantitative Hybrid capture for the detection of DNA CMV. We present results for 200 immunocompromised collected from 1999 to 2003 (122 men and 78 women, whom mean age was 35 years). Our results showed that 25% of women and 36% of men were positif for hybrid capture DNA CMV. This simple test (cold probe) provide quantitative and fast results. Also the efficacity of anti-CMV therapy can be followed. More over, in contrary with pp65-antigenemia assay and CMV PCR, this test can be managed on biopsy sample.

  1. Transfusion in CMV seronegative T-depleted allogeneic stem cell transplant recipients with CMV-unselected blood components results in zero CMV transmissions in the era of universal leukocyte reduction: a U.K. dual centre experience.

    Science.gov (United States)

    Hall, S; Danby, R; Osman, H; Peniket, A; Rocha, V; Craddock, C; Murphy, M; Chaganti, S

    2015-12-01

    To establish rates of cytomegalovirus (CMV) transmission with use of CMV-unselected (CMV-U), leukocyte-reduced blood components transfused to CMV-seronegative patient/CMV-seronegative donor (CMV neg/neg) allogeneic stem cell transplantation (SCT) recipients including those receiving T-depleted grafts. CMV infection remains a major cause of morbidity following SCT. CMV-seronegative SCT recipients are particularly at risk of transfusion transmitted CMV (TT-CMV) and until recently they have received blood components from CMV-seronegative donors with significant resource implications. Although leukocyte reduction of blood components is reported to minimise risk of TT-CMV, its efficacy in high-risk situations, such as in T-depleted transplant recipients, is unknown. We retrospectively analysed the incidence of TT-CMV in CMV neg/neg allogeneic SCT recipients transfused with CMV-U, leukocyte-reduced blood components in two transplantation centres in the UK. Patients were monitored for CMV infection by weekly CMV polymerase chain reaction testing. Leukocyte reduction of blood components was in accordance with current UK standards. Among 76 patients, including 59 receiving in vivo T-depletion, no episodes of CMV infection were detected. Patients were transfused with 1442 CMV-unselected, leukocyte-reduced components, equating to 1862 donor exposures. Our findings confirm the safety of leukocyte reduction as a strategy in preventing TT-CMV in high-risk allogeneic SCT recipients. © 2015 British Blood Transfusion Society.

  2. Characterization of specific antibodies against cytomegalovirus (CMV)-encoded interleukin 10 produced by 28 % of CMV-seropositive blood donors

    DEFF Research Database (Denmark)

    de Lemos Rieper, Carina; Galle, Pia Søndergaard; Pedersen, Bente Klarlund

    2011-01-01

    Cytomegalovirus (CMV) has evolved multiple immunological evasion strategies, including the encoding of viral interleukin (IL)-10 homologues (cmvIL-10). In this study, cmvIL-10 bound avidly to the same receptors on blood mononuclear cells and was as bio-potent as native human IL-10. Seventeen...

  3. Diagnostic moléculaire du Cytomégalovirus (CMV), de l’herpès virus humain de type 6 (HHV6) et d’Epstein-Barr virus (EBV) par PCR en temps réel chez les femmes enceintes VIH séropositives et séronégatives à Ouagadougou, Burkina Faso

    OpenAIRE

    2016-01-01

    Introduction Les herpès virus EBV, CMV et HHV-6 sont des virus qui évoluent sous le modèle pandémique et sont responsables d’infections congénitales pouvant provoquer des séquelles graves chez les nouveau-nés. L’objectif de cette étude était de déterminer les prévalences de CMV, EBV et HHV-6 chez les femmes enceintes VIH(+) et VIH(-) à Ouagadougou. Méthodes Dans cette étude 200 échantillons de plasma sanguin de femmes enceintes dont 100 femmes VIH(+) et 100 femmes VIH(-) ont été diagnostiqués...

  4. CMV-hFasL transgenic mice prevent from experimental autoimmune thyroiditis

    Institute of Scientific and Technical Information of China (English)

    ZHANG Zhen-lin; LIN Bo; YU Lu-yang; GUO Li-he

    2005-01-01

    Background Previous studies showed that the role of Fas ligand (FasL) is not consistent in the pathogenesis of autoimmune thyroiditis. This study was designed to investigate the effects of FasL on the pathogenesis of experimental autoimmune thyroiditis (EAT) using CMV-human FasL (hFasL) transgenic mice. Methods Transgenic mice ubiquitously expressing hFasL were used as an animal model of EAT by injection of porcine thyroglobulin (pTg). Expression of hFasL was detected by RT-PCR and Western blot. The activity of hFasL transgenic thyrocytes killing Jurket cells was determined. CMV-hFasL transgenic mice and wild type (WT) mice were immunized with pTg and killed 28 days later to evaluate the lymphocytic infiltration of their thyroids. The number of CD4+ and CD8+ lymphocytes from the spleen was detected using FACS. The serum interferon-γ (IFN-γ) concentration was measured by ELISA. Results hFasL expression in the thyroid of CMV-hFasL transgenic mice was confirmed. After co-incubation of Jurket thymocytes with thyroid tissues of CMV-hFasL transgenic mice, the percentage of apoptotic cells in the CMV-hFasL transgenic thyroid group was significantly higher than that of the control WT thyroid group [(23.4±4.3)% vs (6.6±2.5)%, P<0.01]. On day 28 after immunization with pTg, the infiltration index of lymphocytes in thyroids of the CMV-hFasL transgenic mice was significantly lower than that of the WT mice [(1.0±0.5) vs (2.1±0.7), P<0.001]. Moreover, the number of CD4+ and CD8+ lymphocytes of the spleen and serum IFN-γ concentration were significantly decreased in the CMV-hFasL transgenic mice. Conclusions FasL plays an important role in the pathogenesis of autoimmune thyroiditis. Transgenic mice ubiquitously expressing hFasL may strongly inhibit lymphocytic infiltration of the thyroid of EAT and ameliorate the course of this disease.

  5. Circular β ensembles, CMV representation, characteristic polynomials

    Institute of Scientific and Technical Information of China (English)

    SU ZhongGen

    2009-01-01

    In this note we first briefly review some recent progress in the study of the circular β ensemble on the unit circle, where 0 > 0 is a model parameter. In the special cases β = 1,2 and 4, this ensemble describes the joint probability density of eigenvalues of random orthogonal, unitary and sympletic matrices, respectively. For general β, Killip and Nenciu discovered a five-diagonal sparse matrix model, the CMV representation. This representation is new even in the case β = 2; and it has become a powerful tool for studying the circular β ensemble. We then give an elegant derivation for the moment identities of characteristic polynomials via the link with orthogonal polynomials on the unit circle.

  6. CMV retinitis in China and SE Asia: the way forward

    Directory of Open Access Journals (Sweden)

    Heiden David

    2011-11-01

    Full Text Available Abstract AIDS-related CMV retinitis is a common clinical problem in patients with advanced HIV/AIDS in China and Southeast Asia. The disease is causing blindness, and current clinical management, commonly characterized by delayed diagnosis and inadequate treatment, results in poor clinical outcomes: 21% - 36% of eyes with CMV retinitis are already blind at the time the diagnosis is first established by an ophthalmologist. CMV retinitis also identifies a group of patients at extraordinary risk of mortality, and the direct or indirect contribution of extra-ocular CMV disease to AIDS-related morbidity and mortality is currently unmeasured and clinically often overlooked. The obvious way to improve clinical management of CMV retinitis is to screen all patients with CD4 counts

  7. Significance of Epstein-Barr virus (HHV-4 and CMV (HHV-5 infection among subtype-C human immunodeficiency virus-infected individuals

    Directory of Open Access Journals (Sweden)

    J Sachithanandham

    2014-01-01

    Full Text Available Purpose: Opportunistic viral infections are one of the major causes of morbidity and mortality in HIV infection and their molecular detection in the whole blood could be a useful diagnostic tool. Objective: The frequency of opportunistic DNA virus infections among HIV-1-infected individuals using multiplex real-time PCR assays was studied. Materials and Methods: The subjects were in two groups; group 1: Having CD4 counts 350 cells/µl (n = 173. Individuals were classified by WHO clinical staging system. Samples from 70 healthy individuals were tested as controls. In-house qualitative multiplex real-time PCR was standardised and whole blood samples from 291 were tested, followed by quantitative real-time PCR for positives. In a proportion of samples genotypes of Epstein-Barr virus (EBV and CMV were determined. Results: The two major viral infections observed were EBV and CMV. The univariate analysis of CMV load showed significant association with cryptococcal meningitis, oral hairy leukoplakia (OHL, CMV retinitis, CD4 counts and WHO staging (P < 0.05 while the multivariate analysis showed an association with OHL (P = 0.02 and WHO staging (P = 0.05. Univariate analysis showed an association of EBV load with CD4 counts and WHO staging (P < 0.05 and multivariate analysis had association only with CD4 counts. The CMV load was significantly associated with elevated SGPT and SGOT level (P < 0.05 while the EBV had only with SGOT. Conclusion: This study showed an association of EBV and CMV load with CD4+ T cell counts, WHO staging and elevated liver enzymes. These viral infections can accelerate HIV disease and multiplex real-time PCR can be used for the early detection. Genotype 1 and 2 of EBV and genotype gB1 and gB2 of CMV were the prevalent in the HIV-1 subtype C-infected south Indians.

  8. Highly quantitative serological detection of anti-cytomegalovirus (CMV antibodies

    Directory of Open Access Journals (Sweden)

    Alter Harvey J

    2009-05-01

    Full Text Available Abstract Background Human cytomegalovirus infection is associated with a variety of pathological conditions including retinitis, pneumonia, hepatitis and encephalitis that may be transmitted congenitally, horizontally and parenterally and occurs both as a primary infection and as reactivation in immunocompromised individuals. Currently, there is a need for improved quantitative serological tests to document seropositivity with high sensitivity and specificity. Methods Here we investigated whether luciferase immunoprecipitation systems (LIPS would provide a more quantitative and sensitive method for detecting anti-CMV antibodies. Four protein fragments of immunodominant regions of CMV antigens pp150 and pp65 were generated as Renilla luciferase (Ruc fusion proteins and used in LIPS with two cohorts of CMV positive and negative sera samples previously tested by ELISA. Results Analysis of the antibody responses to two of these antigen fragments, pp150-d1 and pp150-d2, revealed geometric mean antibody titers in the first cohort that were 100–1000 fold higher in the CMV positive sera compared to the CMV negative samples (p rs = 0.93, p Conclusion These results suggest that LIPS provides a highly robust and quantitative method for studying anti-CMV antibodies and has the potential to more accurately document CMV infection than standard ELISA.

  9. 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.

  10. Construction of recombinant baculovirus Ac-CMV-hSox9 for gene therapy of intervertebral disc degeneration

    Institute of Scientific and Technical Information of China (English)

    LIU Xiao-yun; YANG Shu-hua; LIANG Chang-yong; SONG Jian-hua; LI Kang-hua; CHEN Xin-wen

    2007-01-01

    Objective: To construct the recombinant baculovirus Ac-cytomegalovirus (CMV)-hSox9 for gene therapy of intervertebral disc degeneration. Methods: Bac-to-Bac system was used for the construction of baculovirus Ac-CMV-hSox9. The cDNA of hSox9 was first cloned into a plasmid vector under the control of CMV promotor to generate the donor plasmid pFastBacDul-green fluorescene protein (GFP)-CMV (pFGC)-hSox9.The resultant plasmid was transformed into DH10Bac cells and then the transformation mixture was spread on Luria-Bertani (LB) agarose culture medium containing isopropyl-β-D-thiogalactoside (IPTG), X-gal, gentamicin, kanamycin and tetracycline.The white colonies were selected and cultured for amplification, and the hSox9Bacmid DNA was extracted. After verification, recombinant baculovirus Ac-CMV-hSox9 was obtained through transfecting Sf 21 cells.The expression of hSox9 gene in the intervertebral disc cells in rabbits was determined by Western blotting and immunohistochemical staining.Results: Polymerase chain reaction (PCR) confirmed the presence of hSox9 gene in the recombinant baculovirus and the Sf 21 cells transfected by the baculovirus showed the expression of fluorescence protein.Western blotting and immunohistochemical staining analysis indicated that exogenous hSox9 gene was expressed in the disc cells.Conclusions: The successful construction of the recombinant baculovirus Ac-CMV-hSox9 and the confirmation of the target gene expression provides a novel expression vector system for basic research and clinical treatment of intervertebral degenerative disc disease.

  11. Recombination with coat protein transgene in a complemen-tation system based on Cucumber mosaic virus (CMV)

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    In order to study the feasibility of Cucumber mosaic virus (CMV) as an expression vector, the full-length cDNA of RNA 3 from strain SD was cloned and the sequence around the start codon of the coat protein (CP) gene was modified to create an NsiⅠ site for insertion of foreign genes. The CP gene was replaced by the green fluorescent protein (GFP) gene. The cDNAs of Fny RNAs 1 and 2 and the chimeric SD RNA 3 were cloned between the modified 35S promoter and terminator. Tobacco protoplasts were transfected with a mixture of the viral cDNAs containing 35S promoter and terminator as a replacement vector and expressed GFP. A complementation system was established when the replacement vector was inoculated onto the transgenic tobacco plants ex-pressing SD-CMV CP. GFP was detected in the inoculated leaves in 5 of 18 tested plants and in the first upper systemic leaf of one of the 5 plants ten days after inoculation. However, no GFP could be detected in all the plants one month after inoculation. Recombination between the CMV vector and the CP transgene was proved by retro-transcriptional polymerase chain reaction (RT-PCR) and verified by DNA sequencing. Our results argue against the feasibility of the CMV-based replace-ment vector trans-complemented by the CP transgene, and at the same time, enlighten ways to improve the CMV-based expression vector and the biosafety of CMV CP-mediated virus resistant transgenic plants.

  12. AdCMV-TK和AdCMV-P53联合治疗胶质瘤的体外实验%Combining AdCMV-TK with AdCMV-p53 for gene therapy of glioma in vitro

    Institute of Scientific and Technical Information of China (English)

    王金环; 房欣; 史国利; 蔡宝立

    2002-01-01

    目的为了探讨联合应用单纯疱疹病毒-胸苷激酶和野生型p53两种基因治疗胶质瘤的可能性.方法构建了含CMV启动子和单纯疱疹病毒胸苷激酶基因(HSV-TK)的重组腺病毒AdCMV-TK和含CMV启动子及野生型p53的重组腺病毒AdCMfV-p53.以感染复数(MOI)为100的病毒剂量,用AdCMV-TK和AdCMV-p53同时感染人胶质母细胞瘤细胞系TJ905,并以100MOI的AdCMV-TK和AdCMV-p53分别感染另外两组TJ905细胞.除感染Ad-CMV-p53的细胞外,其他两组均于感染后加入100 μmol/L GCV.结果感染AdCMV-p53的TJ905细胞,其生长受到抑制,于感染后第5 d,细胞存活率下降为36.9%,感染AdCMV-TK的细胞,其生长抑制程度与感染AdCMV-p53的TJ905细胞相似,用药后第5 d,细胞存活率为30.1%,而同时感染两种重组腺病毒的细胞,细胞生长的抑制程度更明显,应用100μmol/L GCV后第5 d,细胞存活率仅为8.8%.结论联合应用AdCMV-TK和AdCMV-p53基因治疗胶质瘤是可行的.

  13. Preface of the Special Issue: “Recent CMV Research”

    Directory of Open Access Journals (Sweden)

    Kayla Dufrene

    2014-01-01

    Full Text Available This Viruses Special Issue on Recent Cytomegalovirus (CMV Research is dedicated to the patients who have suffered CMV infection and to their parents, families and caregivers. We are including as a Preface to this issue the insights of a young college student, Kayla Dufrene, who suffered congenital CMV infection and contacted me and Dr. Roberta DeBiasi, to interview us to learn more about CMV. As I was just returning to the DC area from the 4th Congenital CMV Conference in San Francisco, I was particularly receptive to her request. When we met Kayla, we were both impressed with her personal strength and ability to cope with her disabilities and needed medical treatments. Despite it all, Kayla has an exceptionally positive outlook on life, feeling even lucky. She has not only coped, but has transcended her difficulties. I am proud to say that she was on the Dean’s List (Figure 1 at Gallaudet University. Ultimately, her hope lies in our fields’ efforts to develop a vaccine to prevent CMV disease in other children.

  14. Serological and molecular detection of an isolate of Cucumber Mosaic Virus (CMV infecting cucumber (Cucumis sativus and cloning of its coat protein gene

    Directory of Open Access Journals (Sweden)

    Prashant Shetti

    2012-08-01

    Full Text Available Cucumber mosaic virus (CMV is a widely prevalent plant virus infecting important vegetable, plantation and flower crops. Methods for early detection of viruses in plants and vectors transmitting them play a critical role in plant virus disease management. Direct plate and Dot- Enzyme Linked Immunosorbent Assay (ELISA was standardized for detection of CMV. Optimum OD of 1.249 (1.9 ng/μl and 1.242 (1.52 ng/μl was observed in 1:20 and 1:50 dilution of crude and ultrapurified antigen respectively, at a dilution of 1:1000 of both primary and secondary antibody. Polymerase Chain Reaction (PCR using CMV coat protein (CMV CP gene specific primers amplified a 657 base pair (bp fragment, which was then  cloned in pTZ57R/T cloning vector and positive clones were identified by band shift assay and colony PCR. This will aid in developing field diagnostic kits for detection of CMV in different crops and also in developing transgenics with the CP gene. 

  15. Humoral and cellular CMV responses in healthy donors; identification of a frequent population of CMV-specific, CD4+ T cells in seronegative donors

    DEFF Research Database (Denmark)

    Loeth, Nina; Assing, Kristian; Madsen, Hans O;

    2012-01-01

    .e., T lymphocyte) assays. Here, we have analyzed the CMV status of 100 healthy blood bank donors using both serology and cellular assays. About half (56%) were found to be CMV seropositive, and they all mounted strong CD8+ and/or moderate CD4+ T cell responses ex vivo against the immunodominant CMV...

  16. The value of CMV IgG avidity and immunoblot for timing the onset of primary CMV infection in pregnancy.

    Science.gov (United States)

    Enders, Gisela; Daiminger, Anja; Bäder, Ursula; Exler, Simone; Schimpf, Yvonne; Enders, Martin

    2013-02-01

    Primary CMV infections in pregnancy are usually asymptomatic and only detected by serology. Estimating the onset of infection is a major diagnostic goal, since primary infections around conception and in early gestation hold a higher risk for congenital disease than those in later pregnancy. To assess the ability of serological supplementary CMV assays to date the onset of primary infection. From our routine diagnosis we identified 61 pregnant women (n=188 serum samples) with precisely determined onset of CMV primary infection either by IgG seroconversion (n=24) or by significant IgG antibody rise (n=37). One hundred and forty-seven sera were investigated using the VIDAS(®) CMV IgG avidity EIA (BioMèrieux) and 83 sera using the recomBlot CMV IgG with avidity (Mikrogen). Both assays proofed to be reliable in terms of timing the onset of CMV primary infection. An avidity index (AI) in the VIDAS avidity EIA of <40% indicated primary infection within the last 20 weeks (positive predictive value 93.4%; 99/106), whereas an intermediate AI excluded primary infection within the last 12 weeks (negative predictive value 88.2%; 15/17). The recomBlot showed high reliability (PPV 96.9%; 31/33) for timing the onset of infection within the last 14 weeks. Avidity testing by blot however could not be interpreted in 11 of 47 sera (23.4%). For timing the onset of infection (before or in early pregnancy) CMV avidity testing is most helpful if carried out within the first trimester up to the beginning of second trimester. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Serological cross reactivity to CMV and EBV causes problems in the diagnosis of acute hepatitis E virus infection.

    Science.gov (United States)

    Hyams, Catherine; Mabayoje, Diana A; Copping, Ruth; Maranao, Desmond; Patel, Mauli; Labbett, Wendy; Haque, Tanzina; Webster, Daniel P

    2014-03-01

    Hepatitis E virus (HEV) infection is an important public health concern as a major cause of enterically-transmitted hepatitis worldwide. The detectable window of viraemia is narrow, and HEV IgM and IgG rise simultaneously in acute infection. Furthermore, previous investigators have shown HEV IgM false positive reactions occur against EBV, CMV and potentially hepatitis A. A retrospective analysis of HEV serology testing was performed at a London tertiary referral hospital over a 3-year period. A thousand four hundred and twenty three serum samples were tested for HEV serology, with 33 samples HEV IgM positive and 28 HEV IgM equivocal. One hundred and eleven samples were HEV IgG positive but IgM negative suggesting past infection. No patients with HEV IgM positivity had false positive reactions against hepatitis A. A high degree of EBV and CMV cross reactivity was noted, with 33.3% and 24.2% of HEV IgM positive samples also testing positive for EBV and CMV IgM, respectively. HEV RNA was detected in four HEV IgM positive samples, indicating true positivity, although three demonstrated cross reactivity against EBV. Only 13.3% of samples with positive HEV IgM were HEV PCR positive, highlighting a low positive predictive value of serology testing. Overall a high level of HEV, EBV and CMV IgM cross reactivity was demonstrated, indicating that serology is unreliable in the diagnosis of acute viral hepatitis. It is concluded that that the diagnosis of viral hepatitis should be based on clinical features, raised transaminases, serology, and confirmatory PCR testing.

  18. Cytomegalovirus (CMV) Infection: A Guide for Patients and Families After Stem Cell Transplant

    Science.gov (United States)

    ... Infection: A Guide for Patients and Families after Stem Cell Transplant What is cytomegalovirus (CMV)? Cytomegalovirus (CMV), a ... weakened by medicines that you must take after stem cell transplant and by the transplant itself. Your body ...

  19. 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.

  20. Replication of CMV in the gut of HIV-infected individuals and epithelial barrier dysfunction

    Science.gov (United States)

    Somsouk, Ma; Hunt, Peter W.

    2017-01-01

    Although invasive cytomegalovirus (CMV) disease is uncommon in the era of antiretroviral therapy (ART), asymptomatic CMV coinfection is nearly ubiquitous in HIV infected individuals. While microbial translocation and gut epithelial barrier dysfunction may promote persistent immune activation in treated HIV infection, potentially contributing to morbidity and mortality, it has been unclear whether CMV replication in individuals with no symptoms of CMV disease might play a role in this process. We hypothesized that persistent CMV replication in the intestinal epithelium of HIV/CMV-coinfected individuals impairs gut epithelial barrier function. Using a combination of state-of-the-art in situ hybridization technology (RNAscope) and immunohistochemistry, we detected CMV DNA and proteins and evidence of intestinal damage in rectosigmoid samples from CMV-positive individuals with both untreated and ART-suppressed HIV infection. Two different model systems, primary human intestinal cells differentiated in vitro to form polarized monolayers and a humanized mouse model of human gut, together demonstrated that intestinal epithelial cells are fully permissive to CMV replication. Independent of HIV, CMV disrupted tight junctions of polarized intestinal cells, significantly reducing transepithelial electrical resistance, a measure of monolayer integrity, and enhancing transepithelial permeability. The effect of CMV infection on the intestinal epithelium is mediated, at least in part, by the CMV-induced proinflammatory cytokine IL-6. Furthermore, letermovir, a novel anti-CMV drug, dampened the effects of CMV on the epithelium. Together, our data strongly suggest that CMV can disrupt epithelial junctions, leading to bacterial translocation and chronic inflammation in the gut and that CMV could serve as a target for therapeutic intervention to prevent or treat gut epithelial barrier dysfunction during HIV infection. PMID:28241080

  1. Monitoraggio di infezione da Citomegalovirus (CMV nel paziente sottoposto a trapianto di intestino

    Directory of Open Access Journals (Sweden)

    Giulia Nardini

    2005-03-01

    Full Text Available Six intestinal transplanted patients were monitored for CMV reactivation by pp65 antigenemia. In addition, the CMV viral load of intestinal biopsies was quantified to verify on a possible clinical meaning of this viral marker in order to the organ reject. No relationship emerged between this event and high amounts of CMV genomes in intestinal tissues.

  2. Humoral and cellular CMV responses in healthy donors; identification of a frequent population of CMV-specific, CD4+ T cells in seronegative donors.

    Science.gov (United States)

    Loeth, Nina; Assing, Kristian; Madsen, Hans O; Vindeløv, Lars; Buus, Soren; Stryhn, Anette

    2012-01-01

    CMV status is an important risk factor in immune compromised patients. In hematopoeitic cell transplantations (HCT), both donor and recipient are tested routinely for CMV status by serological assays; however, one might argue that it might also be of relevance to examine CMV status by cellular (i.e., T lymphocyte) assays. Here, we have analyzed the CMV status of 100 healthy blood bank donors using both serology and cellular assays. About half (56%) were found to be CMV seropositive, and they all mounted strong CD8+ and/or moderate CD4+ T cell responses ex vivo against the immunodominant CMV protein, pp65. Of the 44 seronegative donors, only five (11%) mounted ex vivo T cell responses; surprisingly, 33 (75%) mounted strong CD4+ T cell responses after a brief in vitro peptide stimulation culture. This may have significant implications for the analysis and selection of HCT donors.

  3. CMV-hFasL transgenic mice are sensitive to low doses of streptozotocin-induced type Ⅰ diabetes mellitus%CMV-hFasL转基因小鼠容易被小剂量的链尿菌素诱导至糖尿病

    Institute of Scientific and Technical Information of China (English)

    林波; 章振林; 余路阳; 郭礼和

    2003-01-01

    AIM: To investigate the role of Fas-FasL pathway in the pathogenesis of streptozotocin (STZ)-induced type I diabetes mellitus. METHODS: Low dose injections of STZ were used to induce type I diabetes mellitus in the CMV-hFasL transgenic mice. Blood glucose concentration was measured with Glucotrand Plus blood glucose test strips. Expression of hFasL was detected by RT-PCR and Western blotting. The severity of insulitis was determined by histologicalexamination. Expressions of IL- 1 β and TNF-α mRNA in the pancreas were detected by semi-quantitative RT-PCR analysis. Fas expression in apoptotic RIN-5F cells was also confirmed by RT-PCR in vitro. RESULTS: hFasL was expressed in the islets of CMV-hFasL transgenic mice. The transgenic mice were sensitive to diabetic induction than the control WT mice. IL-1 β and TNF-α expressions in the pancreas of CMV-hFasL transgenic mice were far more than that in WT mice. We also found STZ and IL-1β could both induce higher expression of Fas in RIN-5F. The combining of Fas-FasL could lead to the apoptosis of β cells in the CMV-hFasL transgenic mice. CONCLUSION: Fas-FasL interaction plays a significant role in the pathogenic mechanism of type I diabetes mellitus.

  4. Murine model for congenital CMV infection and hearing impairment

    Directory of Open Access Journals (Sweden)

    Tao Liu

    2011-02-01

    Full Text Available Abstract Background Congenital cytomegalovirus (CMV infection is the leading cause of sensorineural hearing loss (SNHL, and SNHL is the most frequent sequela of congenital CMV infection. But the pathogenic mechanism remains unknown, and there is no ideal CMV intrauterine infection animal model to study the mechanisms by which SNHL develops. Methods We established the congenital murine cytomegalovirus (MCMV infection model by directly injecting the virus into the placenta on day 12.5 of gestation. Then, we observed the development and the MCMV congenital infection rate of the fetuses on the day they were born. Furthermore, we detected the auditory functions, the conditions of the MCMV infection, and the histological change of the inner ears of 28-day-old and 70-day-old offspring. Results Both the fetal loss rate and the teratism rate of offspring whose placentas were inoculated with MCMV increased, and their body length, head circumference, and weight decreased. The hearing level of offspring both decreased at both 28- and 70-days post birth; the 70-day-old mice developed lower hearing levels than did the 28-day old mice. No significant inflammatory changes in the cochleae of the mice were observed. MCMV DNA signals were mainly detected in the spiral ganglion neurons and the endolymph area, but not in the perilymph area. The number of neurons decreased, and their ultrastructures changed. Moreover, with age, the number of neurons dramatically decreased, and the ultrastructural lesions of neurons became much more severe. Conclusions The results suggest that the direct injection of MCMV into the placenta may efficiently cause fetal infection and disturb the intrauterine development of the fetus, and placental inoculation itself has no obvious adverse effects on offspring. The reduction in the number of spiral ganglion neurons and the ultrastructural lesions of the neurons may be the major cause of congenital CMV infection-induced progressive SNHL.

  5. Medical image of the week: CMV cytopathic effect

    Directory of Open Access Journals (Sweden)

    Sam A

    2014-12-01

    Full Text Available No abstract available. Article truncated at 150 words. Bronchoalveolar lavage (BAL was performed on a 45-year old man with a history of treated mycosis fungoides and Sézary syndrome, who presented with fever and pulmonary infiltrates. BAL Papanicolaou stain (Figure 1, 400x showed single cells (lymphocytes, arrows and alveolar macrophages, stars and a small cluster of 3 large cells, most likely infected type II pneumocytes, with a single prominent red stained nuclear inclusion surrounded by a clear halo. Nuclear chromatin was marginated on the nuclear membrane creating this “owl’s eye” appearance. In vitro, infected cells show cytomegalovirus (CMV virions within the nuclear inclusion (Figure 2, small black dots encircled, 8,800x The "owl's eye" appearance (Figure 1 is the “cytopathic effect” needed to definitively diagnose active CMV infection. While cells infected with adenovirus or herpesvirus may have nuclear inclusions, the cells typically are much smaller. CMV was cultured from the BAL, and no other pathogen was identified by cytology or ...

  6. 黄疸婴幼儿巨细胞病毒检测及临床意义%The clinical sense of testing CMV in jaundice infants

    Institute of Scientific and Technical Information of China (English)

    刘怡; 路白杨; 陈卫斌

    2013-01-01

    目的 探讨黄疸婴幼儿检测巨细胞病毒的临床意义.方法 将74例黄疸婴幼患儿分为两组,其中阳性组为34例CMV阳性患儿;对照组为40例CMV阴性患儿.采用酶联免疫吸附实验(ELISA)法,检测血清CMV-IgM;用FQ-PCR法检测尿CMV-DNA;血清肝功能检查.对两组肝功能测定结果比较分析.结果 CMV阳性组和对照组的丙氨酸氨基转移酶(ALT)、门冬氨酸氨基转移酶(AST)、碱性磷酸酶(ALP)、总胆红素(TB)、直接胆红素(DB)、总胆汁酸(TBA),检测结果均有统计学意义(P<0.01).阳性组ALT、AST、ALP、TBA检测结果明显高于对照组.结论 婴幼儿黄疸,CMV感染不容忽视,肝功能受损,治疗困难,重在预防.母婴传播是小儿CMV感染的主要途径,育龄妇女在怀孕前做CMV检查,对优生优育,有重要作用.%Objective:To investigate the clinical sense of testing CMV in Jaundice infants.Methods:Divided 74 Jaundice infants into two groups,while the positive group included 34 examples and the contrast group were 40 negative infants.Employing the Enzyme linked immunosorbent assay (ELISA) to test sertum CMV-IgM,adopting FQ-PCR to detect CMV-DNA in urine,applying Serum liver function tests to analysis comparatively the results of two groups.Results:The test results of ALT,AST,ALP,TB,DB,TBA of two groups were rich in statistical significance (P < 0.01).The results of ALT,AST,ALP,TBA in positive group were obviously higher than the comparable group.Conclusion:It can not be ignored that Jaundice infants infected CMV were difficult in treatment as a result of liver function was impaired.Mother-to-child transmission is the mainly route in infants infected CMV.Women with childbearing age in CMV examination before pregnancy to birth have an important role in prenatal and postnatal care.

  7. Cytomegalovirus (CMV) IE1- and pp65-specific CD8+ T cell responses broaden over time after primary CMV infection in infants.

    Science.gov (United States)

    Gibson, Laura; Dooley, Sheryl; Trzmielina, Sonia; Somasundaran, Mohan; Fisher, Donna; Revello, Maria Grazia; Luzuriaga, Katherine

    2007-06-15

    Cytomegalovirus (CMV) infection remains a significant cause of morbidity and mortality in young children. We have previously shown that CD8+ T cell responses to CMV pp65 or IE1 protein were readily detectable in children with congenital or postnatal CMV infection. Here, we have further characterized the evolution of the peptide specificity of these responses in 7 infants<6 months of age at the start of the study. Thirteen pp65 and 15 IE1 peptides (median, 5 peptides/infant) were targeted, and most (61%) represented sequences not previously reported. Peptide specificity remained stable or broadened over time despite the clearance of CMV viremia. Loss of peptide recognition was not observed. Responses with the highest functional peptide avidity were not necessarily detected earliest. These data provide additional evidence that young infants can generate diverse CMV-specific CD8+ T cell responses but show that early responses may exhibit relatively focused peptide specificity and lower peptide avidity.

  8. Concentração espermática e tempo de incubação na fecundação in vitro usando-se sêmen de touros da raça Guzerá Sperm concentration and incubation period on in vitro fecundation using sperm of Guzera bulls

    Directory of Open Access Journals (Sweden)

    L.P.B. Dias

    2006-06-01

    Full Text Available Estudou-se o efeito da concentração espermática e período de incubação e da interação dessas características sobre a fecundação in vitro (FIV usando-se sêmen de touros Guzerá. Oócitos (n=1146 maturados in vitro foram divididos em tratamentos objetivando a FIV, em esquema fatorial 3×2×2 (três touros - A, B e C, duas concentrações espermáticas - 2 e 4×10(6 espermatozóides/ml e dois tempos de incubação 12 e 18 horas. Utilizaram-se espermatozóides viáveis obtidos por swin-up. A FIV foi realizada em meio fert-talp com heparina, em incubadora com 5% de CO2 e 95% de umidade, a 38,5°C. Após incubação, 50% dos oócitos foram fixados e corados para determinação das taxas de penetração, fecundação monoespermática e poliespermia. O restante foi co-cultivado com células da granulosa em meio CR2aa por oito dias, avaliando-se a taxa de clivagem e a produção de blastocisto. Houve maior taxa de penetração (P0,05 entre os demais tratamentos. A taxa de poliespermia foi maior (PThe effects of sperm concentration, incubation period and their interaction on in vitro fertilization (IVF using sperm of Guzera bulls were evaluated. In vitro matured oocytes (n=1146 were allotted to IVF treatments in a factorial scheme 3×2×2 - three bulls (A, B and C, two sperm concentrations (2 and 4×10(6 spermatozoa/ml and two incubation periods (12 and 18h. Viable spermatozoa were obtained by swim-up. The IVF was performed using in Fert-Talp medium with heparin, on incubator with 5% CO2 and 95% humidity, at 38.5ºC. After the incubation, 50% of oocytes were fixed and stained to determine penetration, monospermic fecundation and polyspermy rates. The remainder was co-cultured with granulosa cells in CR2 medium for eight days to evaluate cleavage and embryo production rates. The penetration rate was higher (P0.05 among remainder treatments were observed. The polyspermic rate (P<0.05 was higher for higher sperm concentration and incubation

  9. Clinical evaluation of the Roche Elecsys CMV IgG Avidity assay.

    Science.gov (United States)

    Vauloup-Fellous, C; Lazzarotto, T; Revello, M G; Grangeot-Keros, L

    2014-08-01

    Congenital cytomegalovirus (CMV) infection has potentially severe consequences in newborns. The testing of pregnant women for CMV-specific antibodies may be useful for the identification of women at risk of transmitting the infection to the fetus. The determination of CMV IgG avidity helps to establish the timing of infection as IgG avidity matures during the course of infection. This study examines the performance of the Elecsys CMV IgG Avidity assay using preselected samples from patients at different phases of CMV infection. The Elecsys CMV IgG Avidity assay was tested at three sites using sequential samples from patients with recent primary CMV infection, as well as single samples from patients with recent primary or past CMV infection. The Elecsys assay discriminated well between early (low avidity) and late (high avidity) phases of infection in sequential serum samples. Overall, 98.8% of low-avidity samples corresponded to infection onset 90 days before sampling. The assay's sensitivity was 90-97%, with specificity ranging from 89 to 100%, depending on the consideration of gray-zone avidity values. Single samples from recent primary or past infection showed similar distributions of avidity results. The Elecsys CMV IgG Avidity assay results are in agreement with preselected samples from patients with primary or past CMV infection, showing that the test is an adequate predictor of the phase of infection.

  10. Allergenicity assessment of genetically modified cucumber mosaic virus (CMV) resistant tomato (Solanum lycopersicon).

    Science.gov (United States)

    Lin, Chih-Hui; Sheu, Fuu; Lin, Hsin-Tang; Pan, Tzu-Ming

    2010-02-24

    Cucumber mosaic virus (CMV) has been identified as the causal agent of several disease epidemics in most countries of the world. Insect-mediated virus diseases, such as those caused by CMV, caused remarkable loss of tomato (Solanum lycopersicon) production in Taiwan. With expression of the CMV coat protein gene (Cmvcp) in a local popular tomato cultivar L4783, transgenic tomato line R8 has showed consistent CMV resistance through T(0) to T(8). In this report, the allergenicity of the CMV coat protein (CMV cp) expressed in transgenic tomato R8 was assessed by investigation of the expression of the transgene source of protein, sequence similarity with known allergens, and resistance to pepsin hydrolysis. There is no known account for either the CMV or its coat protein being an allergen. The result of a bioinformatic search also showed no significant homology between CMV cp and any known allergen. The pepsin-susceptible property of recombinant CMV cp was revealed by a simulated gastric fluid (SGF) assay. Following the most recent FAO/WHO decision tree, all results have indicated that CMV cp was a protein with low possibility to be an allergen and the transgenic tomato R8 should be considered as safe as its host.

  11. Treatment of Cytomegalovirus Infection with Cidofovir and CMV Immune Globulin in a Lung Transplant Recipient

    Directory of Open Access Journals (Sweden)

    Heinrike Wilkens

    2016-01-01

    Full Text Available Cytomegalovirus (CMV infection after lung transplantation is associated with increased risk for pneumonitis and bronchiolitis obliterans as well as allograft rejection and opportunistic infections. Ganciclovir is the mainstay of prophylaxis and treatment but CMV infections can be unresponsive. Apart from direct antiviral drugs, CMV immunoglobulin (CMVIG preparations may be considered but are only licensed for prophylaxis. A CMV-seronegative 42-year-old man with cystic fibrosis received a lung from a CMV-seropositive donor. Intravenous ganciclovir prophylaxis was delayed until day 12 due to acute postoperative renal failure and was accompanied by five doses of CMVIG (10 g. By day 16, CMV-DNA was detectable and rising; CMV-specific T-cells were undetectable. Switch from ganciclovir to foscarnet prompted a transient decrease in CMV viral load, but after increasing again to reach 3600 copies/mL foscarnet was changed to intravenous cidofovir and CMVIG was restarted. CMV load continued to fluctuate and declined slowly, whereas CMV-specific T-cells were detected five months later and increased thereafter. At last follow-up, the patient was in very good clinical condition with no evidence of bronchiolitis obliterans. No side effects of this treatment were observed. In this hard-to-treat case, the combination of cidofovir with off-label use of CMVIG contributed to a successful outcome.

  12. Detection of EBV, CMV and HSV-1 in subgingival samples of HIV positive and negative patients with chronic periodontitis.

    Directory of Open Access Journals (Sweden)

    Laura Escalona

    2016-06-01

    Full Text Available Objective: To detect the presence of infection by EBV (Epstein-Barr Virus, CMV (Cytomegalovirus and HSV-1 (Herpes Simplex Virus type 1 in subgingival samples from HIV- positive patients under HAART (High Activity Antiretroviral Therapy, HIV- positive patients without HAART, HIV-negative patients with chronic periodontitis and healthy controls. Methodology: Crevicular fluid samples of 11 HIV+ patients on therapy were evaluated, 6 without antiretroviral therapy, 7 HIV- negative subjects with chronic periodontitis and 7 periodontally-healthy controls. PI (Plaque index, GI (Gingival Index, PD (probing depth and CAL (Clinical Attachment Loss were registered at six sites per each tooth in all teeth and subgingival plaque samples of a tooth were collected per quadrant. Nested PCR was used to detect EBV and endpoint PCR to detect infection by CMV and HSV-1. Results: Clinical parameters showed statistically significant differences between HIV-positive patients and subjects with chronic periodontitis compared with the control group (p<0.05. DNA of EBV was detected mainly in HIV-positive patients under HAART, 91% (10/11. DNA of CMV was detected mainly in patients without HAART, 67% (4/6, while HSV-1 was observed in 27% (3/11 of patients under HAART. In the control group no virus was detected. Coinfection was observed in 50% of HIV patients without HAART, 36% of HIV patients with HAART and 14% of HIV-negative with chronic periodontitis. Conclusion: Viral infection was prevalent in HIV patients under HAART and EBV was the primary viral infection detected in HIV-positive patients with chronic periodontitis.

  13. Development and Evidence for Efficacy of CMV Glycoprotein B Vaccine with MF59 Adjuvant

    OpenAIRE

    Pass, Robert F.

    2009-01-01

    A vaccine comprised of recombinant cytomegalovirus (CMV) envelope glycoprotein B (gB) with MF59 adjuvant developed in the 1990s recently was recently found to have efficacy for prevention of CMV infection in a phase 2 clinical trial in young mothers. This review briefly considers the rationale for gB as a vaccine antigen, the history of this CMV gB vaccine and the data supporting vaccine efficacy.

  14. DETECCIÓN DE LOS VIRUS AMV, CMV Y PLRV EN CULTIVOS DE TOMATE DE ÁRBOL (SOLANUM BETACEUM CAV. EN ANTIOQUIA, COLOMBIA DETECTION OF VIRUSES AMV, CMV AND PLRV IN TAMARILLO (SOLANUM BETACEUM CAV. ORCHARDS IN ANTIOQUIA, COLOMBIA

    Directory of Open Access Journals (Sweden)

    Margarita Jaramillo Zapata

    2011-06-01

    Full Text Available El tomate de árbol es uno de los principales frutales cultivados en la región andina de Colombia. Durante los últimos años las áreas dedicadas a su plantación se han reducido drásticamente, debido entre otras razones, al efecto detrimental de la enfermedad conocida como Virosis. A pesar de la importancia económica de esta enfermedad, el nivel de conocimiento que se tiene en el país, sobre la identidad taxonómica de los virus asociados a su ocurrencia es precario y por tanto, se carece de buena parte de la información necesaria para el diseño de estrategias de manejo efectivas contra esta enfermedad. En este trabajo se evaluó la presencia e incidencia de los virus isométricos AMV, CMV y PLRV en cultivos de tomate de árbol establecidos en el departamento de Antioquia, mediante el empleo de pruebas de ELISA, RT-PCR y secuenciación de la cápside viral. Las pruebas serológicas permitieron detectar la presencia de los tres virus, aunque las secuencias generadas sólo fueron confirmatorias para el virus PLRV, lo que posiblemente sugiere que existen diferencias en las secuencias dirigidas a los sitios de alineamiento de los cebadores para AMV y CMV. La incidencia de los virus CMV y PLRV en las muestras analizadas fue de un 57 y 41% respectivamente, mientras que para AMV ésta solo llegó a un nivel del 2%. Los hallazgos obtenidos permiten confirmar la presencia de un complejo viral asociado a la enfermedad de la Virosis del tomate de árbol en el territorio mencionado.Tamarillo is one of the main fruit crops in the Colombian andean region. However, during the past years, there has been a drastic reduction in the total crop area due to the detrimental effect of a disease known as Virosis. In spite of the economic impact of this disease, there is little knowledge about the taxonomical identity of its associated viruses; therefore, no information is available for the design of appropriate control strategies. In this work, was evaluated

  15. Seronegative invasive gastro-intestinal cytomegalovirus disease in renal allograft recipients a diagnostic dilemma! - Tissue PCR the saviour?

    Directory of Open Access Journals (Sweden)

    A Kaul

    2015-01-01

    Full Text Available Seronegative Invasive Gastro-intestinal cytomegalovirus disease in renal allograft recipients Background -CMV as oppurtunistic infection affecting the gastrointerstinal tract is the most common cause for tissue invasive CMV disease occuring in 10-30% of organ transplant recepients. Gastrointerstinal CMV disease can be diagnosed in presence of clinical suspecion along with histopathological findings (CMV inclusions and presence of mucosal lesion(s on endoscopic examination with collaborative evidences via molecular technique. Aims-Few cases of CMV infection affecting the gastrointerstinal tract show no evidences of dissemintion despite use of highly sensitive molecular techniques. We encountered 6 cases where in despite strong clinical suspecion of Gastrointerstinal CMV disease there were seronegative and endoscopic negative evidences for CMV, blind tissue biopsy yeilded positive results for CMV disease with excellent improvement with antiviral therapy. Conclusions-Blind biopsy specimen for tissue PCR could serve as saviour in an immunocompromised individiual who has a strong clinical symptomatology for GI-CMV disease in absence of viremia, normal endoscopy and histopathology, so that the early therapeutic interventions could help in excellent patient and graft survival.

  16. Transgenic accumulation of a defective cucumber mosaic virus (CMV) replicase derived double stranded RNA modulates plant defence against CMV strains O and Y in potato.

    Science.gov (United States)

    Ntui, Valentine Otang; Kynet, Kong; Azadi, Pejman; Khan, Raham Sher; Chin, Dong Poh; Nakamura, Ikuo; Mii, Masahiro

    2013-12-01

    Cucumber mosaic virus is an important plant pathogen with a broad host range encompassing many plant species. This study demonstrates the production of transgenic potato lines exhibiting complete resistance to cucumber mosaic virus strain O and Y by post transcriptional gene silencing. Two constructs were used, one, pEKH2IN2CMVai, contains inverted repeat of 1,138 bp fragment of a defective CMV replicase gene derived from RNA2 of cucumber mosaic virus strain O (CMV-O), while the other, TRV-based VIGS vector (pTRV2CMVai), contains the same fragment of the replicase gene, but without inverted repeat. These constructs were used to produce transgenic potato lines of cultivar 'Danshaku', a susceptible genotype to CMV. Transgenic lines derived from pEKH2IN2CMVai accumulated small interfering RNA (siRNA) before and after virus challenge, whereas those derived from pTRV2CMVai showed siRNA expression after virus challenge. When transgenic lines were challenged with CMV-O or CMV-Y, four lines exhibited complete (100%) resistance to both strains, whereas the other lines had high levels of resistance. Infectivity of CMV-O was lower than that of CMV-Y in the highly resistant plants. There were no significant differences with regard to resistance between plants derived from pEKH2IN2CMVai and those obtained from pTRV2CMVai. The presence of CMV-specific siRNA in the resistant phenotypes indicates that the resistance was acquired through RNA silencing.

  17. Circular β ensembles,CMV representation,characteristic polynomials

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    In this note we first briefly review some recent progress in the study of the circular β ensemble on the unit circle,where β > 0 is a model parameter.In the special cases β = 1,2 and 4,this ensemble describes the joint probability density of eigenvalues of random orthogonal,unitary and sympletic matrices,respectively.For general β,Killip and Nenciu discovered a five-diagonal sparse matrix model,the CMV representation.This representation is new even in the case β = 2;and it has become a powerful tool for studying the circular β ensemble.We then give an elegant derivation for the moment identities of characteristic polynomials via the link with orthogonal polynomials on the unit circle.

  18. Comparison between valganciclovir and aciclovir/valaciclovir for CMV prophylaxis in pediatric renal transplantation

    Directory of Open Access Journals (Sweden)

    M Fila

    2015-01-01

    Full Text Available Prophylaxis has dramatically decreased the occurrence of cytomegalovirus (CMV infection after renal transplantation. Optimal regimens of treatment remain controversial, especially in pediatric recipients. The aim of this study was to evaluate the effectiveness of valganciclovir (VGC versus aciclovir/valaciclovir (ACV in a pediatric renal transplant population. Data from 101 renal transplantations were retrospectively analyzed. Except those with R-/Dstatus, all patients received prophylaxis either with ACV, n = 39 or VGC, n = 38. Incidences of positive CMV antigenemia and disease, as well as the delay in relation to the prophylaxis, were collected during at least 12 months after the end of treatment. Positive CMV antigenemia was reported in 34 patients (ACV: 16, VGC: 16, no prophylaxis: 2. CMV disease occurred in 15 patients (ACV: 5; VGC: 8 (ns. For the majority of patients under VGC, positive CMV antigenemia occurred within the year following the withdrawal of prophylaxis (VGC: 14; ACV: 5, P <0.05, whereas it occurred during prophylaxis in 11 patients under ACV versus two under VGC (P <0.05. The over-all incidence of positive CMV antigenemia was similar between ACV and VGC prophylaxis. However, VGC was more efficient to prevent early CMV infection while patients treated with ACV had less CMV infection or disease after the end of the prophylaxis.

  19. Comparison between valganciclovir and aciclovir/valaciclovir for CMV prophylaxis in pediatric renal transplantation.

    Science.gov (United States)

    Fila, M; Dechartes, A; Maisin, A; Dossier, C; Zhao, W; Deschênes, G; Baudouin, V

    2015-01-01

    Prophylaxis has dramatically decreased the occurrence of cytomegalovirus (CMV) infection after renal transplantation. Optimal regimens of treatment remain controversial, especially in pediatric recipients. The aim of this study was to evaluate the effectiveness of valganciclovir (VGC) versus aciclovir/valaciclovir (ACV) in a pediatric renal transplant population. Data from 101 renal transplantations were retrospectively analyzed. Except those with R-/Dstatus, all patients received prophylaxis either with ACV, n = 39 or VGC, n = 38. Incidences of positive CMV antigenemia and disease, as well as the delay in relation to the prophylaxis, were collected during at least 12 months after the end of treatment. Positive CMV antigenemia was reported in 34 patients (ACV: 16, VGC: 16, no prophylaxis: 2). CMV disease occurred in 15 patients (ACV: 5; VGC: 8) (ns). For the majority of patients under VGC, positive CMV antigenemia occurred within the year following the withdrawal of prophylaxis (VGC: 14; ACV: 5, P ACV versus two under VGC (P ACV and VGC prophylaxis. However, VGC was more efficient to prevent early CMV infection while patients treated with ACV had less CMV infection or disease after the end of the prophylaxis.

  20. A Modified Peptide Stimulation Method for Efficient Amplification of Cytomegalovirus (CMV)-Specific CTLs

    Institute of Scientific and Technical Information of China (English)

    Guangping Ruan; Li Ma; Qian Wen; Wei Luo; Mingqian Zhou; Xiaoning Wang

    2008-01-01

    CMV-specific immunity is essential for control of human cytomegalovirus (HCMV) infection. Stem cell trans- plantation is used widely in the management of a range of diseases of the hemopoietic system. Patients are immunosuppressed profoundly in the early posttransplant period, and reactivation of cytomegalovirus (CMV) remains a significant cause of morbidity and mortality. Adoptive transfer of CMV-specific CD8+ T cell clones has been shown to reduce the rate of viral reactivation; however, the ex vivo production of cells for adoptive transfer is labor intensive and expensive. We report here a modified peptide stimulation method using CMV-specific epitope peptides to stimulate PBMCs for generation of CMV-specific CTLs. This method permits efficient amplification of CMV-specific CTLs and provides a large number of cells for FACS analysis from a single blood sample. Significantly, it achieves high frequencies of tetramer staining of CD8+ T cells allowing the data of different individuals to be easily compared and sequentially evaluated. Thus, this approach expands and selects HLA- restricted CMV-pp65-reactive T-cell lines of high specificity for potential adoptive immunotherapy. Cellular & Molecular Immunology. 2008;5(3):197-201.

  1. Construction and expression of recombinant plasmid pENTR-CMV-EGFP -hsa-mir-16-1/15a%重组质粒pENTR-CMV-EGFP-hsa-mir-16-1/15a的构建与表达

    Institute of Scientific and Technical Information of China (English)

    方潇碧; 张春鸿; 黄亚; 林森; 黄振校; 吴丽萍; 施清圆; 李文峰; 廖志苏

    2011-01-01

    目的:构建针对人鼻咽癌CNE-2Z细胞Bcl-2基因pENTR-CMV-EGFP-hsa-mir-16.1/15a真核表达质粒,转染至CNE-2Z细胞并检测其表达.方法:采用PCR法从重组质粒PGH-16-1/15a中获得16-1/15a-X2370G全长序列,在T4 DNA Ligase连接酶作用下连接入重组载体pENTR-CMV-EGFP.重组质粒经酶切及测序鉴定.将构建成功的重组质粒转染入人鼻咽癌细胞株CNE-2Z,用荧光显微镜观察转染结果.结果:重组质粒pENTR-CMV-EGFP-hsa-mir-16-1/15a经酶切与测序证实构建成功,转染至鼻咽癌CNE-2Z细胞后,荧光显微镜观察证实该重组质粒能在CNE-2Z中表达.结论:成功构建真核表达质粒DENTR-CMV-EGFP-hsa-mir-16-1/15a,并在鼻咽癌CNE-2Z细胞中得到表达,可用于进一步检测其抗肿瘤机制.%Objective To construct the eukaryotic expression plasmid pENTR-CMV-EGFP-hsa-mir-16-1/15a of human Bcl-2 gene,transfect it into nasopharyngeal carcinoma cells CNE-2Z and detect its expression.Methods The full-length cDNA of 16-1/15a-X2370G gene was amplified by PCR from the recombinant plasmid PCH-16-1/15a, then was linked with the recombinant vector pENTR-CMV-EGFP by T4 DNA Ligase. The recomhinant plasmid was identified by restriction enzyme digestion and sequence analvsis. Then the plasmid was correctly transfected into NPC cell CNE-2Z , and the effect of transfection was directly observed under fluorescence microscope. Results The green fluorescent protein was mainly detectd in the CNE-2Z cell , which suggested that the recomhinant plasmid was constructed successfully. Conclusion The eukaryotic expression plasmid pENTR-CMV-EGFP-hsa-mir-I6-1/15a was constructed successfully and expressed in NPC cell CNE-2Z. Its mechanism of antitumor will be further investigated.

  2. Genetic Influence on the Peripheral Blood CD4+ T-cell Differentiation Status in CMV Infection

    DEFF Research Database (Denmark)

    Goldeck, David; Larsen, Lisbeth Aagaard; Christiansen, Lene

    2016-01-01

    A latent infection with cytomegalovirus (CMV), a ubiquitous beta herpesvirus, is associated with an accumulation of late-differentiated memory T-cells, often accompanied by a reciprocal reduced frequency of early-differentiated cells (commonly also referred to as "naïve"). However, this impact...... heritability analysis confirmed a substantial contribution of genetics to the differentiation status of T-cells in CMV infection. The humoral (IgG) response to different CMV antigens also seems to be genetically influenced, suggesting that a similar degree of immune control of the virus in MZ twins might...

  3. Evaluation of reactivation of HSV1, HHV6, CMV and EBV in a population of patients undergoing allogeneic bone marrow transplantation

    Directory of Open Access Journals (Sweden)

    Caterina Matinato

    2011-06-01

    Full Text Available Herpes viruses are recognized as important pathogens as a result of viral reactivation in immunocompromised hosts, especially in patients undergoing bone marrow transplantation. Objectives of this study were the assessment of the reactivation of herpes virus HSV1, HHV6, CMV and EBV and the correlation between viral reactivation and progression of transplantation in a population of patients undergoing allogeneic bone marrow transplantation at Fondazione IRCCS Ospedale Maggiore Ca’ Granda Policlinico, Milan. Viral DNA was detected and quantified by Real Time PCR in a population of 35 patients undergoing allogeneic bone marrow transplantation. The viral reactivation was observed in 7 patients for HSV1 (20%, 6 patients for HHV6 (17.1%, 11 patients for CMV (31.4% and 4 patients for EBV (11.4%. Difference in the incidence of aGVDH between patients with viral reactivation versus those for which there was no reactivation was statistically significant. These data confirm the importance of monitoring viral load for the management of antiviral therapy in order to prevent CMV disease and complications related to herpes viruses reactivation.

  4. DETECTION OF CYTOMEGALOVIRUS(CMV) IMMEDIATE EARLY ANTIGEN IN KIDNEY BIOPSIES AND TRANSPLANT NEPHRECTOMIES

    Institute of Scientific and Technical Information of China (English)

    燕航; 薛武军; 田普训; 郭奇; 何晓丽

    2004-01-01

    Objective To investigate the relationship between CMV infection and renal allograft rejection. Methods 39 kidney biopsies and transplant nephrectomies were collected and investigated for CMV immediate early antigen by immunohistochemistry. Results In 14 out of 39 tissue specimens CMV immediate early antigen were found. 8 biopsies from normal donor kidneys were negative; only 1 (10%) in 10 tissue specimens with early stage acute rejection was positive; 5(55.6%) in 9 biopsies with late stage acute rejection and 8 (66.7%) in 12 tissue blocks with chronic rejection were positive. Compared with normal kidney tissues, the infections in tissues with early stage acute rejection didn't increase obviously, but increased obviously in kidney tissue specimens with late stage rejection and with chronic rejection (P<0.05). Conclusion CMV infection appears to contribute to late stage acute rejection and chronic rejection after renal transplantation.

  5. Antibodies to early EBV, CMV, and HHV6 antigens in systemic lupus erythematosus patients

    DEFF Research Database (Denmark)

    Rasmussen, N S; Draborg, A H; Nielsen, C T

    2015-01-01

    OBJECTIVES: We investigated the antibody levels against early antigens of Epstein-Barr virus (EBV), cytomegalovirus (CMV), and human herpesvirus 6 (HHV6) in systemic lupus erythematosus (SLE) patients and healthy controls, and further correlated these antibodies to haematology...

  6. Combined CMV- and HSV-1 brainstem encephalitis restricted to medulla oblongata.

    Science.gov (United States)

    Katchanov, J; Branding, G; Stocker, H

    2014-04-15

    We report a very rare case of a combined CMV- and HSV-1 isolated brainstem encephalitis restricted to medulla oblongata in a patient with advanced HIV disease. Neither limbic nor general ventricular involvement was detected on neuroimaging. The case highlights the importance of testing for HSV-1 and CMV in HIV-infected patients presenting with an isolated brainstem syndrome. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Risk factors of CMV replication after allogeneic hematopoietic stem cell transplantation in children and adolescents

    Directory of Open Access Journals (Sweden)

    S. N. Shiriaev

    2014-09-01

    Full Text Available Risk factors of CMV replication in early period after allo-HSCT (D0‑D100 were – myeloablative conditioning – HR 3.74 (1.67–8.37, р = 0.001; unrelated donor – HR 2.18 (0.86–5.26, р = 0.10; HLA-matched donor – HR 0.24 (0.05–1.06, р = 0,06. In late posttransplant period (from D+100 significant risk factors of CMV-reactivation were (according to multivariate analysis myeloablative conditioning – HR 13.17 (3.00–57.86, р = 0.001; combination of pretransplant remission of leukemia and using cyclosporine and methotrexate – HR 0.13 (0.03–0.50, р = 0.003; combination of aGVHD and CMV reactivation in early posttransplant period – HR 2.71 (0.86–8.50, р = 0.088; using of bone marrow – HR 0.37 (0.12–1.19, р = 0.095. We revealed the significant association of aGVHD and CMV-reactivation –OR 2.91 (1.07–7.92, р=0.006, and increased rate of cGVHD in patients with CMV replication at third month after allo-HSCT OR – 2.29 (1.03–5.08, р = 0.066. We revealed a tend to decreasing relapse risk in patients who had CMV-replication – HR 0.07 (0.004–1.17, р = 0.06. Cumulative incidence of CMV-disease was 28 %. CMV-disease was lethal in 44 % patients.

  8. Risk factors of CMV replication after allogeneic hematopoietic stem cell transplantation in children and adolescents

    Directory of Open Access Journals (Sweden)

    S. N. Shiriaev

    2014-01-01

    Full Text Available Risk factors of CMV replication in early period after allo-HSCT (D0‑D100 were – myeloablative conditioning – HR 3.74 (1.67–8.37, р = 0.001; unrelated donor – HR 2.18 (0.86–5.26, р = 0.10; HLA-matched donor – HR 0.24 (0.05–1.06, р = 0,06. In late posttransplant period (from D+100 significant risk factors of CMV-reactivation were (according to multivariate analysis myeloablative conditioning – HR 13.17 (3.00–57.86, р = 0.001; combination of pretransplant remission of leukemia and using cyclosporine and methotrexate – HR 0.13 (0.03–0.50, р = 0.003; combination of aGVHD and CMV reactivation in early posttransplant period – HR 2.71 (0.86–8.50, р = 0.088; using of bone marrow – HR 0.37 (0.12–1.19, р = 0.095. We revealed the significant association of aGVHD and CMV-reactivation –OR 2.91 (1.07–7.92, р=0.006, and increased rate of cGVHD in patients with CMV replication at third month after allo-HSCT OR – 2.29 (1.03–5.08, р = 0.066. We revealed a tend to decreasing relapse risk in patients who had CMV-replication – HR 0.07 (0.004–1.17, р = 0.06. Cumulative incidence of CMV-disease was 28 %. CMV-disease was lethal in 44 % patients.

  9. CMV reactivation after allogeneic HCT and relapse risk: evidence for early protection in acute myeloid leukemia.

    Science.gov (United States)

    Green, Margaret L; Leisenring, Wendy M; Xie, Hu; Walter, Roland B; Mielcarek, Marco; Sandmaier, Brenda M; Riddell, Stanley R; Boeckh, Michael

    2013-08-15

    The association between cytomegalovirus (CMV) reactivation and relapse was evaluated in a large cohort of patients with acute myeloid leukemia (AML) (n = 761), acute lymphoblastic leukemia (ALL) (n = 322), chronic myeloid leukemia (CML) (n = 646), lymphoma (n = 254), and myelodysplastic syndrome (MDS) (n = 371) who underwent allogeneic hematopoietic cell transplantation (HCT) between 1995 and 2005. In multivariable models, CMV pp65 antigenemia was associated with a decreased risk of relapse by day 100 among patients with AML (hazard ratio [HR] = 0.56; 95% confidence interval [CI], 0.3-0.9) but not in patients with ALL, lymphoma, CML, or MDS. The effect appeared to be independent of CMV viral load, acute graft-versus-host disease, or ganciclovir-associated neutropenia. At 1 year after HCT, early CMV reactivation was associated with reduced risk of relapse in all patients, but this did not reach significance for any disease subgroup. Furthermore, CMV reactivation was associated with increased nonrelapse mortality (HR = 1.31; 95% CI, 1.1-1.6) and no difference in overall mortality (HR = 1.05; 95% CI, 0.9-1.3). This report demonstrates a modest reduction in early relapse risk after HCT associated with CMV reactivation in a large cohort of patients without a benefit in overall survival.

  10. Coinfection with EBV/CMV and other respiratory agents in children with suspected infectious mononucleosis

    Directory of Open Access Journals (Sweden)

    Wei Cong

    2010-09-01

    Full Text Available Abstract Background Numerous studies have shown that Epstein-Barr virus (EBV and cytomegalovirus (CMV can infect immunocompetent patients simultaneously with other agents. Nonetheless, multiple infections with other agents in EBV/CMV-infected children have received little attention. We conducted a retrospective study of children with suspected infectious mononucleosis. Peripheral blood samples were analyzed by indirect immunofluorescence to detect EBV, CMV and other respiratory agents including respiratory syncytial virus; adenovirus; influenza virus types A and B; parainfluenza virus types 1, 2 and 3; Chlamydia pneumoniae and Mycoplasma pneumoniae. A medical history was collected for each child. Results The occurrence of multipathogen infections was 68.9%, 81.3% and 63.6% in the children with primary EBV, CMV or EBV/CMV, respectively, which was significantly higher than that in the past-infected group or the uninfected group (p C. pneumoniae in children with primary infection was as high as 50%, significantly higher than in the other groups (p Conclusion Our study suggests that there is a high incidence of multipathogen infections in children admitted with EBV/CMV primary infection and that the distribution of these pathogens is not random.

  11. A cucumber mosaic virus (CMV) RNA 1 transgene mediates suppression of the homologous viral RNA 1 constitutively and prevents CMV entry into the phloem.

    Science.gov (United States)

    Canto, T; Palukaitis, P

    2001-10-01

    Resistance to Cucumber mosaic virus (CMV) in tobacco lines transformed with CMV RNA 1 is characterized by reduced virus accumulation in the inoculated leaf, with specific suppression of accumulation of the homologous viral RNA 1, and by the absence of systemic infection. We show that the suppression of viral RNA 1 occurs in protoplasts from resistant transgenic plants and therefore is not due to a host response activated by the cell-to-cell spread of virus. In contrast, suppression of Tobacco rattle virus vectors carrying CMV RNA 1 sequences did not occur in protoplasts from resistant plants. Furthermore, steady-state levels of transgene mRNA 1 were higher in resistant than in susceptible lines. Thus, the data indicate that sequence homology is not sufficient to induce suppression. Grafting experiments using transgenic resistant or susceptible rootstocks and scions demonstrated that the resistance mechanism exhibited an additional barrier to phloem entry, preventing CMV from moving a long distance in resistant plants. On the other hand, virus from susceptible rootstocks could systemically infect grafted resistant scions via the phloem. Analysis of viral RNA accumulation in the infected scions showed that the mechanism that suppresses the accumulation of viral RNA 1 at the single-cell level was overcome. The data indicate that this transgene-mediated systemic resistance probably is not based on a posttranscriptional gene-silencing mechanism.

  12. CMV infection in a cohort of HIV-exposed infants born to mothers receiving antiretroviral therapy during pregnancy and breastfeeding.

    Science.gov (United States)

    Pirillo, Maria Franca; Liotta, Giuseppe; Andreotti, Mauro; Jere, Haswel; Sagno, Jean-Baptiste; Scarcella, Paola; Mancinelli, Sandro; Buonomo, Ersilia; Amici, Roberta; Marazzi, Maria Cristina; Vella, Stefano; Palombi, Leonardo; Giuliano, Marina

    2017-02-01

    Antiretroviral therapy has been shown to reduce rates of congenital CMV infection. Little information is available on the possible impact of antiretroviral therapy on postnatal breastfeeding-associated CMV infection acquisition. A cohort of 89 HIV-infected mothers and their children was studied. Women received antiretroviral therapy from week 25 of gestation until 6 months postpartum or indefinitely if meeting the criteria for treatment. All women were evaluated for CMV IgG presence and CMV DNA in breast milk. Children were tested for CMV infection by either the presence of IgM or the presence of CMV DNA in plasma at 1, 6 and 12 months and by the presence of IgG at 24 months. All mothers had high titers of CMV DNA in breast milk (5.7 log at Month 1 and 5.1 log at Month 6). Cumulative CMV infection rates were 60.3 % at Month 6, 69 % at Month 12 and 96.4 % at Month 24. There was a significant negative correlation between the duration of antiretroviral treatment during pregnancy and levels of CMV DNA in breast milk at Month 1 (P = 0.033). There was a trend for a correlation between high titers of CMV DNA in breast milk at 6 months and CMV infection at 6 months (P = 0.069). In this cohort, more than 95 % of the children had acquired CMV infection by 2 years of age. Besides breastfeeding, which played a major role, also horizontal transmission between 1 and 2 years was certainly relevant in determining CMV infection acquisition.

  13. Rapid quantitative PCR assays for the simultaneous detection of herpes simplex virus, varicella zoster virus, cytomegalovirus, Epstein-Barr virus, and human herpesvirus 6 DNA in blood and other clinical specimens

    NARCIS (Netherlands)

    Engelmann, I.; Petzold, D. R.; Kosinska, A.; Hepkema, B. G.; Schulz, T. F.; Heim, A.

    Rapid diagnosis of human herpesvirus primary infections or reactivations is facilitated by quantitative PCRs. Quantitative PCR assays with a standard thermal cycling profile permitting simultaneous detection of herpes simplex virus (HSV), varicella zoster virus (VZV), cytomegalovirus (CMV),

  14. Postmortem diagnosis of cytomegalovirus and accompanying other infection agents by real-time PCR in cases of sudden unexpected death in infancy (SUDI).

    Science.gov (United States)

    Yagmur, Gulhan; Ziyade, Nihan; Elgormus, Neval; Das, Taner; Sahin, M Feyzi; Yildirim, Muzaffer; Ozgun, Ayse; Akcay, Arzu; Karayel, Ferah; Koc, Sermet

    2016-02-01

    As an opportunistic pathogen with high mortality rates, Cytomegalovirus (CMV) may lead to fatal disseminated CMV infection of the premature and newborn; thus necessitating the demonstration of CMV-DNA with clinical history and/or histopathological findings of CMV infection and defining other bacterial and viral infection agents with real-time polymerase chain reaction (RT-PCR) in udden unexpected death in infancy (SUDI) cases as we aimed in this study. 314 (144 female, 170 male) SUDI cases were prospectively investigated from January 2013 to January 2015 in Istanbul Forensic Medicine Institution. The study includes 87 tissue samples of 39 cases for post-mortem histopathological examination of interstitial pneumonia, myocarditis, meningitis, encephalitis, hepatitis, colitis or tubulointerstitial nephritis and/or accompanying chronic sialadenitis. CMV-DNA was found positive in 35 (40.2%) salivary gland, 19 (21.8%) lung, 1 (1.1%) tonsil, and 1 (1.1%) brain tissues. CMV sialadenitis and/or CMV pneumonia associated with other viral and/or bacterial agents were detected in 23 (60%) of 39 infant cases. The demonstration of CMV-DNA would significantly clarify the cause of death and collection of epidemiological data in SUDI cases with clinical history and histopathological findings of CMV infection accompanying chronic CMV sialadenitis. Furthermore, CMV suppresses the immune system, and may predispose to other bacterial and/or viral infections in these cases. Post-mortem molecular investigations are useful in explaining cause of death in SUDI with a suspicion of infection in forensic autopsies.

  15. An artemisinin-derived dimer has highly potent anti-cytomegalovirus (CMV and anti-cancer activities.

    Directory of Open Access Journals (Sweden)

    Ran He

    Full Text Available We recently reported that two artemisinin-derived dimers (dimer primary alcohol 606 and dimer sulfone 4-carbamate 832-4 are significantly more potent in inhibiting human cytomegalovirus (CMV replication than artemisinin-derived monomers. In our continued evaluation of the activities of artemisinins in CMV inhibition, twelve artemisinin-derived dimers and five artemisinin-derived monomers were used. Dimers as a group were found to be potent inhibitors of CMV replication. Comparison of CMV inhibition and the slope parameter of dimers and monomers suggest that dimers are distinct in their anti-CMV activities. A deoxy dimer (574, lacking the endoperoxide bridge, did not have any effect on CMV replication, suggesting a role for the endoperoxide bridge in CMV inhibition. Differences in anti-CMV activity were observed among three structural analogs of dimer sulfone 4-carbamate 832-4 indicating that the exact placement and oxidation state of the sulfur atom may contribute to its anti-CMV activity. Of all tested dimers, artemisinin-derived diphenyl phosphate dimer 838 proved to be the most potent inhibitor of CMV replication, with a selectivity index of approximately 1500, compared to our previously reported dimer sulfone 4-carbamate 832-4 with a selectivity index of about 900. Diphenyl phosphate dimer 838 was highly active against a Ganciclovir-resistant CMV strain and was also the most active dimer in inhibition of cancer cell growth. Thus, diphenyl phosphate dimer 838 may represent a lead for development of a highly potent and safe anti-CMV compound.

  16. Breastfeeding and transmission of cytomegalovirus to preterm infants. Case report and kinetic of CMV-DNA in breast milk

    Directory of Open Access Journals (Sweden)

    Rossi Marta J

    2011-01-01

    Full Text Available Abstract Background Breastfeeding has a major impact on CMV epidemiology. Postnatal CMV reactivation's incidence during lactation is nearby the maternal seroprevalence. Although perinatal CMV infection has practically no consequences in term newborn, it may cause, in some cases, a severe symptomatic disease in preterm newborns. The aims of the present study are to evaluate the rate and clinical expression of CMV infection breast milk transmitted in preterm infants and to check the safety of the freezing treated breast milk. Methods The study included fifty-seven preterm infants and their CMV seropositive mothers. Fresh breast milk samples have been collected from 1st to 9th postpartum week. Both fresh breast milk and 72, 96, 120 hours frozen samples have been examined, checking the presence of CMV; urine samples have been tested too. Results 70.2% of tested mothers showed reactivation of the infection, and CMV-positive breast milk during the six weeks postpartum has been found. However, only one infant was infected by CMV, developing hepatic affection concomitantly with a multi-system involvement, as shown CMV DNA detection in urine, saliva, blood, gastric aspirate, and stools. Conclusion Freezing breast milk at -20°C and pasteurization may respectively reduce or eliminate the viral load.

  17. Prevalence of specific IGM due to toxoplasma, rubella, CMV and c.trachomatis infections during pregnancy

    Directory of Open Access Journals (Sweden)

    Yasodhara P

    2001-01-01

    Full Text Available One hundred and seventy five apparently normal asymptomatic pregnant women were studied prospectively and 247 women with different complications of pregnancy were screened at the time of delivery for infections like Chlamydia trachomatis, Toxoplasma, Rubella and cytomegalovirus (CMV. One hundred and forty two women with normal outcome of pregnancy served as controls. Specific IgM due to these agents were determined in the sera using commercial diagnostic kits. Results of the study showed that chlamydial infection was the most prevalent (29.8% followed by Toxoplasmosis (13.1, Rubella (6.5% and CMV (5.8%. Adverse outcome was seen among those seropositive for Chlamydia, Toxoplasmosis and Rubella. CMV showed no association with adverse outcome of pregnancy. Since Chlamydia and Toxoplasmosis are amenable to treatment with antibiotics, screening for these infections and appropriate treatment would improve outcome of pregnancy.

  18. CMV-specific CD8 T Cell Differentiation and Localization: Implications for Adoptive Therapies

    Directory of Open Access Journals (Sweden)

    Corinne J Smith

    2016-09-01

    Full Text Available Human cytomegalovirus (HCMV is a ubiquitous virus that causes chronic infection, and thus is one of the most common infectious complications of immune suppression. Adoptive transfer of HCMV-specific T cells has emerged as an effective method to reduce the risk for HCMV infection and/or reactivation by restoring immunity in transplant recipients. However, the CMV-specific CD8+ T cell response is comprised of a heterogenous mixture of subsets with distinct functions and localization and it is not clear if current adoptive immunotherapy protocols can reconstitute the full spectrum of CD8+ T cell immunity. The aim of this review is to briefly summarize the role of these T cell subsets in CMV immunity and to describe how current adoptive immunotherapy practices might affect their reconstitution in patients. The bulk of the CMV-specific CD8+ T cell population is made up of terminally differentiated effector T cells with immediate effector function and a short life span. Self-renewing memory T cells within the CMV-specific population retain the capacity to expand and differentiate upon challenge and are important for the long-term persistence of the CD8+ T cell response. Finally mucosal organs, which are frequent sites of CMV reactivation, are primarily inhabited by tissue resident memory T cells, which do not recirculate. Future work on adoptive transfer strategies may need to focus on striking a balance between the formation of these subsets to ensure the development of long lasting and protective immune responses that can access the organs affected by CMV disease.

  19. Retinite por citomegalovirus (CMV após terapia imunossupressora para vasculite leucocitoclástica Cytomegalovirus (CMV retinitis after immunossupressive therapy for leukocytoclastic vasculitis

    Directory of Open Access Journals (Sweden)

    Carlos Ewerton Maia Rodrigues

    2009-02-01

    Full Text Available A retinite por citomegalovírus (CMV é uma doença rara que acomete principalmente pacientes com a síndrome da imunodeficiência adquirida (AIDS. No entanto, outros pacientes imunossuprimidos, como os transplantados, os que estão em uso de quimioterápicos, pacientes com lúpus eritematoso sistêmico (LES ou em tratamento com drogas imunossupressoras também podem ser acometidos. O quadro clínico caracteriza-se por visão turva, diminuição da acuidade ou alterações de campo visual, geralmente unilateral, podendo ocorrer deslocamento de retina. A perda visual é progressiva, evolui em ritmo variável até a completa amaurose do olho acometido. O presente relato de caso descreve um paciente com vasculite leucocitoclástica grave submetido à terapia com corticoide em dose imunossupressora que evoluiu com glaucoma, panuveíte por CMV, perda da acuidade visual e infecção bacteriana secundária.Cytomegalovirus (CMV retinitis is a rare disease which mainly affects patients with acquired immunodeficiency syndrome (AIDS. Nevertheless, other immunosuppressed patients, such as the organ transplant recipients, the ones using chemotherapy, patients with systemic lupus erythematosus (SLE or in treatment with immunosuppressive drugs can also be attacked. The clinical characteristics are blurred vision, decrease of the visual acuity or visual field alterations, generally unilateral, with the possibility of retinal detachment. The visual loss is progressive, evolving in a variable rate until complete amaurosis of the attacked eye. The present case report describes a patient with severe leukocytoclastic vasculitis, submitted to corticosteroid therapy in immunosuppressive doses that evolved with glaucoma, panuveitis by CMV, loss of visual acuity and secondary bacterial infection.

  20. qPCR is a sensitive and rapid method for detection of cytomegaloviral DNA in formalin-fixed, paraffin-embedded biopsy tissue.

    Science.gov (United States)

    McCoy, Morgan H; Post, Kristin; Sen, Joyashree D; Chang, Hsim Y; Zhao, Zijin; Fan, Rong; Chen, Shaoxiong; Leland, Diane; Cheng, Liang; Lin, Jingmei

    2014-01-01

    It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

  1. cmv1 is a gate for Cucumber mosaic virus transport from bundle sheath cells to phloem in melon.

    Science.gov (United States)

    Guiu-Aragonés, Cèlia; Sánchez-Pina, María Amelia; Díaz-Pendón, Juan Antonio; Peña, Eduardo J; Heinlein, Manfred; Martín-Hernández, Ana Montserrat

    2016-08-01

    Cucumber mosaic virus (CMV) has the broadest host range among plant viruses, causing enormous losses in agriculture. In melon, strains of subgroup II are unable to establish a systemic infection in the near-isogenic line SC12-1-99, which carries the recessive resistance gene cmv1 from the accession PI 161375, cultivar 'Songwhan Charmi'. Strains of subgroup I overcome cmv1 resistance in a manner dependent on the movement protein. We characterized the resistance conferred by cmv1 and established that CMV-LS (subgroup II) can move from cell to cell up to the veins in the inoculated leaf, but cannot enter the phloem. Immunogold labelling at transmission electron microscopy level showed that CMV-LS remains restricted to the bundle sheath (BS) cells in the resistant line, and does not invade vascular parenchyma or intermediary cells, whereas, in the susceptible line 'Piel de Sapo' (PS), the virus invades all vein cell types. These observations indicate that the resistant allele of cmv1 restricts systemic infection in a virus strain- and cell type-specific manner by acting as an important gatekeeper for virus progression from BS cells to phloem cells. Graft inoculation experiments showed that CMV-LS cannot move from the infected PS stock into the resistant cmv1 scion, thus suggesting an additional role for cmv1 related to CMV transport within or exit from the phloem. The characterization of this new form of recessive resistance, based on a restriction of virus systemic movement, opens up the possibility to design alternative approaches for breeding strategies in melon.

  2. 腺病毒穿梭质粒pShuttle CMV-BMP2+-IRES-hrGFP-1的构建和鉴定%Construction and Identification of Recombinant Adenovirus Shuttle Plasimid pShuttle CMV-BMP2 +-IRES-GFP-1

    Institute of Scientific and Technical Information of China (English)

    张正; 刘丹平; 蒲勤; 郭韬; 张男

    2005-01-01

    目的构建腺病毒穿梭质粒pShuttle CMV-BMP2+-IRES-hrGFP-1,为构建表达具有抗原表位标记的骨形态发生蛋白2(bone morpbogenetic protein 2,BMP-2),并同时表达绿色荧光蛋白(green fluorescent protein,GFP)报告分子的腺病毒真核细胞表达载体打下基础.方法对目的基因供体质粒pcDNA3-BMP2携带的BMP2基因测序和序列内部存在的限制性内切酶识别位点进行分析,利用PCR(polymeras chain reaction,PCR)技术对pcDNA3-BMP2携带的BMP2基因突变,以去除翻译终止密码子后的基因序列并添加新的酶切识别位点Xho Ⅰ.测序检测突变情况,将突变后的BMP2基因(BMP2+基因)定向连入腺病毒穿梭质粒pShuttleCMV-IRES-hrGFP-1,通过限制性内切酶酶切图谱分析鉴定获得的重组质粒.结果重组质粒经双酶切鉴定图谱正确.结论成功构建了pShuttle CMV-BMP2+-IRES-hrGFP-1.

  3. Humoral and cellular responses to Pneumocystis carinii, CMV, and herpes simplex in patients with AIDS and in controls

    DEFF Research Database (Denmark)

    Hofmann, B; Nielsen, P B; Ødum, Niels;

    1988-01-01

    in the second of paired samples taken about 6 months apart, compared with whose with PCP (9%; p = 0.05). All patients had high titers of antibodies to CMV and HSV and normal total concentrations of immunoglobulins. None of the patients responded in lymphocyte transformation to P. carinii, CMV, or HSV antigens...

  4. Resolution of Mild Ganciclovir-Resistant Cytomegalovirus Disease with Reduced-Dose Cidofovir and CMV-Hyperimmune Globulin

    Directory of Open Access Journals (Sweden)

    Samir J. Patel

    2014-01-01

    Full Text Available Ganciclovir-resistant cytomegalovirus (CMV is associated with significant morbidity in solid organ transplant recipients. Management of ganciclovir-resistant CMV may be complicated by nephrotoxicity which is commonly observed with recommended therapies and/or rejection induced by “indirect” viral effects or reduction of immunosuppression. Herein, we report a series of four high serologic risk (donor CMV positive/recipient CMV negative kidney transplant patients diagnosed with ganciclovir-resistant CMV disease. All patients initially developed “breakthrough” viremia while still receiving valganciclovir prophylaxis after transplant and were later confirmed to exhibit UL97 mutations after failing to eradicate virus on adequate dosages of valganciclovir. The patients were subsequently and successfully treated with reduced-dose (1-2 mg/kg cidofovir and CMV-hyperimmune globulin, given in 2-week intervals. In addition, all patients exhibited stable renal function after completion of therapy, and none experienced acute rejection. The combination of reduced-dose cidofovir and CMV-hyperimmune globulin appeared to be a safe and effective regimen in patients with mild disease due to ganciclovir-resistant CMV.

  5. Prevalence of CMV infection among staff in a metropolitan children’s hospital – occupational health screening findings

    Directory of Open Access Journals (Sweden)

    Stranzinger, Johanna

    2016-09-01

    Full Text Available Background: Staff in children’s hospitals may run an increased risk of cytomegalovirus (CMV contact infection leading to a congenital CMV fetopathy during pregnancy. The main risk factor is close contact with inapparent carriers of CMV among infants (<3 years. We therefore examined CMV seroprevalence (SP and possible risk factors for CMV infection among staff at a children’s hospital.Method: In 2014, staff at a metropolitan children’s hospital were offered a CMV antibody test in the context of occupational health screening. Besides of anti-CMV immunoglobulin G (anti-CMV IgG gender, age, profession, number of children and migration background were assessed and used as independent variables in multiple logistic regression. Women without a migration background (MIG were considered as a separate group.Results: The study included 219 employees. Women showed a significant higher risk than men of being CMV-positive (adjusted odds ratio [aOR] 3.0; 95% CI 1.1–7.8. The risk among age groups of 30 and over was double that of the under-30s (aOR 2.0; 95% CI 1.0–3.9; among those aged 40-plus it was aOR 2.3 (95% CI 1.1–4.7. Staff with an MIG tested more often positive than those without an MIG (95.5% versus 45.7%. CMV SP was 47.7% among women without an MIG. In this subgroup the probability of CMV infection increased with age (p=0.08 as well. Conclusion: In the staff group as a whole there was a significant correlation between CMV SP, country of origin and age. We found no significant differences between occupational groups; perhaps our random sample was too small. Given the low CMV SP particularly in those without MIG, women who want to have children in particular must be protected from CMV infection. Follow-up studies should be undertaken to test whether good workplace hygiene offers sufficient protection for pregnant women and could be an alternative to prohibiting certain activities.

  6. High levels of protein expression using different mammalian CMV promoters in several cell lines.

    Science.gov (United States)

    Xia, Wei; Bringmann, Peter; McClary, John; Jones, Patrick P; Manzana, Warren; Zhu, Ying; Wang, Soujuan; Liu, Yi; Harvey, Susan; Madlansacay, Mary Rose; McLean, Kirk; Rosser, Mary P; MacRobbie, Jean; Olsen, Catherine L; Cobb, Ronald R

    2006-01-01

    With the recent completion of the human genome sequencing project, scientists are faced with the daunting challenge of deciphering the function of these newly found genes quickly and efficiently. Equally as important is to produce milligram quantities of the therapeutically relevant gene products as quickly as possible. Mammalian expression systems provide many advantages to aid in this task. Mammalian cell lines have the capacity for proper post-translational modifications including proper protein folding and glycosylation. In response to the needs described above, we investigated the protein expression levels driven by the human CMV in the presence or absence of intron A, the mouse and rat CMV promoters with intron A, and the MPSV promoter in plasmid expression vectors. We evaluated the different promoters using an in-house plasmid vector backbone. The protein expression levels of four genes of interest driven by these promoters were evaluated in HEK293EBNA and CHO-K1 cells. Stable and transient transfected cells were utilized. In general, the full-length human CMV, in the presence of intron A, gave the highest levels of protein expression in transient transfections in both cell lines. However, the MPSV promoter resulted in the highest levels of stable protein expression in CHO-K1 cells. Using the CMV driven constitutive promoters in the presence of intron A, we have been able to generate >10 microg/ml of recombinant protein using transient transfections.

  7. CMV matrices in random matrix theory and integrable systems: a survey

    Energy Technology Data Exchange (ETDEWEB)

    Nenciu, Irina [Courant Institute, 251 Mercer St, New York, NY 10012 (United States)

    2006-07-14

    We present a survey of recent results concerning a remarkable class of unitary matrices, the CMV matrices. We are particularly interested in the role they play in the theory of random matrices and integrable systems. Throughout the paper we also emphasize the analogies and connections to Jacobi matrices.

  8. CMV promoter is repressed by p53 and activated by JNK pathway.

    Science.gov (United States)

    Rodova, Marianna; Jayini, Renuka; Singasani, Reddy; Chipps, Elizabeth; Islam, M Rafiq

    2013-05-01

    Viral promoters are widely utilized in commercial and customized vectors to drive expression of genes of interest including reporter, effector and transfection control, because of their high transcription efficiency in a variety of primary and transformed cell lines. However, we observed altered rate of transcription for these promoters under conditions such as presence of an effector protein. These variations in viral promoter driven expressions can potentially lead to incorrect conclusion, especially in comparative and quantitative experiments. We found significantly reduced viral promoter activity in cells overexpressing tumor suppressor protein p53, whereas markedly induced transcription in cells overexpressing MAP/ERK kinase kinase 1 (Mekk 1). Using deletion constructs generated from the CMV promoter, we found the transcription reduction by p53 is possibly mediated through the TATA motif present in proximal CMV promoter. The activation of the CMV promoter by Mekk 1, on the other hand, is attributed to the proximal CRE binding site in the promoter. These findings may be of interest to investigators who use CMV (or other viral) promoter driven vectors for either comparative or quantitative gene expression, or effect on promoter activity. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Development of modulation strategies for NPC converter addressing DC link voltage balancing and CMV reduction

    DEFF Research Database (Denmark)

    Boian, D.; Biris, C.; Teodorescu, Remus

    2012-01-01

    in insulation breakdown and bearing failures. By the use of this type of converters, both Electromagnetic Interference (EMI) and harmonic distortions are improved. This paper proposes two modulation strategies for Three Level Neutral Point Clamped Converter (3L-NPC). The main focus of these modulation...... strategies is to reduce the Common Mode Voltage (CMV) and balance the DC Link Voltage....

  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. 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.

  12. Case report: persistent cytomegalovirus (CMV) infection after haploidentical hematopoietic stem cell transplantation using in vivo alemtuzumab: emergence of resistant CMV due to mutations in the UL97 and UL54 genes.

    Science.gov (United States)

    Oshima, Kumi; Kanda, Yoshinobu; Kako, Shinichi; Asano-Mori, Yuki; Watanabe, Takuro; Motokura, Toru; Chiba, Shigeru; Shiraki, Kimiyasu; Kurokawa, Mineo

    2008-10-01

    Addition of in vivo alemtuzumab to the conditioning regimen enabled 2- or 3-locus-mismatched hematopoietic stem cell transplantation with an acceptable incidence of graft-versus-host-disease. However, the procedure was associated with a high incidence of cytomegalovirus (CMV) reactivation. Although preemptive therapy with ganciclovir prevented successfully severe CMV diseases and CMV-related mortality, a patient developed persistent positive CMV antigenemia for more than 1 year after transplantation and CMV disease, despite the use of ganciclovir and foscarnet. The in vitro susceptibility assay showed that the clinical isolate was resistant to foscarnet, moderately resistant to ganciclovir, but sensitive to cidofovir. Therefore, cidofovir was administered. CMV antigenemia became negative within 2 weeks and never developed again. Nucleotide sequence of the UL54 and UL97 of the clinical isolate showed 4 amino acid substitutions (V11L, Q578H, S655L, and G874R) in UL54 and 2 mutations (A140V and A594V) in UL97 compared with the Towne and AD169 strains. Ganciclovir resistance was suspected to be caused by both A594V of UL97 and Q578H of UL54, whereas foscarnet resistance was due mainly to Q578H of UL54. In conclusion, the in vitro susceptibility assay as well as nucleotide sequence of clinical isolate is important to choose appropriate antiviral agents for patients who have persistent CMV reactivation after stem cell transplantation.

  13. Analysis of HIV-1- and CMV-specific memory CD4 T-cell responses during primary and chronic infection.

    Science.gov (United States)

    Harari, Alexandre; Rizzardi, G Paolo; Ellefsen, Kim; Ciuffreda, Donatella; Champagne, Patrick; Bart, Pierre-Alexandre; Kaufmann, Daniel; Telenti, Amalio; Sahli, Roland; Tambussi, Giuseppe; Kaiser, Laurent; Lazzarin, Adriano; Perrin, Luc; Pantaleo, Giuseppe

    2002-08-15

    CD4 T-cell-specific memory antiviral responses to human immunodeficiency virus type 1 (HIV-1) and cytomegalovirus (CMV) were investigated in 16 patients with documented primary HIV-1 infection (4 of the 16 subjects also had primary CMV infection) and compared with those observed in patients with chronic HIV-1 and CMV coinfection. Virus-specific memory CD4 T cells were characterized on the basis of the expression of the chemokine receptor CCR7. HIV-1- and CMV-specific interferon-gamma-secreting CD4 T cells were detected in patients with primary and chronic HIV-1 and CMV coinfection and were mostly contained in the cell population lacking expression of CCR7. The magnitude of the primary CMV-specific CD4 T-cell response was significantly greater than that of chronic CMV infection, whereas there were no differences between primary and chronic HIV-1-specific CD4 T-cell responses. A substantial proportion of CD4(+)CCR7(-) T cells were infected with HIV-1. These results advance the characterization of antiviral memory CD4 T-cell response and the delineation of the potential mechanisms that likely prevent the generation of a robust CD4 T-cell immune response during primary infection.

  14. Human bone marrow as a source to generate CMV-specific CD4+ T cells with multifunctional capacity.

    Science.gov (United States)

    Na, Il-Kang; Letsch, Anne; Guerreiro, Manuel; Bauer, Sandra; Noack, Ines; Geginat, Jens; Reinke, Petra; Loesch, Michael; Kienapfel, Heino; Thiel, Eckhard; Volk, Hans Dieter; Scheibenbogen, Carmen

    2009-01-01

    The bone marrow (BM) is an important compartment for T cell memory. In cytomegalovirus (CMV)-seropositive individuals peripheral blood (PB) CMV-specific T cells constitute a large fraction of PB T cells but are mostly differentiated effector/effector memory T cells with limited survival and proliferative potential. In this study, we performed a comprehensive analysis of the CMV-specific T cell response in BM studying both CD4+ and CD8+ T cell responses against overlapping peptide pools of the CMV proteins pp65 and immediate early protein-1. CMV-specific T cell responses were characterized ex vivo and after in vitro expansion of paired PB/BM samples by multiparameter flow cytometry determining surface phenotype, cytokine profile, and cytotoxic capability. Comparable frequencies of CMV-specific T cells were found in un-manipulated PB and BM. Both total CD4+ and CD8+ T cells could be more rapidly expanded from BM. Expanded BM T cells contained significantly higher frequencies of CMV-specific CD4+ T cells than PB. Furthermore, higher frequencies of specific CD4+ T cells from BM were multifunctional, characterized by simultaneous production of interferon-gamma, tumor necrosis factor, and interleukin-2. Use of BM may thus facilitate more rapid generation of adoptive T cells with enhanced functionality.

  15. Control de brazo electrónico usando señales electromiográficas

    Directory of Open Access Journals (Sweden)

    Jorge Andrés García-Pinzón

    2015-05-01

    Full Text Available Los trabajos enfocados en la extracción de patrones en señales electromiográficas (SEMG han venido creciendo debido a sus múltiples aplicaciones. En este artículo se presenta una aplicación en la cual se implementa un sistema electrónico para el registro de las SEMG de la extremidad superior en un sujeto, con el fin de controlar de forma remota un brazo electrónico. Se realizó una etapa de preprocesamiento de las señales registradas, para eliminar información poco relevante, y reconocimiento de zonas de interés; enseguida se extraen los patrones y se clasifican. Las técnicas utilizadas fueron: análisis wavelet (AW, análisis de componentes principales (ACP, transformada de fourier (TF, transformada del coseno discreta (TDC, energía, máquinas de soporte vectorial (MSV o SVM y redes neuronales (RNA. En este artículo se demuestra que la metodología planteada permite realizar un proceso de clasificación con un rendimiento superior al 95%. Se registraron más de 4000 señales.

  16. Screening of Turkish Melon Accessions for Resistance to ZYMV, WMV and CMV

    Directory of Open Access Journals (Sweden)

    Ercan EKBIC

    2010-03-01

    Full Text Available In the Çukurova University Department of Horticulture more than 350 melon accessions were collected from different ecological parts of Turkey which is located on the secondary genetic diversification center of this crop, and their characterization studies are near completion. Furthermore, evaluation studies of these materials have started. In the present study 67 melon accessions, sampled from this germplasm, were tested for resistance to zucchini yellow mosaic virus (ZYMV, Cucumber mosaic virus (CMV and watermelon mosaic virus (WMV. After resistance tests made by mechanical inoculation, four accessions (‘CU 100’, ‘CU 287’, ‘CU 305’ and ‘CU 328’ were found resistant to ZYMV and three accessions (‘CU 305’, ‘C 264’, and ‘C 276’ to WMV. No resistant genotype was found to CMV.

  17. Effect of a 14-day course of foscarnet on cytomegalovirus (CMV) blood markers in a randomized study of human immunodeficiency virus-infected patients with persistent CMV viremia. Agence National de Recherche du SIDA 023 Study Group.

    Science.gov (United States)

    Salmon-Céron, D; Fillet, A M; Aboulker, J P; Gérard, L; Houhou, N; Carrière, I; Ostinelli, J; Vildé, J L; Brun-Vézinet, F; Leport, C

    1999-04-01

    A randomized open-label phase 2 trial compared the virological and clinical effects on cytomegalovirus (CMV) infection of a 14-day course of intravenous foscarnet (100 mg/[kg x 12 h]) or no treatment in 42 HIV-infected patients with virus load rapidly increased. The probability of CMV disease at 6 months was 43% in both groups. Patients who had or who achieved a negative blood culture at any time had a reduced risk of CMV disease (RR = 2.64; 95% CI = 1.24-5.62; P = .02). This study suggests that sequential courses of intravenous foscarnet might not be a good strategy for preemptive therapy in this population and that in patients with a positive blood marker, treatment able to induce and maintain negative CMV blood cultures could constitute an effective intervention.

  18. Proteomic analysis of the plant-virus interaction in cucumber mosaic virus (CMV) resistant transgenic tomato.

    Science.gov (United States)

    Di Carli, Mariasole; Villani, Maria Elena; Bianco, Linda; Lombardi, Raffaele; Perrotta, Gaetano; Benvenuto, Eugenio; Donini, Marcello

    2010-11-05

    Cucumber mosaic virus (CMV), a member of the Cucumovirus genus, is the causal agent of several plant diseases in a wide range of host species, causing important economic losses in agriculture. Because of the lack of natural resistance genes in most crops, different genetic engineering strategies have been adopted to obtain virus-resistant plants. In a previous study, we described the engineering of transgenic tomato plants expressing a single-chain variable fragment antibody (scFv G4) that are specifically protected from CMV infection. In this work, we characterized the leaf proteome expressed during compatible plant-virus interaction in wild type and transgenic tomato. Protein changes in both inoculated and apical leaves were revealed using two-dimensional gel electrophoresis (2-DE) coupled to differential in gel electrophoresis (DIGE) technology. A total of 2084 spots were detected, and 50 differentially expressed proteins were identified by nanoscale liquid chromatographic-electrospray ionization-ion trap-tandem mass spectrometry (nLC-ESI-IT-MS/MS). The majority of these proteins were related to photosynthesis (38%), primary metabolism (18%), and defense activity (14%) and demonstrated to be actively down regulated by CMV in infected leaves. Moreover, our analysis revealed that asymptomatic apical leaves of transgenic inoculated plants had no protein profile alteration as compared to control wild type uninfected plants demonstrating that virus infection is confined to the inoculated leaves and systemic spread is hindered by the CMV coat protein (CP)-specific scFv G4 molecules. Our work is the first comparative study on compatible plant-virus interactions between engineered immunoprotected and susceptible wild type tomato plants, contributing to the understanding of antibody-mediated disease resistance mechanisms.

  19. A Cmv2 QTL on chromosome X affects MCMV resistance in New Zealand male mice.

    Science.gov (United States)

    Rodriguez, Marisela R; Lundgren, Alyssa; Sabastian, Pearl; Li, Qian; Churchill, Gary; Brown, Michael G

    2009-07-01

    NK cell-mediated resistance to viruses is subject to genetic control in humans and mice. Here we used classical and quantitative genetic strategies to examine NK-mediated murine cytomegalovirus (MCMV) control in genealogically related New Zealand white (NZW) and black (NZB) mice. NZW mice display NK cell-dependent MCMV resistance while NZB NK cells fail to limit viral replication after infection. Unlike Ly49H(+) NK resistance in C57BL/6 mice, NZW NK-mediated MCMV control was Ly49H-independent. Instead, MCMV resistance in NZW (Cmv2) involves multiple genetic factors. To establish the genetic basis of Cmv2 resistance, we further characterized a major chromosome X-linked resistance locus (DXMit216) responsible for innate MCMV control in NZW x NZB crosses. We found that the DXMit216 locus affects early MCMV control in New Zealand F(2) crosses and demonstrate that the NZB-derived DXMit216 allele enhances viral resistance in F(2) males. The evolutionary conservation of the DXMit216 region in mice and humans suggests that a Cmv2-related mechanism may affect human antiviral responses.

  20. Provision of cellular blood components to CMV-seronegative patients undergoing allogeneic stem cell transplantation in the UK: survey of UK transplant centres.

    Science.gov (United States)

    Morton, S; Peniket, A; Malladi, R; Murphy, M F

    2017-09-15

    To identify current UK practice with regards to provision of blood components for cytomegalovirus (CMV)-seronegative, potential, allogeneic stem cell recipients of seronegative grafts. Infection with CMV remains a major cause of morbidity and mortality after allogeneic stem cell transplantation (aSCT). CMV transmission has been a risk associated with the transfusion of blood components from previously exposed donors, but leucocyte reduction has been demonstrated to minimise this risk. In 2012, the UK Advisory Committee for the Safety of Tissues and Organs (SaBTO) recommended that CMV-unselected components could be safely transfused without increased risk of CMV transmission. We surveyed UK aSCT centres to establish current practice. Fifteen adult and seven paediatric centres (75%) responded; 22·7% continue to provide components from CMV-seronegative donors. Reasons cited include the continued perceived risk of CMV transmission by blood transfusion, its associated morbidity and concerns regarding potential for ambiguous CMV serostatus in seronegative potential transplant recipients due to passive antibody transfer from CMV-seropositive blood donors, leading to erroneous donor/recipient CMV matching at transplant. The survey demonstrated a surprisingly high rate (22.7%) of centres continuing to provide blood components from CMV-seronegative donors despite SaBTO guidance. © 2017 British Blood Transfusion Society.

  1. Identification a coat protein region of cucumber mosaic virus (CMV) essential for long-distance movement in cucumber.

    Science.gov (United States)

    Salánki, Katalin; Kiss, László; Gellért, Akos; Balázs, Ervin

    2011-12-01

    To characterise the long-distance movement determinant of cucumoviral coat proteins (CPs), five mutants were engineered into the CMV CP bearing the corresponding tomato aspermy virus (TAV) loops exposed on the surface of the virion. Both viruses can move long-distance in Nicotiana clevelandii, but only CMV can move long-distance in cucumber. Investigation of the CMV chimeras identified three amino acids of the βB-βC loop that were essential for the CMV long-distance movement in cucumber. Introducing these mutations into the TAV CP was not sufficient for long-distance movement, indicating that this is not the sole region causing long-distance movement deficiency.

  2. Ongoing burden of disease and mortality from HIV/CMV coinfection in Africa in the antiretroviral therapy era

    Directory of Open Access Journals (Sweden)

    Emily eAdland

    2015-09-01

    Full Text Available Human Cytomegalovirus (CMV is a well recognised pathogen in the context of HIV infection, but since the roll out of ART, clinical and scientific interest in the problem of HIV/CMV coinfection has diminished. However, CMV remains a significant cofactor in HIV disease, with an influence on HIV acquisition, disease progression, morbidity and mortality. Disease manifestations may be a result of direct interplay between the two viruses, or may arise as a secondary consequence of immune dysregulation and systemic inflammation. The problem is most relevant when the rates of coinfection are high, most notably in sub-Saharan Africa, and in children at risk of acquiring both infections early in life. Understanding the interplay between these viruses and developing strategies to diagnose, treat and prevent CMV should be a priority.

  3. Amino acid 1-209 is essential for PDX-1-mediated repression of human CMV IE promoter activity

    Institute of Scientific and Technical Information of China (English)

    Jing CHEN; Lei CHEN; Ge LI; Lu CHENG; Yin HUANG; Jia-xin ZHANG; Wei-wei FAN; Da-ru LU

    2006-01-01

    Aim: To explore the different roles of pancreatic duodenal homeobox factors-1 (PDX-1) domains in PDX-1 mediated repression of human cytomegalovirus immediately early (CMV IE) promoter. Methods: A series of truncated PDX-1 mutants were constructed. The binding of PDX-1 and CMV IE promoter was identified by electrophoretic mobility shift assay (EMSA). The dual-reporter assay was applied to examine the repression activities of PDX-1 mutants on CMV IE promoter. In addition, RNAi technology was used to specifically knock down the endogenous PDX-1 expression. Results: The reporter assay indicated that compared to the mock controls (pEGFP-N2), overexpression of PDX-1 resulted in a 41% decrease of CMV IE promoter activity in the 293 cells (P<0.05) and 43% decrease in HeLa cells (P<0.05), and the repression levels of various truncated mutants played on CMV IE promoter were different. Specific knock down of the endogenous PDX-1 expression significantly restored the activity of CMV IE promoter. EMS A demonstrated that domain 3 is necessary for nuclear localization and DNA binding activity of PDX-1. However, binding of PDX-1 alone to CMV IE promoter was not sufficient to inhibit its transcriptional activity, and other domains of PDX-1 presented were also required. Conclusion: Our data suggested that the DNA binding activity of PDX-1 domain 3 and the cooperative binding of PDX-1 domain 1/2 with other proteins were required for PDX-1 mediated repression of CMV IE promoter.

  4. Rapid genotyping of cytomegalovirus in dried blood spots by multiplex real-time PCR assays targeting the envelope glycoprotein gB and gH genes.

    Science.gov (United States)

    de Vries, Jutte J C; Wessels, Els; Korver, Anna M H; van der Eijk, Annemiek A; Rusman, Lisette G; Kroes, Aloys C M; Vossen, Ann C T M

    2012-02-01

    Genotyping of cytomegalovirus (CMV) is useful to examine potential differences in the pathogenicity of strains and to demonstrate coinfection with multiple strains involved in CMV disease in adults and congenitally infected newborns. Studies on genotyping of CMV in dried blood spots (DBS) are rare and have been hampered by the small amount of dried blood available. In this study, two multiplex real-time PCR assays for rapid gB and gH genotyping of CMV in DBS were developed. Validation of the assays with 39 CMV-positive plasma samples of transplant recipients and 21 urine specimens of congenitally infected newborns was successful in genotyping 100% of the samples, with gB1 and gB3 being the most prevalent genotypes. Multiple gB and gH genotypes were detected in 36% and 33% of the plasma samples, respectively. One urine sample from a newborn with symptomatic congenital CMV was positive for gB1 and gB2. DBS of congenitally infected newborns (n = 41) were tested using 9 μl of dried blood, and genotypes were detected in 81% (gB) and 73% (gH) of the samples, with gB3 being the most prevalent genotype. No clear association of specific genotypes with clinical outcome was observed. In conclusion, the CMV gB and gH PCR assays were found to be rapid, sensitive for detecting mixed infections, and suitable for direct usage on DBS. These assays are efficient tools for genotyping of CMV in DBS of congenitally infected newborns.

  5. Intravaginal cytomegalovirus (CMV challenge elicits maternal viremia and results in congenital transmission in a guinea pig model

    Directory of Open Access Journals (Sweden)

    Schleiss Mark R

    2011-03-01

    Full Text Available Abstract Background The objective of this study was to compare intravaginal (ivg and subcutaneous (sc administration of the guinea pig cytomegalovirus (GPCMV in pregnant and non-pregnant guinea pigs. These studies tested the hypotheses that ivg infection would elicit immune responses, produce maternal viremia, and lead to vertical transmission, with an efficiency similar to the traditionally employed sc route. Results Four groups of age- and size-matched guinea pigs were studied. Two groups were pregnant, and two groups were not pregnant. Animals received 5x105 plaque-forming units (PFU of a GPCMV reconstituted from an infectious bacterial artificial chromosome (BAC construct containing the full-length GPCMV genome. Seroconversion was compared by IgG ELISA, and viremia (DNAemia was monitored by PCR. In both pregnant and non-pregnant animals, sc inoculation resulted in significantly higher serum ELISA titers than ivg inoculation at 8 and 12 weeks post-infection. Patterns of viremia (DNAemia were similar in animals inoculated by either sc or ivg route. However, in pregnant guinea pigs, animals inoculated by both routes experienced an earlier onset of DNAemia than did non-pregnant animals. Neither the percentage of dead pups nor the percentage of GPCMV positive placentas differed by inoculation route. Conclusions In the guinea pig model of congenital CMV infection, the ivg route is as efficient at causing congenital infection as the conventional but non-physiologic sc route. This finding could facilitate future experimental evaluation of vaccines and antiviral interventions in this highly relevant animal model.

  6. Construction of pGEX-4T-2-PLEKHQ1 prokaryotic plasmid and pCMV-Myc-PLEKHQ1 eukaryotic plasmid and their protein expression%原核质粒pGEX-4T-2-PLEKHQ1及真核质粒pCMV-Myc-PLEKHQ1的构建与蛋白表达

    Institute of Scientific and Technical Information of China (English)

    陆琤; 周晨辰; 张鹏飞; 张硌

    2015-01-01

    目的:构建基因PLEKHQ1的原核质粒及真核质粒。方法:全长编码基因PLEKHQ1的聚合酶链反应(PCR)产物经EcoRⅠ和Kpn1双酶切后,分别与双酶切后的载体pGEX-4T-2及载体pCMV-Myc相连,在pGEX-4T-2-PLEKHQ1转化E.coli DH5α提取质粒后,转化E.coli BL21中诱导GST-Q1融合蛋白表达,利用谷胱甘肽琼脂糖4B纯化诱导的融合蛋白;而pCMV-Myc-PLEKHQ1则瞬转至293TX细胞;用蛋白质印迹法(Western blot)检测GST-Q1和Myc-Q1融合蛋白表达。结果:将获得的重组质粒进行双酶切鉴定,得到约1500 bp的目的片段,符合预计大小。质粒经测序分析正确后,Western blot检测到诱导及纯化后的GST-Q1和转染293TX后的Myc-Q1蛋白。结论:成功构建的pGEX-4T-2-PLEKHQ1和pCMV-Myc-PLEKHQ1重组质粒,为深入研究PLEKHQ1功能奠定良好的基础。%Objective:To construct prokaryotic plasmid and eukaryotic plasmid of PLEKHQ1 gene. Methods: The PCR product of the PLEKHQ1 coding sequence, which was digested with EcoR1 and Kpn1 restriction enzymes, was taped into the plasmid pGEX-4T-2 and pCMV-Myc. Then pGEX-4T-2-PLEKHQ1 was transformed into E.coli DH5α and plasmid DNA was extracted. After that, the expression of GST-Q1 fusion protein was induced in BL21 and was purified by Glutathione Sepharose 4B. While the pCMV-Myc-PLEKHQ1 was transfected into TX cells. The expression of both GST-Q1 and Myc-Q1 was detected by Western blot. Results:The restriction enzyme digestion showed target fragment of 1500bp, which was as expected. The recombinant plasmid was sequenced right and GST-Q1 protein and Myc-Q1 protein could be detected by Western blot. Conclusion:The recombinant prokaryotic plasmid and eukaryotic expression plasmid were successfully constructed, which laid a foundation for further research for PLEKHQ1.

  7. Cytomegalovirus (CMV) seroprevalence in pregnant women, bone marrow donors and adolescents in Germany, 1996-2010.

    Science.gov (United States)

    Enders, Gisela; Daiminger, Anja; Lindemann, Lisa; Knotek, Frank; Bäder, Ursula; Exler, Simone; Enders, Martin

    2012-08-01

    In Germany, studies on the IgG seroprevalence in pregnancy and in women of childbearing age are rare. Therefore, we retrospectively evaluated the CMV IgG seropositive rate in 40,324 pregnant women as well as in 31,093 female and male bone marrow donors over 15 consecutive years (1996-2010). Furthermore, the result of a study conducted in 1999 investigating 1,305 healthy adolescents with known ethnicity was included. The overall CMV IgG seroprevalence in pregnant women (15-50 years) was 42.3%. Age-dependent analysis revealed a significantly higher seropositive rate (55.6%) in young women (15-25 years) than in those aged 26-40 years (37-42%) and in women older than 40 years (48.3%). Over the study period of 15 years, the rate of seroprevalence in pregnant women declined significantly (χ(2) test < 0.01) from 44.3% in the first interval period (1996-2000), to 42.8% (2001-2005) and to 40.9% (2006-2010). The most influencing factor on CMV seropositivity appeared to be the socioeconomic status (SES), which we characterized by type of health insurance: Seroprevalence in women with low, middle and upper SES was 91.8, 46.9 and 33.7%, respectively. Female bone marrow donors of childbearing age (15-45 years) showed a significantly higher seropositive rate of 36.5% than age-matched male donors (28.6%). In adolescents aged 13-16 years, no gender-specific differences were recognized. Concerning ethnicity, youngsters with German descent had a significantly lower seroprevalence (29.9%) than those with non-German descent (67.4%).

  8. CMVpp65 Vaccine Enhances the Antitumor Efficacy of Adoptively Transferred CD19-Redirected CMV-Specific T Cells.

    Science.gov (United States)

    Wang, Xiuli; Wong, ChingLam W; Urak, Ryan; Mardiros, Armen; Budde, Lihua E; Chang, Wen-Chung; Thomas, Sandra H; Brown, Christine E; La Rosa, Corinna; Diamond, Don J; Jensen, Michael C; Nakamura, Ryotaro; Zaia, John A; Forman, Stephen J

    2015-07-01

    T cells engineered with chimeric antigen receptors (CAR) recognizing CD19 can induce complete remission of B-cell malignancies in clinical trials; however, in some disease settings, CAR therapy confers only modest clinical benefit due to attenuated persistence of CAR T cells. The purpose of this study was to enhance persistence and augment the antitumor activity of adoptively transferred CD19CAR T cells by restimulating CAR(+) T cells through an endogenous cytomegalovirus (CMV)-specific T-cell receptor. CMV-specific T cells from CMV seropositive healthy donors were selected after stimulation with pp65 protein and transduced with clinical-grade lentivirus expressing the CD19R:CD28:ζ/EGFRt CAR. The resultant bispecific T cells, targeting CMV and CD19, were expanded via CD19 CAR-mediated signals using CD19-expressing cells. The bispecific T cells proliferated vigorously after engagement with either endogenous CMVpp65 T-cell receptors or engineered CD19 CARs, exhibiting specific cytolytic activity and IFNγ secretion. Upon adoptive transfer into immunodeficient mice bearing human lymphomas, the bispecific T cells exhibited proliferative response and enhanced antitumor activity following CMVpp65 peptide vaccine administration. We have redirected CMV-specific T cells to recognize and lyse tumor cells via CD19CARs, while maintaining their ability to proliferate in response to CMV antigen stimulation. These results illustrate the clinical applications of CMV vaccine to augment the antitumor activity of adoptively transferred CD19CAR T cells in patients with B-cell malignancies. ©2015 American Association for Cancer Research.

  9. PCR thermocycler

    Science.gov (United States)

    Benett, William J.; Richards, James B.

    2003-01-01

    A sleeve-type silicon polymerase chain reaction (PCR) chamber or thermocycler having improved thermal performance. The silicon sleeve reaction chamber is improved in thermal performance by etched features therein that reduce thermal mass and increase the surface area of the sleeve for cooling. This improved thermal performance of the thermocycler enables an increase in speed and efficiency of the reaction chamber. The improvement is accomplished by providing grooves in the faces of the sleeve and a series of grooves on the interior surfaces that connect with grooves on the faces of the sleeve. The grooves can be anisotropically etched in the silicon sleeve simultaneously with formation of the chamber.

  10. Real-time PCR in infectious uveitis as an alternative diagnosis

    Directory of Open Access Journals (Sweden)

    Fabio Felipe dos Santos

    2011-08-01

    Full Text Available PURPOSE: Uveitis is a major visual impairment disease affecting parts or the entire uveal tract and occasionally the sclera, the cornea or the optic nerve. The disease is a major cause of ocular morbidity and blindness in immunocompetent and immunocompromised patients. In this work we analyzed the sensitivity and specificity of real-time PCR to detect the etiological agent from blood, plasma, vitreous and aqueous humor and compared with the diagnostic hypothesis. METHODS: Twenty-seven patients (13 male were studied and Real-time PCR method was used for the detection of herpes simplex virus 1 (HSV-1, herpes simplex virus 2 (HSV-2, varicella zoster virus (VZV, cytomegalovirus (CMV, Mycobacterium tuberculosis (TB and Toxoplasma gondii (Toxo in the aqueous humor as well as in the vitreous, blood and plasma. RESULTS: Our results showed the presence of Toxo, CMV, VZV or HSV-2 in 19.2% of aqueous humor samples, and in 30% of vitreous humor samples. In plasma and blood samples, only CMV was detected (11.1% and 3.7%, respectively. CONCLUSION: Real-time PCR was able to detect and to confirm diagnostic hypothesis in uveitis. Our data also confirms that vitreous humor is the best source for molecular diagnosis of infectious uveitis but indicates aqueous humor samples that are easier to obtain may also be appropriate to be tested by Real-time PCR.

  11. Effect of Cytomegalovirus (CMV) and Ageing on T-Bet and Eomes Expression on T-Cell Subsets

    Science.gov (United States)

    Hassouneh, Fakhri; Lopez-Sejas, Nelson; Campos, Carmen; Sanchez-Correa, Beatriz; Tarazona, Raquel; Pera, Alejandra; Solana, Rafael

    2017-01-01

    The differential impact of ageing and cytomegalovirus (CMV) latent infection on human T-cell subsets remains to some extent controversial. The purpose of this study was to analyse the expression of the transcription factors T-bet and Eomes and CD57 on CD4+, CD4hiCD8lo and CD8+ T-cell subsets in healthy individuals, stratified by age and CMV serostatus. The percentage of CD4+ T-cells expressing T-bet or Eomes was very low, in particular in CD4+ T-cells from young CMV-seronegative individuals, and were higher in CMV-seropositive older individuals, in both CD57− and CD57+ CD4+ T-cells. The study of the minor peripheral blood double-positive CD4hiCD8lo T-cells showed that the percentage of these T-cells expressing both Eomes and T-bet was higher compared to CD4+ T-cells. The percentage of CD4hiCD8lo T-cells expressing T-bet was also associated with CMV seropositivity and the coexpression of Eomes, T-bet and CD57 on CD4hiCD8lo T-cells was only observed in CMV-seropositive donors, supporting the hypothesis that these cells are mature effector memory cells. The percentage of T-cells expressing Eomes and T-bet was higher in CD8+ T-cells than in CD4+ T-cells. The percentages of CD8+ T-cells expressing Eomes and T-bet increased with age in CMV-seronegative and -seropositive individuals and the percentages of CD57− CD8+ and CD57+ CD8+ T-cells coexpressing both transcription factors were similar in the different groups studied. These results support that CMV chronic infection and/or ageing are associated to the expansion of highly differentiated CD4+, CD4hiCD8lo and CD8+ T-cells that differentially express T-bet and Eomes suggesting that the expression of these transcription factors is essential for the generation and development of an effector-memory and effector T lymphocytes involved in conferring protection against chronic CMV infection. PMID:28661443

  12. Cmv2b-AGO interaction is required for the suppression of RDR-dependent antiviral silencing in Arabidopsis

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    Yuan-Yuan Fang

    2016-08-01

    Full Text Available Using a transient plant system, it was previously found that the suppression of Cucumber mosaic virus (CMV 2b protein relies on its double-strand (ds RNA binding capacity, but it is independent of its interaction with ARGONAUTE (AGO proteins. Thus, the biological meaning of the 2b-AGO interaction in the context of virus infection remains elusive. In this study, we created infectious clones of CMV mutants that expressed the 2b functional domains of dsRNA or AGO binding and tested the effect of these CMV mutants on viral pathogenicity. We found that the mutant CMV2b(1-76 expressing the 2b dsRNA-binding domain exhibited the same virulence as wild-type CMV in infection with either wild-type Arabidopsis or rdr1/6 plants with RDR1- and RDR6-deficient mutations. However, remarkably reduced viral RNA levels and increased virus (vsiRNAs were detected in CMV2b(1-76-infected Arabidopsis in comparison to CMV infection, which demonstrated that the 2b(1-76 deleted AGO-binding domain failed to suppress the RDR1/RDR6-dependent degradation of viral RNAs. The mutant CMV2b(8-111 expressing mutant 2b, in which the N-terminal 7 amino acid (aa was deleted, exhibited slightly reduced virulence, but not viral RNA levels, in both wild-type and rdr1/6 plants, which indicated that 2b retained the AGO-binding activity acquired the counter-RDRs degradation of viral RNAs. The deletion of the N-terminal 7 aa of 2b affected virulence due to the reduced affinity for long dsRNA. The mutant CMV2b(18-111 expressing mutant 2b lacked the N-terminal 17 aa but retained its AGO-binding activity greatly reduced virulence and viral RNA level. Together with the instability of both 2b(18-111-EGFP and RFP-AGO4 proteins when co-expressed in Nicotiana benthamiana leaves, our data demonstrates that the effect of 2b-AGO interaction on counter-RDRs antiviral defense required the presence of 2b dsRNA-binding activity. Taken together, our findings demonstrate that the dsRNA-binding activity of the

  13. Comparison of pp65 antigenemia, quantitative PCR and DNA hybrid capture for detection of cytomegalovirus in transplant recipients and AIDS patients.

    Science.gov (United States)

    Mhiri, Leila; Kaabi, Belhassen; Houimel, Mehdi; Arrouji, Zakia; Slim, Amine

    2007-07-01

    The cytomegalovirus (CMV) antigenemia assay has been used frequently for rapid diagnosis of CMV infection, and antigenemia threshold values are recommended for triggering preemptive therapy. Hybrid capture of CMV's DNA and quantitative polymerase chain reaction (qPCR) are increasingly being adopted for early detection of CMV. The performance of the antigenemia assay, qPCR in plasma and hybrid capture in leukocytes were compared in 110 immunocompromised patients (38 bone-marrow transplants, 50 renal transplants and 22 AIDS patients). The most sensitive test was hybrid capture for transplants, while antigenemia and the qPCR showed similar performance for patients with AIDS. QPCR and hybrid capture thresholds requiring antiviral therapy were calculated using a receiver-operating-characteristic curve for antigenemia values corresponding to 2 positive cells for bone-marrow transplants and to 10 positive cells for renal transplants and AIDS patients. These threshold values varied with the group of patients considered, with corresponding sensitivities higher than 86% and specificities higher than 76% for hybrid capture, and sensitivities higher than 61% and specificities higher than 75% for qPCR in plasma. Hybrid capture in leukocytes can substitute for antigenemia in the case of transplants, and qPCR in plasma can substitute for it in the case of AIDS patients.

  14. Acute confusional state: A manifestation of toxoplasma and CMV co-infection in HIV patient

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    Waqas Jehangir

    2014-01-01

    Full Text Available Context: When dealing with a patient with HIV that presents with an altered mental status, there are various infections and disease etiologies a physician has to rule out that may play a role in complicating the inherent complex nature of HIV. Toxoplasma gondii (T. gondii and cytomegalovirus (CMV affect a large part of the world′s population and lead to a varied and broad symptomatology depending upon the severity of HIV, the CD4 count and how early the infection is diagnosed. Case Report: We report an HIV+ patient in his early 50s and with a low CD4 count that presented with severe lethargy and confusion. Imaging studies that were performed after stabilizing the patient revealed a ring-enhancing lesion in the brain and after further testing, a diagnosis of reactivated T. gondii with co-infection with CMV was made. Patients infected with T.gondii that are already immune-compromised deteriorate rapidly and the disease diagnosis poses several challenges. Conclusion: Clinicians have to be extremely careful about making a prompt diagnosis and initiate treatment without delay before the infection takes a deadly toll on the patient. Since our patient was not on the required prophylactic medication to prevent infection with T. gondii, it was imperative to start treatment in a timely manner and to monitor the patient for any further decline in functioning.

  15. Monitoring of trough plasma ganciclovir levels and peripheral blood cytomegalovirus (CMV)-specific CD8+ T cells to predict CMV DNAemia clearance in preemptively treated allogeneic stem cell transplant recipients.

    Science.gov (United States)

    Giménez, Estela; Solano, Carlos; Azanza, José Ramón; Amat, Paula; Navarro, David

    2014-09-01

    It is uncertain whether monitoring plasma ganciclovir (GCV) levels is useful in predicting cytomegalovirus (CMV) DNAemia clearance in preemptively treated allogeneic stem cell transplant recipients. In this observational study, including 13 episodes of CMV DNAemia treated with intravenous (i.v.) GCV or oral valganciclovir, we showed that monitoring trough plasma GCV levels does not reliably predict response to therapy. Rather, immunological monitoring (pp65 and immediate-early [IE]-1-specific gamma interferon [IFN-γ]-producing CD8+ T cells) appeared to perform better for this purpose.

  16. Autocrine production of beta-chemokines protects CMV-Specific CD4 T cells from HIV infection.

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    Joseph P Casazza

    2009-10-01

    Full Text Available Induction of a functional subset of HIV-specific CD4+ T cells that is resistant to HIV infection could enhance immune protection and decrease the rate of HIV disease progression. CMV-specific CD4+ T cells, which are less frequently infected than HIV-specific CD4+ T cells, are a model for such an effect. To determine the mechanism of this protection, we compared the functional response of HIV gag-specific and CMV pp65-specific CD4+ T cells in individuals co-infected with CMV and HIV. We found that CMV-specific CD4+ T cells rapidly up-regulated production of MIP-1alpha and MIP-1beta mRNA, resulting in a rapid increase in production of MIP-1alpha and MIP-1beta after cognate antigen stimulation. Production of beta-chemokines was associated with maturational phenotype and was rarely seen in HIV-specific CD4+ T cells. To test whether production of beta-chemokines by CD4+ T cells lowers their susceptibility to HIV infection, we measured cell-associated Gag DNA to assess the in vivo infection history of CMV-specific CD4+ T cells. We found that CMV-specific CD4+ T cells which produced MIP-1beta contained 10 times less Gag DNA than did those which failed to produce MIP-1beta. These data suggest that CD4+ T cells which produce MIP-1alpha and MIP-1beta bind these chemokines in an autocrine fashion which decreases the risk of in vivo HIV infection.

  17. Impact of HIV on CD8+ T cell CD57 expression is distinct from that of CMV and aging.

    Directory of Open Access Journals (Sweden)

    Sulggi A Lee

    Full Text Available Chronic antigenic stimulation by cytomegalovirus (CMV is thought to increase "immunosenesence" of aging, characterized by accumulation of terminally differentiated CD28- CD8+ T cells and increased CD57, a marker of proliferative history. Whether chronic HIV infection causes similar effects is currently unclear.We compared markers of CD8+ T cell differentiation (e.g., CD28, CD27, CCR7, CD45RA and CD57 expression on CD28- CD8+ T cells in healthy HIV-uninfected adults with and without CMV infection and in both untreated and antiretroviral therapy (ART-suppressed HIV-infected adults with asymptomatic CMV infection.Compared to HIV-uninfected adults without CMV (n=12, those with asymptomatic CMV infection (n=31 had a higher proportion of CD28-CD8+ T cells expressing CD57 (P=0.005. Older age was also associated with greater proportions of CD28-CD8+ T cells expressing CD57 (rho: 0.47, P=0.007. In contrast, untreated HIV-infected CMV+ participants (n=55 had much lower proportions of CD28- CD8+ cells expressing CD57 than HIV-uninfected CMV+ participants (P<0.0001 and were enriched for less well-differentiated CD28- transitional memory (TTR CD8+ T cells (P<0.0001. Chronically HIV-infected adults maintaining ART-mediated viral suppression (n=96 had higher proportions of CD28-CD8+ T cells expressing CD57 than untreated patients (P<0.0001, but continued to have significantly lower levels than HIV-uninfected controls (P=0.001. Among 45 HIV-infected individuals initiating their first ART regimen, the proportion of CD28-CD8+ T cells expressing CD57 declined (P<0.0001, which correlated with a decline in percent of transitional memory CD8+ T cells, and appeared to be largely explained by a decline in CD28-CD57- CD8+ T cell counts rather than an expansion of CD28-CD57+ CD8+ T cell counts.Unlike CMV and aging, which are associated with terminal differentiation and proliferation of effector memory CD8+ T cells, HIV inhibits this process, expanding less well

  18. Everolimus-treated renal transplant recipients have a more robust CMV-specific CD8+ T-cell response compared with cyclosporine- or mycophenolate-treated patients.

    Science.gov (United States)

    Havenith, Simone H C; Yong, Si La; van Donselaar-van der Pant, Karlijn A M I; van Lier, René A W; ten Berge, Ineke J M; Bemelman, Fréderike J

    2013-01-15

    In renal transplant recipients, mammalian target of rapamycin (mTOR) inhibitors have been reported to protect against cytomegalovirus (CMV) disease. Here, we questioned whether mTOR inhibitors specifically influence human CMV-induced T-cell responses. We studied renal transplant recipients treated with prednisolone, cyclosporine A (CsA), and mycophenolate sodium (MPS) for the first 6 months after transplantation followed by double therapy consisting of prednisolone/everolimus, which is an mTOR inhibitor (P/EVL; n=10), prednisolone/CsA (P/CsA; n=7), or prednisolone/MPS (P/MPS; n=9). All patients were CMV-IgG positive before transplantation. CMV reactivation was detectable in the first 6 months after transplantation and not thereafter. None of the patients included in this study suffered from CMV disease. Both CD27CD8 and CD27CD28CD4 effector-type T-cell counts, known to be associated with CMV infection, were measured before transplantation and at 6 and 24 months after transplantation. Additionally, we determined both number and function of CMV-specific CD8 T cells at these time points. The number of total CD8 T cells, CD27CD8 T cells, and CD28CD4 T cells increased significantly after switch to therapy with P/EVL but not after switch to P/CsA or P/MPS. Specifically, CMV-specific CD8 T-cell counts significantly increased after switch to therapy with P/EVL. Furthermore, the mTOR inhibitor sirolimus strongly inhibited alloresponses in vitro, whereas it did not affect CMV-specific responses. We observed a significant increase in (CMV-specific) effector-type CD8 and CD4 T-cell counts in everolimus-treated patients. These findings may at least in part explain the reported low incidence of CMV-related pathology in everolimus-treated patients.

  19. Partial corrosion casting to assess cochlear vasculature in mouse models of presbycusis and CMV infection.

    Science.gov (United States)

    Carraro, Mattia; Park, Albert H; Harrison, Robert V

    2016-02-01

    Some forms of sensorineural hearing loss involve damage or degenerative changes to the stria vascularis and/or other vascular structures in the cochlea. In animal models, many methods for anatomical assessment of cochlear vasculature exist, each with advantages and limitations. One methodology, corrosion casting, has proved useful in some species, however in the mouse model this technique is difficult to achieve because digestion of non vascular tissue results in collapse of the delicate cast specimen. We have developed a partial corrosion cast method that allows visualization of vasculature along much of the cochlear length but maintains some structural integrity of the specimen. We provide a detailed step-by-step description of this novel technique. We give some illustrative examples of the use of the method in mouse models of presbycusis and cytomegalovirus (CMV) infection.

  20. Detection of human CMV PP65 protein in glioma brain tumors with immunohistochemistry method

    Directory of Open Access Journals (Sweden)

    MR. Jabbari

    2015-08-01

    Full Text Available Background: Human cytomegalovirus (HCMV may play a role in the development of glioma disease that is one of the most common brain tumors. Objective: The aim of this study was to detect human CMV in patients with glioma in Imam Khomeini hospital, Tehran. Methods: This experimental study was conducted on paraffin-embedded tumor samples of 18 patients referred to Imam Khomeini hospital in 2012. Immunohistochemistry (IHC was performed with monoclonal antibody specific for HCMV PP65 protein and the samples were assessed using a light microscope. Findings: Of 18 patients, 13 (72.2% were positive for HCMV PP65 protein and four of them expired. Conclusion: With regards to the results, more comprehensive studies are recommended for detection of HCMV in patients with glioma using different diagnostic methods.

  1. CMV infection attenuates the disease course in a murine model of multiple sclerosis.

    Directory of Open Access Journals (Sweden)

    Istvan Pirko

    Full Text Available Recent evidence in multiple sclerosis (MS suggests that active CMV infection may result in more benign clinical disease. The goal of this pilot study was to determine whether underlying murine CMV (MCMV infection affects the course of the Theiler's murine encephalitis virus (TMEV induced murine model of MS. A group of eight TMEV-infected mice were co-infected with MCMV at 2 weeks prior to TMEV infection while a second group of TMEV-infected mice received MCMV two weeks post TMEV. We also used 2 control groups, where at the above time points MCMV was replaced with PBS. Outcome measures included (1 monthly monitoring of disability via rotarod for 8 months; (2 in vivo MRI for brain atrophy studies and (3 FACS analysis of brain infiltrating lymphocytes at 8 months post TMEV infection. Co-infection with MCMV influenced the disease course in mice infected prior to TMEV infection. In this group, rotarod detectable motor performance was significantly improved starting 3 months post-infection and beyond (p≤0.024. In addition, their brain atrophy was close to 30% reduced at 8 months, but this was only present as a trend due to low power (p = 0.19. A significant reduction in the proportion of brain infiltrating CD3+ cells was detected in this group (p = 0.026, while the proportion of CD45+ Mac1+ cells significantly increased (p = 0.003. There was also a strong trend for a reduced proportion of CD4+ cells (p = 0.17 while CD8 and B220+ cell proportion did not change. These findings support an immunomodulatory effect of MCMV infection in this MS model. Future studies in this co-infection model will provide insight into mechanisms which modulate the development of demyelination and may be utilized for the development of novel therapeutic strategies.

  2. Antenatal interventions for preventing the transmission of cytomegalovirus (CMV) from the mother to fetus during pregnancy and adverse outcomes in the congenitally infected infant.

    LENUS (Irish Health Repository)

    McCarthy, Fergus P

    2012-01-31

    BACKGROUND: Cytomegalovirus (CMV) is a herpesvirus and the most common cause of congenital infection in developed countries. Congenital CMV infection can have devastating consequences to the fetus. The high incidence and the serious morbidity associated with congenital CMV infection emphasise the need for effective interventions to prevent the antenatal transmission of CMV infection. OBJECTIVES: The aim of this review was to assess the benefits and harms of interventions used during pregnancy to prevent mother to fetus transmission of CMV infection. SEARCH STRATEGY: We searched the Cochrane Pregnancy and Childbirth Group\\'s Trials Register (31 December 2010). SELECTION CRITERIA: All randomised controlled trials (RCTs) and quasi RCTs investigating antenatal interventions for preventing the transmission of CMV from the mother to fetus during pregnancy and adverse outcomes in the congenitally infected infant. DATA COLLECTION AND ANALYSIS: Two review authors independently assessed studies for inclusion. MAIN RESULTS: We identified six studies from the search. None of these studies met the pre-defined criteria for inclusion in this review. AUTHORS\\' CONCLUSIONS: To date, no RCTs are available that examine antenatal interventions for preventing the transmission of CMV from the infected mother to fetus during pregnancy and adverse outcomes in the congenitally infected infant. Further research is needed to assess the efficacy of interventions aimed at preventing the transmission of CMV from the mother to fetus during pregnancy including a long-term follow-up of exposed infants and a cost effective analysis.

  3. Breakage of resistance to Cucumber mosaic virus by co-infection with Zucchini yellow mosaic virus: enhancement of CMV accumulation independent of symptom expression.

    Science.gov (United States)

    Wang, Y; Lee, K C; Gaba, V; Wong, S M; Palukaitis, P; Gal-On, A

    2004-02-01

    Resistance to the cucumovirus Cucumber mosaic virus (CMV) in cucumber cv. Delila was manifested as a very low level of accumulation of viral RNA and capsid protein, and an absence of CMV-induced symptoms. In addition, resistance was observed at the single cell level, with a reduction in accumulation of CMV RNAs, compared to accumulation in cells of the susceptible cucumber cv. Bet Alpha. Resistance to CMV in cv. Delila was broken by co-infection with the potyvirus Zucchini yellow mosaic virus (ZYMV). Resistance breakage in cv. Delila plants was manifested by an increase in the accumulation of (+) and (-) CMV RNA as well as CMV capsid protein, with no increase in the level of accumulation of ZYMV. Resistance breakage in the resistant cultivar by ZYMV also occurred at the single cell level. Thus, synergistic interactions known to occur between a potyvirus and a cucumovirus led to resistance breakage during a double infection. However, resistance breakage was not accompanied by an increase in disease symptoms beyond those induced by ZYMV itself. On co-inoculation with an asymptomatic variant of ZYMV-AG an enhancement of CMV infection occurred without disease manifestation. Consequently, intensification of viral RNA and capsid protein accumulation can occur without a corresponding increase in disease development, suggesting that different host genes regulate viral accumulation and disease development in the CMV-resistant cucumber plants.

  4. Pregnancy Outcomes of Mothers with Detectable CMV-Specific IgM Antibodies: A Three-Year Review in a Large Irish Tertiary Referral Maternity Hospital

    Science.gov (United States)

    Drew, Richard J.; Stapleton, Patrick; Abu, Hala; Healy, Eibhlín; Ferguson, Wendy; De Gascun, Cillian; O'Gorman, Joanne; Eogan, Maeve

    2015-01-01

    A retrospective audit was performed for all obstetric patients who had positive CMV IgM results between January 2012 and December 2014 in the Rotunda Hospital, Ireland. In total, 622 CMV IgM positive tests were performed on samples from 572 patients. Thirty-seven patients had a positive CMV IgM result (5.9%) on the Architect system as part of the initial screening. Three patients were excluded as they were not obstetric patients. Of the 34 pregnant women with CMV IgM positive results on initial screening, 16 (47%) had CMV IgM positivity confirmed on the second platform (VIDAS) and 18 (53%) did not. In the 16 patients with confirmed positive CMV IgM results, four (25%) had acute infection, two (12.5%) had infection of uncertain timing, and ten (62.5%) had infection more than three months prior to sampling as determined by the CMV IgG avidity index. Two of the four neonates of women with low avidity IgG had CMV DNA detected in urine. Both these cases had severe neurological damage and the indication for testing their mothers was because the biparietal diameter (BPD) was less than the 5th centile at the routine 20-week gestation anomaly scan. PMID:26696757

  5. CMV latent infection improves CD8+ T response to SEB due to expansion of polyfunctional CD57+ cells in young individuals.

    Directory of Open Access Journals (Sweden)

    Alejandra Pera

    Full Text Available Cytomegalovirus (CMV latent infection has a deleterious effect on the efficacy of influenza vaccination in the elderly, suggesting that CMV restricts immunological diversity impairing the immune system functionality in old age. Polyfunctional T cells produce multiple cytokines and higher amounts than mono-functional T cells. High number of polyfunctional T cells correlates with better prognosis during infection. Thus, the efficiency of T cell response associates with quality (polyfunctionality rather than with quantity (percentage of T cells. We analyze the effect of CMV infection on CD8+ T cells polyfunctionality --degranulation (CD107a, IFN-gamma and TNF-alpha production--, from young CMV-seropositive and CMV-seronegative individuals and in middle age CMV-seropositive donors, in response to Staphylococcal Enterotoxin B (SEB. Our results show a higher percentage of polyfunctional CD8+ T cells in young CMV-seropositive individuals compared to CMV-seronegative. Also, we find an expansion of CD8+CD57+ T cells in CMV-seropositive individuals, which are more polyfunctional than CD8+CD57- cells. In middle age individuals there is a higher frequency of SEB-responding CD8+ T cells, mainly TNF-alpha or TNF-alpha/IFN-gamma producers, whereas the percentage of polyfunctional cells (IFN-gamma/TNF-alpha/CD107a is similar to the percentages found in young CMV-seropositive. Therefore, whereas it has been shown that CMV latent infection can be detrimental for immune response in old individuals, our results indicate that CMV-seropositivity is associated to higher levels of polyfunctional CD8+ T cells in young and middle age donors. This increase in polyfunctionality, which can provide an immunological advantage in the response to other pathogens, is due to a CD8+CD57+ T cell expansion in CMV-seropositive individuals and it is independent of age. Conversely, age could contribute to the inflammation found in old individuals by increasing the percentage of cells

  6. A Significant Increase of RNAi Efficiency in Human Cells by the CMV Enhancer with a tRNAlys Promoter

    Directory of Open Access Journals (Sweden)

    Ma Weiwei

    2009-01-01

    Full Text Available RNA interference (RNAi is the process of mRNA degradation induced by double-stranded RNA in a sequence-specific manner. Different types of promoters, such as U6, H1, tRNA, and CMV, have been used to control the inhibitory effect of RNAi expression vectors. In the present study, we constructed two shRNA expression vectors, respectively, controlled by tRNAlys and CMV enhancer-tRNAlys promoters. Compared to the vectors with tRNAlys or U6 promoter, the vector with a CMV enhancer-tRNAlys promoter silenced pokemon more efficiently on both the mRNA and the protein levels. Meanwhile, the silencing of pokemon inhibited the proliferation of MCF7 cells, but the induction of apoptosis of MCF7 cells was not observed. We conclude that the CMV enhancer-tRNAlys promoter may be a powerful tool in driving intracellular expression of shRNA which can efficiently silence targeted gene.

  7. 重庆市辣椒TMV和CMV的鉴定%Identification of TMV and CMV in Peper in Chongqing Area

    Institute of Scientific and Technical Information of China (English)

    林清; 吕中华; 黄任中; 黄启中; 雷蕾; 史思茹

    2001-01-01

    1997~1999年在重庆市郊辣椒产区采集病毒病标样162个,采用生物学鉴定、血清学鉴定及稳定性试验进行检测,结果表明,重庆市辣椒病毒病主要由CMV和TMV复合侵染所致,其检出率分别为79.0 %和54.2 %。TMV单独为害检出率较低(7.5 %),CMV单独侵染的频率较高(32.3 %)。%In 1997~1999,162 pepper samples with typical virus symptoms collected from fields in Chongqing were identified by the diagnostic hosts,stability tests and indirect ELISA. The result showed that the pepper virus diseases were mainly caused by complex infection of TMV and CMV. The infecting percentage of CMV and TMV were 79.0 % and 54.2 % respectively. CMV and TMV are the predominant causal viruses of pepper crop in Chongqing area. TMV infected pepper indepedently with 7.5 % and CMV with 32.3 %.

  8. CMV+ Serostatus Associates Negatively with CD4:CD8 Ratio Normalization in Controlled HIV-Infected Patients on cART

    Science.gov (United States)

    Poizot-Martin, Isabelle; Allavena, Clotilde; Duvivier, Claudine; Cano, Carla Eliana; Guillouet de Salvador, Francine; Rey, David; Dellamonica, Pierre; Cuzin, Lise; Cheret, Antoine; Hoen, Bruno

    2016-01-01

    Cytomegalovirus (CMV) infection is common among HIV-infected patients but its repercussion on the course of CD4+ and CD8+ T cells after cART initiation remains elusive. The French Dat'AIDS cohort enrolled 5,688 patients on first-line cART, from which we selected patients who achieved HIV suppression for at least 12 months without modification of cART, and for whom CMV serostatus was available. Five hundred and three patients fulfilled the selection criteria (74% male, median age 43 yrs, 15.5% CDC stage C), of whom 444 (88.3%) were seropositive for CMV (CMV+). Multivariate analyses using mixed-linear models adjusted for the time from HIV suppression, sex, age, transmission risk group, duration of HIV follow-up, the interaction between time from HIV suppression and CMV+ serology, and the nadir CD4 count revealed a negative correlation between CMV+ and CD4:CD8 ratio (coeff. = -0.16; p = 0.001). This correlation was also observed among patients displaying optimal CD4 recovery (≥500 cells/mm3 at M12; coeff. = -0.24; p = 0.002). Hence, CMV+ serostatus antagonizes normalization of the CD4:CD8 ratio, although further analyses of the impact of co-morbidities that associate with CMV serostatus, like HCV infection, are needed to elucidate this antagonism formally. However, this might reflect a premature T cell senescence, thus advocating for a close monitoring of T cells in CMV co-infected patients. In addition, our results raise the question of the benefit of treatment for asymptomatic CMV co-infection in HIV-infected patients. PMID:27824907

  9. Analysis of memory T lymphocyte activity following stimulation with overlapping HLA-A*2402, A*0101 and Cw*0402 restricted CMV pp65 peptides

    Directory of Open Access Journals (Sweden)

    Provenzano Maurizio

    2005-05-01

    Full Text Available Abstract The continuous efforts aimed at the identification of new immune epitopes across the MHC system has led to the discovery that more than one peptide may be restricted to the same HLA antigen and function as an immune determinant for that association. The aim of this study was to compare the ability of two overlapping peptides, the nonamer (9-mer cytomegalovirus (CMV pp65341–349 (QYDPVAALF and the decamer (10-mer CMV pp65341–350 (QYDPVAALFF, and the esadecamer (16-mer peptide containing both the 9-mer and 10-mer sequences, CMV pp65340–355 (RQYDPVAALFFFDIDL, to stimulate and maintain over time a T cell immune reactivation by HLA-A*2402, A*0101, and Cw*0402 cells from CMV-seropositive subjects. The 9-mer, 10-mer, and 16-mer peptides effectively stimulated CTLs from HLA-A*2402, HLA-A*0101, and HLA-Cw*0402 CMV seropositive donors. This data confirms that both the 9-mer and the 10-mer peptides are promiscuous and are not restricted to a single HLA antigen. CMV pp65341–349 and CMV pp65341–350 have the ability to produce CMV-specific CTLs in subjects with several different HLA types, presenting a practical advantage over other peptides that are restricted only to a single HLA antigen, and thus being optimal for CMV adoptive immune therapy. Moreover, since the 16-mer peptide encompasses both the 9-mer and 10-mer peptides, it may be better than either of these peptides for CMV adoptive immune therapy.

  10. 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).

  11. Microgravity Analogues of Herpes Virus Pathogenicity: Human Cytomegalovirus (hCMV) and Varicella Zoster (VZV) Infectivity in Human Tissue Like Assemblies (TLAs)

    Science.gov (United States)

    Goodwin, T. J.; McCarthy, M.; Albrecht, T.; Cohrs, R.

    2009-01-01

    The old adage we are our own worst enemies may perhaps be the most profound statement ever made when applied to man s desire for extraterrestrial exploration and habitation of Space. Consider the immune system protects the integrity of the entire human physiology and is comprised of two basic elements the adaptive or circulating and the innate immune system. Failure of the components of the adaptive system leads to venerability of the innate system from opportunistic microbes; viral, bacteria, and fungal, which surround us, are transported on our skin, and commonly inhabit the human physiology as normal and imunosuppressed parasites. The fine balance which is maintained for the preponderance of our normal lives, save immune disorders and disease, is deregulated in microgravity. Thus analogue systems to study these potential Risks are essential for our progress in conquering Space exploration and habitation. In this study we employed two known physiological target tissues in which the reactivation of hCMV and VZV occurs, human neural and lung systems created for the study and interaction of these herpes viruses independently and simultaneously on the innate immune system. Normal human neural and lung tissue analogues called tissue like assemblies (TLAs) were infected with low MOIs of approximately 2 x 10(exp -5) pfu hCMV or VZV and established active but prolonged low grade infections which spanned .7-1.5 months in length. These infections were characterized by the ability to continuously produce each of the viruses without expiration of the host cultures. Verification and quantification of viral replication was confirmed via RT_PCR, IHC, and confocal spectral analyses of the respective essential viral genomes. All host TLAs maintained the ability to actively proliferate throughout the entire duration of the experiments as is analogous to normal in vivo physiological conditions. These data represent a significant advance in the ability to study the triggering

  12. Microgravity Analogues of Herpes Virus Pathogenicity: Human Cytomegalovirus (hCMV) and Varicella Zoster (VZV) Infectivity in Human Tissue Like Assemblies (TLAs)

    Science.gov (United States)

    Goodwin, T. J.; McCarthy, M.; Albrecht, T.; Cohrs, R.

    2009-01-01

    The old adage we are our own worst enemies may perhaps be the most profound statement ever made when applied to man s desire for extraterrestrial exploration and habitation of Space. Consider the immune system protects the integrity of the entire human physiology and is comprised of two basic elements the adaptive or circulating and the innate immune system. Failure of the components of the adaptive system leads to venerability of the innate system from opportunistic microbes; viral, bacteria, and fungal, which surround us, are transported on our skin, and commonly inhabit the human physiology as normal and imunosuppressed parasites. The fine balance which is maintained for the preponderance of our normal lives, save immune disorders and disease, is deregulated in microgravity. Thus analogue systems to study these potential Risks are essential for our progress in conquering Space exploration and habitation. In this study we employed two known physiological target tissues in which the reactivation of hCMV and VZV occurs, human neural and lung systems created for the study and interaction of these herpes viruses independently and simultaneously on the innate immune system. Normal human neural and lung tissue analogues called tissue like assemblies (TLAs) were infected with low MOIs of approximately 2 x 10(exp -5) pfu hCMV or VZV and established active but prolonged low grade infections which spanned .7-1.5 months in length. These infections were characterized by the ability to continuously produce each of the viruses without expiration of the host cultures. Verification and quantification of viral replication was confirmed via RT_PCR, IHC, and confocal spectral analyses of the respective essential viral genomes. All host TLAs maintained the ability to actively proliferate throughout the entire duration of the experiments as is analogous to normal in vivo physiological conditions. These data represent a significant advance in the ability to study the triggering

  13. Hearing impairment in children with congenital cytomegalovirus (CMV) infection based on distortion product otoacoustic emissions (DPOAE) and brain evoked response audiometry stimulus click (BERA Click) examinations

    Science.gov (United States)

    Airlangga, T. J.; Mangunatmadja, I.; Prihartono, J.; Zizlavsky, S.

    2017-08-01

    Congenital cytomegalovirus (congenital CMV) infection is a leading factor of nongenetic sensorineural hearing loss in children. Hearing loss caused by CMV infection does not have a pathognomonic configuration hence further research is needed. The development of knowledge on hearing loss caused by congenital CMV infection is progressing in many countries. Due to a lack of research in the context of Indonesia, this study assesses hearing impairment in children with congenital CMV infection in Indonesia, more specifically in the Cipto Mangunkusumo Hospital. Our objective was to profile hearing impairment in children 0-5 years of age with congenital CMV infection using Distortion Product Otoacoustic Emissions (DPOAE) and Brain Evoked Response Audiometry Stimulus Click (BERA Click) examinations. This cross-sectional study was conducted in the Cipto Mangunkusum Hospital from November, 2015 to May 2016 with 27 children 0-5 years of age with congenital CMV infection. Of individual ears studied, 58.0% exhibited sensorineural hearing loss. There was a significant relationship between developmental delay and incidence of sensorineural hearing loss. Subjects with a developmental delay were 6.57 times more likely (CI 95%; 1.88-22.87) to experience sensorineural hearing loss. Congenital CMV infection has an important role in causing sensorineural hearing loss in children.

  14. Four sequence positions of the movement protein of Cucumber mosaic virus determine the virulence against cmv1-mediated resistance in melon.

    Science.gov (United States)

    Guiu-Aragonés, Cèlia; Díaz-Pendón, Juan Antonio; Martín-Hernández, Ana Montserrat

    2015-09-01

    The resistance to a set of strains of Cucumber mosaic virus (CMV) in the melon accession PI 161375, cultivar 'Songwhan Charmi', is dependent on one recessive gene, cmv1, which confers total resistance, whereas a second set of strains is able to overcome it. We tested 11 strains of CMV subgroups I and II in the melon line SC12-1-99, which carries the gene cmv1, and showed that this gene confers resistance to strains of subgroup II only and that restriction is not related to either viral replication or cell-to-cell movement. This is the first time that a resistant trait has been correlated with CMV subgroups. Using infectious clones of the CMV strains LS (subgroup II) and FNY (subgroup I), we generated rearrangements and viral chimaeras between both strains and established that the determinant of virulence against the gene cmv1 resides in the first 209 amino acids of the movement protein, as this region from FNY is sufficient to confer virulence to the LS clone in the line SC12-1-99. A comparison of the sequences of the strains of both subgroups in this region shows that there are five main positions shared by all strains of subgroup II, which are different from those of subgroup I. Site-directed mutagenesis of the CMV-LS clone to substitute these residues for those of CMV-FNY revealed that a combination of four of these changes [the group 64-68 (SNNLL to HGRIA), and the point mutations R81C, G171T and A195I] was required for a complete gain of function of the LS MP in the resistant melon plant.

  15. Transgenic peppers that are highly tolerant to a new CMV pathotype.

    Science.gov (United States)

    Lee, Yun Hee; Jung, Min; Shin, Sun Hee; Lee, Ji Hee; Choi, Soon Ho; Her, Nam Han; Lee, Jang Ha; Ryu, Ki Hyun; Paek, Kee Yoeup; Harn, Chee Hark

    2009-02-01

    The CMV (cucumber mosaic virus) is the most frequently occurring virus in chili pepper farms. A variety of peppers that are resistant to CMVP0 were developed in the middle of 1990s through a breeding program, and commercial cultivars have since been able to control the spread of CMVP0. However, a new pathotype (CMVP1) that breaks the resistance of CMVP0-resistant peppers has recently appeared and caused a heavy loss in productivity. Since no genetic source of this new pathotype was available, a traditional breeding method cannot be used to generate a CMVP1-resistant pepper variety. Therefore, we set up a transformation system of pepper using Agrobacterium that had been transfected with the coat protein gene, CMVP0-CP, with the aim of developing a new CMVP1-resistant pepper line. A large number of transgenic peppers (T(1), T(2) and T(3)) were screened for CMVP1 tolerance using CMVP1 inoculation. Transgenic peppers tolerant to CMVP1 were selected in a plastic house as well as in the field. Three independent T(3) pepper lines highly tolerant to the CMVP1 pathogen were found to also be tolerant to the CMVP0 pathogen. These selected T(3) pepper lines were phenotypically identical or close to the non-transformed lines. However, after CMVP1 infection, the height and fruit size of the non-transformed lines became shorter and smaller, respectively, while the T(3) pepper lines maintained a normal phenotype.

  16. Small bowel perforation due to CMV enteritis infection in an HIV-positive patient

    Directory of Open Access Journals (Sweden)

    Michalopoulos Nick

    2013-02-01

    Full Text Available Abstract Background Cytomegalovirus infection of the gastrointestinal tract is common and is more often seen in patients with acquired immunodeficiency syndrome (AIDS. Although small bowel infection is less common than infection of other parts of the gastrointestinal system, it may lead to perforation, an acute complication, with dreadful results. Case presentation This article reports a case of Cytomegalovirus ileitis with multiple small bowel perforations in a young man with human immunodeficiency virus (HIV infection. The patient developed abdominal pain with diarrhea and fever, and eventually acute abdomen with pneumoperitoneum. The patient had poor prognosis and deceased despite the prompt surgical intervention and the antiviral therapy he received. At pathology a remarkable finding was the presence of viral inclusions in smooth muscle fibers. The destruction of muscle cells was the main cause of perforation. Conclusion Morbidity and mortality associated with perforation from CMV enteritis in AIDS patients are high and the life expectancy is short. Cytomegalovirus disease is multifocal; therefore, excision of one portion of the gastrointestinal tract may be followed by a complication elsewhere. Our case elucidate that muscle cell destruction by the virus is a significant cause leading to perforation.

  17. A New FOC Approach of Induction Motor Drive Using DTC Strategy for the Minimization of CMV

    Directory of Open Access Journals (Sweden)

    Kola Satya Narayana

    2013-02-01

    Full Text Available This paper presents a New FOC Approach of Induction Motor Drive using DTC Strategy for the Minimization of CMV (common mode voltage with the switching tables for the generation of PWM signals. High performance induction motor drives require a better transient and steady state performance. To achieve high performance, there are two control strategies namely, field oriented control (FOC and direct torque control (DTC for induction motor drives. Though these two methods give better transient performance, the FOC needs reference frame transformations and DTC gives large steady state ripples. To overcome these drawbacks, this paper presents a novel FOC algorithm for induction motor drives, which combines the principles of both FOC and DTC. The proposed method uses a predetermined switching table instead of a much more time consuming pulse width modulation (PWM procedure. This approach gives a quick torque response like DTC and gives reduced ripple like FOC. The switching table is based on the conventional DTC principle, which gives good performance with reduced common mode voltage variations. To validate the proposed method numerical simulations have been carried out and compared with the existing algorithms. The simulation results confirm the effectiveness of the proposed method.

  18. Detection and quantification of EBV, HHV-6 and CMV DNA in the gastrointestinal tract of HIV-positive patients.

    Science.gov (United States)

    Falasca, F; Maida, P; Gaeta, A; Verzaro, S; Mezzaroma, I; Fantauzzi, A; Donato, G; Bonci, E; Castilletti, C; Antonelli, G; Turriziani, O

    2014-12-01

    Human herpes viruses (HHVs) have been frequently detected in the gastrointestinal (GI) tract and may contribute to the development of gastric cancer. In the present study, the detection rate and viral load of Epstein Barr virus (EBV), HHV-6 and Cytomegalovirus (CMV) were assessed in the GI tract of human immunodeficiency virus (HIV) positive patients and of uninfected patients. The analysis revealed a significantly higher detection rate of EBV and HHV-6 in HIV-infected individuals than in uninfected subjects (88.5 vs 63%; p = 0.03). Moreover, EBV DNA load was significantly higher in the stomach of HIV patients than in controls. These data suggest that the HIV infection status may increase the persistence of these viruses in the GI compartment. Intriguingly, CMV DNA was undetectable in all biopsy specimens analyzed.

  19. Sex Determination Using PCR

    Science.gov (United States)

    Kima, Peter E.; Rasche, Madeline E.

    2004-01-01

    PCR has revolutionized many aspects of biochemistry and molecular biology research. In the following exercise, students learn PCR by isolating their own DNA, amplifying specific segments of the X and Y chromosomes, and estimating the sizes of the PCR products using agarose gel electrophoresis. Based on the pattern of PCR products, students can…

  20. BnSGS3 Has Differential Effects on the Accumulation of CMV, ORMV and TuMV in Oilseed Rape

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    Quan Chen

    2015-07-01

    Full Text Available Virus diseases greatly affect oilseed rape (Brassica napus production. Investigating antiviral genes may lead to the development of disease-resistant varieties of oilseed rape. In this study, we examined the effects of the suppressor of gene silencing 3 in Brassica napus (BnSGS3, a putative antiviral gene with different genus viruses by constructing BnSGS3-overexpressing (BnSGS3-Ov and BnSGS3-silenced (BnSGS3-Si oilseed rape (cv. Zhongshuang No. 6 plants. These three viruses are Oilseed rape mosaic virus (ORMV, Turnip mosaic virus (TuMV and Cucumber mosaic virus (CMV. The native BnSGS3 expressed in all examined tissues with the highest expression in siliques. All three viruses induced BnSGS3 expression, but ORMV induced a dramatic increase in the BnSGS3-Ov plants, followed by TuMV and CMV. Upon inoculation with three different viruses, transcript abundance of BnSGS3 gene follows: BnSGS3-Ov > non-transgenic plants > BnSGS3-Si. The accumulation quantities of ORMV and TuMV exhibited a similar trend. However, CMV accumulation showed an opposite trend where virus accumulations were negatively correlated with BnSGS3 expression. The results suggest that BnSGS3 selectively inhibits CMV accumulation but promotes ORMV and TuMV accumulation. BnSGS3 should be used in different ways (up- and down-regulation for breeding virus-resistant oilseed rape varieties.

  1. ELIMINATION OF Cucumber Mozaic Virus (CMV FROM A RANGE OF CHRYSANTHEMUM CULTIVARS THROUGH MERISTEM CULTURE FOLLOWING HEAT TREATMENT

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    Kurniawan Budiarto

    2011-11-01

    Full Text Available Cucumber Mosaic Virus (CMV is one of pathogenic viruses that commonly found and has caused significant losses in many chrysanthemum growers in Indonesia.  Efforts have been made to get healthy plant source through eliminating virus from infected plants and one of the promising methods was the combination of thermotherapy and meristem culture. The research was conducted to find out the effect of meristem culture following heat treatments on the existence of CMV in infected chrysanthemum plantlets. The experiment was carried out in the laboratory of tissue culture and virology at The Indonesian Ornamental Crops Research Institute (IOCRI from August 2007 until June 2008. A complete factorial with ten replications was designed to accomplish the combination of two factors.  The first factor was three chrysanthemum cultivars, namely Stroika, Dewi Sartika and White Fiji, while the second dealt with the durations of heat treatment i.e. one, two and three weeks.  The results showed that plantlet survival decreased, yet improved plantlet performance by faster bud initiation with lengthened heat duration.  The percentage of virus-free planlets also increased along with the duration of treatment and three weeks heat treatment followed by meristem culture effectively eliminated CMV from infected planlets.

  2. Lower CD28+ T cell proportions were associated with CMV-seropositivity in patients with Hashimoto’s thyroiditis

    Science.gov (United States)

    2013-01-01

    Background Alterations in the naive T cell subpopulations have been demonstrated in patients with T cell mediated autoimmune disorders, reminiscent of immunological changes found in the elderly during immunosenescence, including the switch from CD45RA + to CD45RO + T cells and decreased thymic function with increased compensatory proliferative mechanisms, partly associated with latent Cytomegalovirus (CMV) infection. The present study was aimed to investigate proportions of lymphocytes, their relation to CMV-seropositivity and the replicative history of CD45RA + expressing T cells in Hashimoto’s thyroiditis (HT, n = 18) and healthy controls (HC, n = 70). Methods Proportions of peripheral T cells were investigated by flow cytometry. The replicative history was assessed by T cell receptor excision circles (TRECs) and relative telomere length (RTL). Expression of CD62L was analyzed by immunohistochemistry in thyroid sections. The role of CMV was assessed by serology, ELISPOT assay and in situ hybridization. Results Our results demonstrated a significant increase of CD28-negative T cells, associated with CMV-seropositivity in HT patients. HT showed abundant CD45RO + T cells with peripheral loss of CD62L-expressing CD8 + CD45RA + T cells, the latter mainly depending on disease duration. CD62L was expressed in thyroid lymphocyte infiltrations. The diagnosis of HT and within the HT group CMV-seropositivity were the main determinants for the loss of CD28 expression. RTL was not different between HC and HT. HT showed significantly lower TRECs in CD4 + CD45RA + T cells compared to HC. Conclusions Patients with HT display a peripheral T cell phenotype reminiscent of findings in elderly persons or other autoimmune disorders. Whether these mechanisms are primary or secondary to the immunological alterations of autoimmune conditions should be investigated in longitudinal studies which may open research on new therapeutic regimes for

  3. The effect of Chrysoperla carnea (Neuroptera: Chrysopidae) and Adalia bipunctata (Coleoptera: Coccinellidae) on the spread of cucumber mosaic virus (CMV) by Aphis gossypii (Hemiptera: Aphididae).

    Science.gov (United States)

    Garzón, A; Budia, F; Medina, P; Morales, I; Fereres, A; Viñuela, E

    2015-02-01

    The effects of two aphidophagous predators, the larvae of Chrysoperla carnea and adults of Adalia bipunctata, on the spread of cucumber mosaic virus (CMV) transmitted in a non-persistent manner by the cotton aphid Aphis gossypii were studied under semi-field conditions. Natural enemies and aphids were released inside insect-proof cages (1 m × 1 m × 1 m) with a central CMV-infected cucumber plant surrounded by 48 healthy cucumber seedlings, and the spatiotemporal dynamics of the virus and vector were evaluated in the short and long term (1 and 5 days) in the presence and absence of the natural enemy. The spatial analysis by distance indices methodology together with other indices measuring the dispersal around a single focus was used to assess the spatial pattern and the degree of association between the virus and its vector. Both natural enemies significantly reduced the number of aphids in the CMV-source plant after 5 days but not after 1 day. The CMV transmission rate was generally low, especially after 1 day, due to the limited movement of aphids from the central CMV-source plant, which increased slightly after 5 days. Infected plants were mainly located around the central virus-infected source plant, and the percentage of aphid occupation and CMV-infected plants did not differ significantly in absence and presence of natural enemies. The distribution patterns of A. gossypii and CMV were only coincident close to the central plant. The complexity of multitrophic interactions and the role of aphid predators in the spread of CMV are discussed.

  4. Insertion of core CpG island element into human CMV promoter for enhancing recombinant protein expression stability in CHO cells.

    Science.gov (United States)

    Mariati; Yeo, Jessna H M; Koh, Esther Y C; Ho, Steven C L; Yang, Yuansheng

    2014-01-01

    The human cytomegalovirus promoter (hCMV) is susceptible to gene silencing in CHO cells, most likely due to epigenetic events, such as DNA methylation and histone modifications. The core CpG island element (IE) from the hamster adenine phosphoribosyltransferase gene has been shown to prevent DNA methylation. A set of modified hCMV promoters was developed by inserting one or two copies of IE in either forward or reverse orientations either upstream of the hCMV enhancer, between the enhancer and core promoter (CP), or downstream of the CP. The modified hCMV with one copy of IE inserted between the enhancer and core promoter in reverse orientation (MR1) was most effective at enhancing expression stability without compromising expression level when compared with the wild-type (WT) hCMV. A third of 18 EGFP expressing clones generated using MR1 retained 70% of their starting expression level after 8 weeks of culture in the absence of selection pressure, while none of 18 WT hCMV generated clones had expression above 50%. MR1 also improved antibody expression stability of methotrexate (MTX) amplified CHO cell lines. Stably transfected pools generated using MR1 maintained 62% of their original monoclonal antibody titer after 8 weeks of culture in the absence of MTX, compared to only 37% for WT hCMV pools. Low levels of CpG methylation within both WT hCMV and MR1 were observed in all the analyzed cell lines and the methylation levels did not correlate to the expression stability, suggesting IE enhances expression stability by other mechanisms other than preventing methylation. © 2014 American Institute of Chemical Engineers.

  5. CODEHOP PCR and CODEHOP PCR primer design.

    Science.gov (United States)

    Staheli, Jeannette P; Boyce, Richard; Kovarik, Dina; Rose, Timothy M

    2011-01-01

    While PCR primer design for the amplification of known sequences is usually quite straightforward, the design, and successful application of primers aimed at the detection of as yet unknown genes is often not. The search for genes that are presumed to be distantly related to a known gene sequence, such as homologous genes in different species, paralogs in the same genome, or novel pathogens in diverse hosts, often turns into the proverbial search for the needle in the haystack. PCR-based methods commonly used to address this issue involve the use of either consensus primers or degenerate primers, both of which have significant shortcomings regarding sensitivity and specificity. We have developed a novel primer design approach that diminishes these shortcomings and instead takes advantage of the strengths of both consensus and degenerate primer designs, by combining the two concepts into a Consensus-Degenerate Hybrid Oligonucleotide Primer (CODEHOP) approach. CODEHOP PCR primers contain a relatively short degenerate 3' core and a 5' nondegenerate clamp. The 3' degenerate core consists of a pool of primers containing all possible codons for a 3-4 aminoacid motif that is highly conserved in multiply aligned sequences from known members of a protein family. Each primer in the pool also contains a single 5' nondegenerate nucleotide sequence derived from a codon consensus across the aligned aminoacid sequences flanking the conserved motif. During the initial PCR amplification cycles, the degenerate core is responsible for specific binding to sequences encoding the conserved aminoacid motif. The longer consensus clamp region serves to stabilize the primer and allows the participation of all primers in the pool in the efficient amplification of products during later PCR cycles. We have developed an interactive web site and algorithm (iCODEHOP) for designing CODEHOP PCR primers from multiply aligned protein sequences, which is freely available online. Here, we describe the

  6. Are female daycare workers at greater risk of cytomegalovirus infection? A secondary data analysis of CMV seroprevalence between 2010 and 2013 in Hamburg, Germany

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    Stranzinger, Johanna

    2016-04-01

    Full Text Available Background: Close contact with asymptomatic children younger than three years is a risk factor for a primary cytomegalovirus (CMV infection. In pregnant women, such primary infection increases the risk of CMV-induced feto- or embryopathy. Daycare providers have therefore implemented working restrictions for pregnant daycare workers (DCWs in accordance with legislation and guidelines for maternity protection. However, little is known about the infection risk for DCWs. We therefore compared the prevalence of CMV antibodies of pregnant DCWs to that of female blood donors (BDs.Method: In a secondary data analysis, the prevalence of anti-CMV IgG among pregnant DCWs (N=509 in daycare centers (DCCs was compared to the prevalence of female first-time BDs (N=14,358 from the greater region of Hamburg, Germany. Data collection took place between 2010 and 2013. The influence of other risk factors such as age, pregnancies and place of residence was evaluated using logistic regression models. Results: The prevalence of CMV antibodies in pregnant DCWs was higher than in female BDs (54.6 vs 41.5%; OR 1.6; 95%CI 1.3–1.9. The subgroup of BDs who had given birth to at least one child and who lived in the city of Hamburg (N=2,591 had a prevalence of CMV antibodies similar to the prevalence in pregnant DCWs (53.9 vs 54.6%; OR 0.9; 95%CI 0.8–1.2. Age, pregnancy history and living in the center of Hamburg were risk factors for CMV infections.Conclusion: The comparison of pregnant DCWs to the best-matching subgroup of female first-time BDs with past pregnancies and living in the city of Hamburg does not indicate an elevated risk of CMV infection among DCWs. However, as two secondary data sets from convenience samples were used, a more detailed investigation of the risk factors other than place of residence, age and maternity was not possible. Therefore, the CMV infection risk in DCWs should be further studied by taking into consideration the potential preventive

  7. Extracellular matrix synthesis and ultrastructural changes of degenerative disc cells transfected by Ad/CMV-hTGF-β1

    Institute of Scientific and Technical Information of China (English)

    谭延斌; 胡有谷; 谭江威

    2003-01-01

    Objective To determine whether the synthesis of proteoglycan, collagen and associated ultrastructure are related to the adenovirus-mediated gene transferred to adult degenerative cells.Methods Adenovirus/cytomegalovirus human transforming growth fector-β1 (Ad/CMV-hTGF-β1) was used to transfect degenerative cells. Antonopulos method, Miamine method and transmission electrion microscopy were conducted to study the synthesis of proteoglycan, collagen, and ultrastructure, respectively. Cell cultures were established from the nucleus pulpous and annulus fibrosus tissues, which were taken from surgery.Results Nucleus pulpous and annulus fibrosus cells were efficiently transduced by the adenoviral vector carrying hTGF-β1 gene. The synthesis of proteoglycan and collagen increased compared with the control group (P<0.05). The metabolism of cells was slightly improved. No significant toxic effects were found.Conclusions Expression of hTGF-β1 gene is efficient to accelerates proteoglycan synthesis and thus accelerates the improvement of collagen. The function and structure of degenerative cells are improved. Ad/CMV-hTGF-β1 may be suitable for treating disc degeneration.

  8. Immunotherapy with Donor T-cells Sensitized with Overlapping Pentadecapeptides for Treatment of Persistent CMV Infection or Viremia

    Science.gov (United States)

    Koehne, Guenther; Hasan, Aisha; Doubrovina, Ekaterina; Prockop, Susan; Tyler, Eleanor; Wasilewski, Gloria; O’Reilly, Richard J.

    2015-01-01

    We conducted a Phase I trial of allogeneic T-cells sensitized in vitro against a pool of 15-mer peptides spanning the sequence of CMVpp65 for adoptive therapy of 17 allogeneic hematopoietic cell transplant recipients with CMV viremia or clinical infection persisting despite prolonged treatment with antiviral drugs. All but three of the patients had received T-cell depleted transplants without GVHD prophylaxis with immunosuppressive drugs post transplant. The CMVpp65-specific T-cells (CMVpp65CTLs) generated were oligoclonal and specific for only 1–3 epitopes, presented by a limited set of HLA class I or II alleles. T-cell infusions were well tolerated without toxicity or GVHD. Of 17 patients treated with transplant donor (N=16) or third party (N=1) CMVpp65CTLs, 15 cleared viremia including 3 of 5 with overt disease. In responding patients, the CMVpp65CTLs infused consistently proliferated and could be detected by T-cell receptor (TCR) Vβ usage in CMVpp65/HLA tetramer+ populations for period of 120 days to up to 2 years post infusion. Thus, CMVpp65CTLs generated in response to synthetic 15-mer peptides of CMVpp65 are safe and can clear persistent CMV infections in the post transplant period. PMID:26028505

  9. Evaluation of a Probe-Based PCR-ELISA System for Simultaneous Semi Quantitative Detection and Genotyping of Human Cytomegalovirus (HCMV) Infection in Clinical Specimens.

    Science.gov (United States)

    Talkhabifard, Majid; Javid, Naeme; Moradi, Abdolvahab; Ghaemi, Amir; Tabarraei, Alijan

    2017-01-01

    Human cytomegalovirus (HCMV) is a common opportunistic pathogen that causes serious complications in immunosuppressed patients and infected newborns. In this study, PCR-ELISA was optimized for semi-quantitative detection of infection in clinical specimens and simultaneous genotyping of glycoprotein B for 4 major genotypes, due to its significance. During DIG-labeling PCR, a pair of primers amplifies a fragment of variable region of the glycoprotein B encoding sequence. Under optimized conditions, labeled Target amplicons hybridize to biotinated specific probes and are detected in an ELISA system. PCR-ELISA system showed specific performance with detection limit of approximately 100 copies of CMV DNA. The linear correlation was observed between the PCR-ELISA results (OD) and logarithmic scale of CMV (r=0.979). Repeatability of PCR-ELISA detection system for intra-assay and inter-assay was evaluated for negative and positive samples. In optimized conditions of hybridization, differentiation between genotypes of glycoprotein B was feasible using genotype-specific probes in PCR-ELISA genotyping system. In comparison with sequencing method, genotyping system was confirmed with kappa index of 1. PCR-ELISA is proposed as an applicable and reliable technique for semi-quantitative diagnosis and typing of the infection. This technique is flexible to apply in a variety of molecular fields.

  10. CMV retinitis screening and treatment in a resource-poor setting: three-year experience from a primary care HIV/AIDS programme in Myanmar

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    Tun NiNi

    2011-08-01

    Full Text Available Abstract Background Cytomegalovirus retinitis is a neglected disease in resource-poor settings, in part because of the perceived complexity of care and because ophthalmologists are rarely accessible. In this paper, we describe a pilot programme of CMV retinitis management by non-ophthalmologists. The programme consists of systematic screening of all high-risk patients (CD4 3 by AIDS clinicians using indirect ophthalmoscopy, and treatment of all patients with active retinitis by intravitreal injection of ganciclovir. Prior to this programme, CMV retinitis was not routinely examined for, or treated, in Myanmar. Methods This is a retrospective descriptive study. Between November 2006 and July 2009, 17 primary care AIDS clinicians were trained in indirect ophthalmoscopy and diagnosis of CMV retinitis; eight were also trained in intravitreal injection. Evaluation of training by a variety of methods documented high clinical competence. Systematic screening of all high-risk patients (CD4 3 was carried out at five separate AIDS clinics throughout Myanmar. Results A total of 891 new patients (1782 eyes were screened in the primary area (Yangon; the majority of patients were male (64.3%, median age was 32 years, and median CD4 cell count was 38 cells/mm3. CMV retinitis was diagnosed in 24% (211/891 of these patients. Bilateral disease was present in 36% of patients. Patients with active retinitis were treated with weekly intravitreal injection of ganciclovir, with patients typically receiving five to seven injections per eye. A total of 1296 injections were administered. Conclusions A strategy of management of CMV retinitis at the primary care level is feasible in resource-poor settings. With appropriate training and support, CMV retinitis can be diagnosed and treated by AIDS clinicians (non-ophthalmologists, just like other major opportunistic infections.

  11. Associations between EBV and CMV Seropositivity, Early Exposures, and Gut Microbiota in a Prospective Birth Cohort: A 10-Year Follow-up

    Science.gov (United States)

    Carvalho-Queiroz, Claudia; Johansson, Maria A.; Persson, Jan-Olov; Jörtsö, Evelina; Kjerstadius, Torbjörn; Nilsson, Caroline; Saghafian-Hedengren, Shanie; Sverremark-Ekström, Eva

    2016-01-01

    Early-life infections with persistent Epstein–Barr virus (EBV) and cytomegalovirus (CMV) are delayed in affluent countries, probably due to alterations in early environmental exposures, such as maternal age, siblings, and day-care attendance. We have previously reported that the timing of EBV and CMV contraction is related both to allergic sensitization and changes in functional competence of immune cells, while the presence/absence of lactobacilli [Lactobacillus (L.) casei, L. paracasei, and L. rhamnosus] or Staphylococcus (S.) aureus in feces is related to the risk for allergy. Here, we used the same prospective longitudinal birth cohort of children to investigate early-life environmental exposures and their influence on EBV and CMV contraction over time. Since gut microbes also belong to this category of early exposures, we investigated their association with herpesvirus contraction. Our results show that these two viruses are acquired with different kinetics and that EBV and CMV seroprevalence at 10 years of age was 47 and 57%, respectively. We also observed that a delayed EBV or CMV infection was associated with older maternal age [time ratio (TR) 1.14, 95% confidence interval (CI) 1.07–1.21, Padj < 0.001 and TR 1.09, CI 1.03–1.16, Padj = 0.008, respectively]. Further, we present the novel finding that S. aureus colonization reduced the time to CMV acquisition (TR 0.21, CI 0.06–0.78, Padj = 0.02). Together, these findings suggest that there is a relationship between timing of herpesvirus acquisition and early-life immune modulating exposures, which interestingly also includes the early infant gut microbiota. PMID:27630978

  12. Construction of pCMV-Myc-RIG-I-2CARD vector and its expression and localization in HeLa cells%RIG-I蛋白2CARD结构域真核表达载体的构建及其在HeLa细胞中的表达定位

    Institute of Scientific and Technical Information of China (English)

    张丽丽; 程丽鑫; 张华; 曹宏伟; 杨琦; 闫江翠; 张雅婷; 陈宁; 丁筠; 邹德华; 李兴志; 郑亮

    2016-01-01

    Objective To construct pCMV-Myc-RIG-I-2CARD vector and give its expression and localization in HeLa cells.Methods Retinoic acid inducible gene-I (RIG-I) as the pattern recognition receptor, can participate in the MAVS-mediated NF-κB innate anti-viral immune response. Caspase activation and recruitment domain (CARD) is two repeatable caspase activation recruiting areas on the N-terminal of RIG-I. In the processes virus infection, 2CARD domain plays important roles in recognizing and priming immune response. In this study, 2CARD gene was amplified with PCR method, and then was inserted into pCMV-Myc plasmid. The obtained pCMV-Myc-RIG-I-2CARD was transfected into HeLa cells, and the expression of 2CARD gene was confirmed by Western Blot and immunofluorescent. Results The result showed that the 2CARD fusion protein was successfully expressed in HeLa cells. Conclusions This research constructs the pCMV-Myc-RIG-I-2CARD vector, through 2CARD fusion protein successfully expressed in HeLa cells, establishes a foundation to further studies of interactions between virus and RIG-I.%目的:构建 RIG-I 蛋白2CARD 结构域真核表达载体及其在 HeLa 细胞中的表达定位。方法 RIG-I是一种模式识别受体,它能够参与由 MAVS 介导的 NF-κB 天然抗病毒免疫反应。2CARD (Caspase activation and recruitment domain,CARD)结构域是视黄酸诱导基因-I(Retinoin acid inducible gene I,RIG-I)N 端的2个串联重复的半胱天冬酶激活招募区,在病毒侵染细胞过程中,2CARD 结构域发挥了识别并启动机体免疫应答的功能。本研究通过 PCR 获取2CARD 片段酶切后,与 pCMV-Myc 载体连接后转染 HeLa 细胞,在 HeLa 细胞中表达,并应用 Western Blot 和免疫荧光进行鉴定。结果2CARD 基因在HeLa 细胞中成功表达。结论本研究成功构建了 pCMV-Myc-RIG-I-2CARD 真核表达载体,通过将 RIG-I分子中负责信号转导的2CARD 在 HeLa 中表达,为研究病毒与 RIG-I 的互作关系奠定一定的基础。

  13. PCR-based approach for qualitative molecular analysis of six neurotropic pathogens.

    Science.gov (United States)

    Ferese, R; Scorzolini, L; Campopiano, R; Albano, V; Griguoli, A M; Giardina, E; Scala, S; Ryskalin, L; D'Alessio, C; Zampatti, S; Fantozzi, R; Storto, M; Fornai, F; Gambardella, S

    2017-01-01

    In the last few years, polymerase chain reaction analysis is frequently required to improve the detection of pathogen infections in central nervous system as a potential cause of neurological disorders and neuropsychiatric symptoms. The goal of this paper is to set up a fast, cheap and reliable molecular approach for qualitative detection of six neurotropic pathogens. A method based on PCR has been designed and implemented to guarantee the qualitative DNA detection of herpes simplex virus types 1 and 2 (HSVI/II), Epstein-Barr virus (EBV), cytomegalovirus (CMV), varicella-zoster virus (VZV), rubella virus (RUBV) and Toxoplasma gondii in the cerebrospinal fluid, where otherwise they are barely detectable. Each PCR assay was tested using dilutions of positive controls, which demonstrated a sensitivity allowing to detect up to 102 copies/ml in PCR and 10 copies/ml in real-time PCR for each pathogen. Once been set up, the protocol was applied to evaluate the cerebrospinal fluid from 100 patients with suspected infectious diseases of the central nervous system and 50 patients without any infection. The method allowed to identify 17 positive cerebrospinal fluid with polymerase chain reaction and 22 with real-time PCR (RT-PCR), respectively. Therefore, application of RT PCR allows a fast and sensitive evaluation of neurotropic DNA pathogens in the course of diagnostic routine within neurological units.

  14. The combination of a synthetic promoter and a CMV promoter improves foreign gene expression efficiency in myocytes.

    Science.gov (United States)

    Jianwei, Dai; Qianqian, Zhang; Songcai, Liu; Mingjun, Zhang; Xiaohui, Ren; Linlin, Hao; Qingyan, Jiang; Yongliang, Zhang

    2012-04-15

    Skeletal muscle is becoming an attractive target tissue for gene therapy. Nevertheless, the low level of gene therapeutic expression in this tissue is the major limitation to it becoming an ideal target for gene transfer. The promoter is important element for gene transcription; however, the gene expression efficiencies and specificities of viral promoters and skeletal muscle-specific promotors are in themselves limiting factors. In this study, we established a dual-promoters system in skeletal muscle using a cytomegalovirus (CMV) promoter and a skeletal muscle-specific synthetic promoter. Mouse myoblast cell line C2C12 cells were transfected with the system. We demonstrated that the dual-promoters system could significantly improve exogenous gene expression rate in vitro when compared with a single CMV promoter system and a skeletal muscle-specific synthetic promoter system in C2C12 cell line, by 69.48% and 41.93%, respectively. Next, we evaluated the system efficiency in vivo, the results showed that the dual-promoters system increased gene expression in mice 1.23-fold and 1.60-fold, respectively compared with expression controlled by the two single promoter vectors. Finally, we tested the dual-promoters system in growth hormone-releasing hormone (GHRH) gene therapy, and revealed that when these two promoters co-drove the GHRH gene expression in vivo animal growth was enhanced significantly. All these results indicate that use of the dual-promoter vector was more efficient for gene expression in skeletal muscle tissue than use of the single promoter vectors. These finding could, hopefully, lead to the development of a high efficiency expression system in myocytes and form an ideal approach for gene therapy. Copyright © 2011 Elsevier B.V. All rights reserved.

  15. Auxin and Cytokinin Metabolism and Root Morphological Modifications in Arabidopsis thaliana Seedlings Infected with Cucumber mosaic virus (CMV or Exposed to Cadmium

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    Adriano Sofo

    2013-03-01

    Full Text Available Arabidopsis thaliana L. is a model plant but little information is available about morphological root changes as part of a phytohormonal common response against both biotic and abiotic stressors. For this purpose, two-week-old Arabidopsis seedlings were treated with 10 µM CdSO4 or infected with CMV. After 12 days the entire aerial parts and the root system were analyzed, and the presence of CMV or the accumulation of Cd were detected. Microscopic analysis revealed that both CMV and Cd influenced root morphology by a marked development in the length of root hairs and an intense root branching if compared to controls. Among the three treatments, Cd-treated seedlings showed a shorter root axis length and doubled their lateral root diameter, while the lateral roots of CMV-infected seedlings were the longest. The root growth patterns were accompanied by significant changes in the levels of indole-3-acetic acid, trans-zeatin riboside, dihydrozeatin riboside, as a probable consequence of the regulation of some genes involved in their biosynthesis/degradation. The opposite role on root development played by the phythormones studied is discussed in detail. The results obtained could provide insights into novel strategies for plant defense against pathogens and plant protection against pollutants.

  16. WSSV ie1 promoter is more efficient than CMV promoter to express H5 hemagglutinin from influenza virus in baculovirus as a chicken vaccine

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    Yu Li

    2008-12-01

    Full Text Available Abstract Background The worldwide outbreak of influenza A (H5N1 viruses among poultry species and humans highlighted the need to develop efficacious and safe vaccines based on efficient and scaleable production. Results White spot syndrome virus (WSSV immediate-early promoter one (ie1 was shown to be a stronger promoter for gene expression in insect cells compared with Cytomegalovirus immediate-early (CMV promoter in luciferase assays. In an attempt to improve expression efficiency, a recombinant baculovirus was constructed expressing hemagglutinin (HA of H5N1 influenza virus under the control of WSSV ie1 promoter. HA expression in SF9 cells increased significantly with baculovirus under WSSV ie1 promoter, compared with CMV promoter based on HA contents and hemagglutination activity. Further, immunization with baculovirus under WSSV ie1 promoter in chickens elicited higher level anti-HA antibodies compared to CMV promoter, as indicated in hemagglutination inhibition, virus neutralization and enzyme-linked immunosorbent assays. By immunohistochemistry, strong HA antigen expression was observed in different chicken organs with vaccination of WSSV ie1 promoter controlled baculovirus, confirming higher efficiency in HA expression by WSSV ie1 promoter. Conclusion The production of H5 HA by baculovirus was enhanced with WSSV ie1 promoter, especially compared with CMV promoter. This contributed to effective elicitation of HA-specific antibody in vaccinated chickens. This study provides an alternative choice for baculovirus based vaccine production.

  17. Comparative analysis of presence of Cytomegalovirus (CMV and Epsteinbarr virus -1 (EBV-1 in cases of chronic periodontitis and aggressive periodontitis with controls

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    Rupali Sharma

    2012-01-01

    Conclusion: Our studyshowed that the prevalence of EBV1 was higher in chronic and aggressive periodontitis subjects compared to controls and the prevalence of CMV was higher in aggressive periodontitis patients. The virus positive sites showed higher pocket depth compared to virus negative sites.

  18. Clinical assessment of anti-viral CD8+ T cell immune monitoring using QuantiFERON-CMV® assay to identify high risk allogeneic hematopoietic stem cell transplant patients with CMV infection complications.

    Directory of Open Access Journals (Sweden)

    Siok-Keen Tey

    Full Text Available The reconstitution of anti-viral cellular immunity following hematopoietic stem cell transplantation (HSCT is crucial in preventing cytomegalovirus (CMV-associated complications. Thus immunological monitoring has emerged as an important tool to better target pre-emptive anti-viral therapies. However, traditional laboratory-based assays are too cumbersome and complicated to implement in a clinical setting. Here we conducted a prospective study of a new whole blood assay (referred to as QuantiFERON-CMV® to determine the clinical utility of measuring CMV-specific CD8+ T-cell responses as a prognostic tool. Forty-one evaluable allogeneic HSCT recipients underwent weekly immunological monitoring from day 21 post-transplant and of these 21 (51.2% showed CMV reactivation and 29 (70.7% developed acute graft-versus-host disease (GvHD. Patients with acute GvHD (grade ≥ 2 within 6 weeks of transplant showed delayed reconstitution of CMV-specific T-cell immunity (p = 0.013 and a higher risk of CMV viremia (p = 0.026. The median time to stable CMV-specific immune reconstitution was 59 days and the incidence of CMV reactivation was lower in patients who developed this than those who did not (27% versus 65%; p = 0.031. Furthermore, a failure to reconstitute CMV-specific immunity soon after the onset of CMV viraemia was associated with higher peak viral loads (5685 copies/ml versus 875 copies/ml; p = 0.002. Hence, QuantiFERON-CMV® testing in the week following CMV viremia can be useful in identifying HSCT recipients at risk of complicated reactivation.

  19. Cytomegalovirus DNA quantification using an automated platform for nucleic acid extraction and real-time PCR assay setup.

    Science.gov (United States)

    Forman, Michael; Wilson, Andy; Valsamakis, Alexandra

    2011-07-01

    Analytical performance characteristics of the QIAsymphony RGQ system with artus cytomegalovirus (CMV) reagents were determined. Measurable range spanned 2.0 to ≥ 7.0 log(10) copies/ml. The detection limit was 23 copies/ml. Intrarun and interrun coefficients of variation were ≤ 2.1% at 3.0 and 5.0 log(10) copies/ml. In clinical specimens, RGQ values were ~0.2 log(10) copies/ml higher than those in an assay using a BioRobot M48 extraction/manual reaction setup/7500 Real-Time PCR instrument. No cross-contamination was observed.

  20. Apta-PCR.

    Science.gov (United States)

    Pinto, Alessandro; Polo, Pedro Nadal; Rubio, Miriam Jauest; Svobodova, Marketa; Lerga, Teresa Mairal; O'Sullivan, Ciara K

    2016-01-01

    Real-time Apta-PCR is a methodology that can be used for a wide variety of applications ranging from food quality control to clinical diagnostics. This method takes advantage of the combination of the sensitivity of nucleic acid amplification with the selectivity of aptamers. Ultra-low detection of target analyte can potentially be achieved, or, improved detection limits can be achieved with aptamers of low-medium affinity. Herein, we describe a generic methodology coined real-time Apta-PCR, using a model target (β-conglutin) and a competitive format, which can be adapted for the detection of any target which an aptamer has been selected for.

  1. Type I Interferon Elevates Co-regulatory Receptor Expression on CMV- and EBV-specific CD8 T cells in Chronic Hepatitis C

    Directory of Open Access Journals (Sweden)

    Solomon eOwusu Sekyere

    2015-06-01

    Full Text Available Hepatitis C virus (HCV readily sets up persistence in a large fraction of infected hosts. Mounting epidemiological and immunological evidence suggest that HCV’s persistence could influence immune responses towards unrelated pathogens and vaccines. Nonetheless, the fundamental contribution of the inflammatory milieu during persistent HCV infection in impacting immune cells specific for common pathogens such as CMV and EBV has not been fully studied. As the co-regulatory receptors PD-1, Tim-3, and 2B4 have all been shown to be vital in regulating CD8+ T cell function, we assessed their expression on CMV/EBV-specific CD8+ T cells from patients with chronic hepatitis C (CHC and healthy controls ex vivo and upon stimulation with virus-specific peptides in vitro. Total and CMV/EBV-specific CD8+ T cells expressing PD-1, Tim-3 and 2B4 were highly enriched in patients with CHC compared to healthy individuals ex vivo. In vitro peptide stimulation further potentiated the differential co-regulatory receptor expression of PD-1, Tim-3 and 2B4 which then culminated in an enhanced functionality of CMV/EBV-specific CD8+ T cells in CHC patients. Comprehensively analyzing plasma cytokines between the two cohorts, we observed that not only was IFNα-2a dominant among 21 other inflammatory mediators elevated in CHC patients, but it also correlated with PD-1 and Tim-3 expressions ex vivo. Importantly, IFNα-2a further caused up-regulation of these markers upon in vitro peptide stimulation. Finally we could prospectively study patients receiving novel IFN-free antiviral therapy. Here we observed that treatment-induced clearance of HCV resulted in a partial reversion of the phenotype of CMV/EBV-specific CD8+ T cells in patients with CHC. These data reveal an alteration of the plasma concentrations of IFNα-2a together with other inflammatory mediators during chronic hepatitis C, which appeared to pervasively influence co-regulatory receptor expression on CMV

  2. Correlation between clinical diagnosis and PCR analysis of serum, aqueous, and vitreous samples in patients with inflammatory eye disease Correlação entre o diagnóstico clínico e análise do PCR de amostras do soro, aquoso e vítreo em pacientes com doença inflamatória ocular

    Directory of Open Access Journals (Sweden)

    Kimble Matos

    2007-02-01

    Full Text Available PURPOSE: To study the applicability (sensitivity, specificity of polymerase chain reaction (PCR tests in the detection of cytomegalovirus (CMV, herpes virus (HSV and varicella zoster (VZV, Epstein-Barr virus (EBV, Mycobacterium sp and Toxoplasma gondii in the diagnosis of patients with or without AIDS, with presumably infectious uveitis, using serum, aqueous humor and vitreous humor samples. METHODS: Twenty individuals with uveitis of presumed infectious origin were evaluated. Sixteen of them had AIDS, four were immunocompetent individuals. We also evaluated 4 normal controls who underwent vitrectomy surgery. Clinical evaluation of the patients was performed together by three clinicians. PCR evaluations of the serum, aqueous, and vitreous humor were performed in a masked fashion by the laboratory staff. RESULTS: Twelve patients had a clinical diagnosis of CMV retinitis. Of these 6 (50% had a positive PCR for CMV in the vitreous, three (25% had a positive PCR for CMV in the serum, and none were positive in the aqueous. Five patients had a clinical diagnosis of acute retinal necrosis (ARN. Three (60% of these had positive PCR for HSV/VZV in the vitreous. One of these patients had a positive PCR reaction for both EBV and HSV/VZV in the vitreous samples. One patient with cutaneous herpes zoster had a positive PCR reaction for HSV/VZV in the serum. Four patients had a presumed diagnosis of ocular toxoplasmosis, one patient (25% had a positive PCR for Toxoplasma gondii in the serum, 3 (75% had positive results in the aqueous, and 2 (50% had positive results in the vitreous. One patient with presumed ocular tuberculosis had a positive PCR reaction both in the serum and in the vitreous samples. Finally, none of the four control individuals revealed any positive PCR reaction. CONCLUSION: PCR is an auxiliary diagnostic procedure that should be evaluated together with ophthalmological aspects of the patient.OBJETIVOS: Avaliar a aplicabilidade

  3. Explanatory chapter: PCR primer design.

    Science.gov (United States)

    Álvarez-Fernández, Rubén

    2013-01-01

    This chapter is intended as a guide on polymerase chain reaction (PCR) primer design (for information on PCR, see General PCR and Explanatory Chapter: Troubleshooting PCR). In the next section, general guidelines will be provided, followed by a discussion on primer design for specific applications. A list of recommended software tools is shown at the end. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Antiphytoviral Activity of Satureja montana L. ssp. variegata (Host P. W. Ball Essential Oil and Phenol Compounds on CMV and TMV

    Directory of Open Access Journals (Sweden)

    Dubravka Cukrov

    2010-09-01

    Full Text Available The essential oil of Satureja montana L. ssp. variegata (Host P. W. Ball (Lamiaceae was characterized by a high concentration of oxygenated monoterpenes (71.3%, among which carvacrol (19.4% and thymol (16.6% were the major compounds. When the essential oil was applied on local hosts Chenopodium amaranticolor Coste & Reyn. and Chenopodium quinoa Willd. simultaneously with the infecting virus, the number of local lesions on both Tobacco mosaic virus (TMV and Cucumber mosaic virus (CMV infected plants was reduced for 29.2% and 24.1%, respectively. When applied individually for each virus, thymol was more effective in reducing CMV infection (33.2%, while carvacrol was more effective in reducing the TMV infection (34.3%. No synergistic effect of both monoterpenes was observed in the antiviral activity of the oil.

  5. Multifunctional cytomegalovirus (CMV)-specific CD8(+) T cells are not restricted by telomere-related senescence in young or old adults.

    Science.gov (United States)

    Riddell, Natalie E; Griffiths, Stephen J; Rivino, Laura; King, David C B; Teo, Guo H; Henson, Sian M; Cantisan, Sara; Solana, Rafael; Kemeny, David M; MacAry, Paul A; Larbi, Anis; Akbar, Arne N

    2015-04-01

    Antigen-specific multifunctional T cells that secrete interferon-γ, interleukin-2 and tumour necrosis factor-α simultaneously after activation are important for the control of many infections. It is unclear if these CD8(+) T cells are at an early or late stage of differentiation and whether telomere erosion restricts their replicative capacity. We developed a multi-parameter flow cytometric method for investigating the relationship between differentiation (CD45RA and CD27 surface phenotype), function (cytokine production) and replicative capacity (telomere length) in individual cytomegalovirus (CMV) antigen-specific CD8(+) T cells. This involves surface and intracellular cell staining coupled to fluorescence in situ hybridization to detect telomeres (flow-FISH). The end-stage/senescent CD8(+)  CD45RA(+)  CD27(-) T-cell subset increases significantly during ageing and this is exaggerated in CMV immune-responsive subjects. However, these end-stage cells do not have the shortest telomeres, implicating additional non-telomere-related mechanisms in inducing their senescence. The telomere lengths in total and CMV (NLV)-specific CD8(+) T cells in all four subsets defined by CD45RA and CD27 expression were significantly shorter in old compared with young individuals in both a Caucasian and an Asian cohort. Following stimulation by anti-CD3 or NLV peptide, similar proportions of triple-cytokine-producing cells are found in CD8(+) T cells at all stages of differentiation in both age groups. Furthermore, these multi-functional cells had intermediate telomere lengths compared with cells producing only one or two cytokines after activation. Therefore, global and CMV (NLV)-specific CD8(+) T cells that secrete interferon-γ, interleukin-2 and tumour necrosis factor-α are at an intermediate stage of differentiation and are not restricted by excessive telomere erosion.

  6. Construction of recombinant p16 Adenovirus plasmid%重组pAd/CMV/V5-DEST-p16质粒的构建

    Institute of Scientific and Technical Information of China (English)

    姚彬; 胡勇; 熊自忠; 李旭

    2006-01-01

    摘要目的构建并鉴定pAd/CMV/V5-DEST-p16重组腺病毒质粒.方法合成人p16-INK4表达基因,构建pENTR1A-p16质粒.通过LR反应,入口克隆pENTR1A-p16质粒的外源性合成的修饰后的p16 cDNA,取代目的质粒pAd/CMV/V5-DEST中的ccdB基因,形成表达克隆pAd/CMV/V5-DEST-p16,测序证实质粒含有目的基因.结果构建了p16重组腺病毒质粒,为研究p16-INK4的作用创造了条件.结论用通路克隆系统构建重组p16腺病毒质粒稳定、可靠、方便.

  7. PCR in forensic genetics

    DEFF Research Database (Denmark)

    Morling, Niels

    2009-01-01

    Since the introduction in the mid-1980s of analyses of minisatellites for DNA analyses, a revolution has taken place in forensic genetics. The subsequent invention of the PCR made it possible to develop forensic genetics tools that allow both very informative routine investigations and still more...... and more advanced, special investigations in cases concerning crime, paternity, relationship, disaster victim identification etc. The present review gives an update on the use of DNA investigations in forensic genetics....

  8. Evaluation of PCR and multiplex PCR in relation to nested PCR for diagnosing Theileria equi

    Directory of Open Access Journals (Sweden)

    Danielle C. Leal

    2011-07-01

    Full Text Available Conventional PCR (PCRTeq for diagnosing Theileria equi and multiplex PCR (M/PCRTeq-Bc for diagnosing T. equi and Babesia caballi were comparatively evaluated with nested PCR (N/PCR-Teq for diagnosing equine piroplasmosis. In DNA sensitivity determinations, in multiple dilutions of equine blood that had tested positive for T. equi, PCR-Teq and N/PCR-Teq detected hemoparasite DNA in the larger dilutions (1:128, but did not differ significantly from the M/PCRTeq-Bc (1:64. In analyses on equine serum tested by ELISA, there was high agreement between this serological test and PCR-Teq (k = 0.780 and moderate agreement with N/PCR-Teq (k = 0.562 and M/PCRTeq-Bc (k = 0.488. PCR-Teq found a higher frequency of T. equi both in extensively and intensively reared horses, but this was not significant in relation to N/PCR-Teq (P>0.05, and both PCRs indicated that there was an endemic situation regarding T. equi in the population of horses of this sample. PCR-Teq was only significantly different from M/PCR-Teq-Bc (P<0.05. PCR-Teq presented high sensitivity and specificity, comparable to N/PCR-Teq, but with the advantage of higher speed in obtaining results and lower costs and risks of laboratory contamination. This accredits PCR-Teq for epidemiological studies and for determinations on affected horses.

  9. Precision and linearity targets for validation of an IFNγ ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides

    Directory of Open Access Journals (Sweden)

    Lyerly Herbert K

    2008-03-01

    Full Text Available Abstract Background Single-cell assays of immune function are increasingly used to monitor T cell responses in immunotherapy clinical trials. Standardization and validation of such assays are therefore important to interpretation of the clinical trial data. Here we assess the levels of intra-assay, inter-assay, and inter-operator precision, as well as linearity, of CD8+ T cell IFNγ-based ELISPOT and cytokine flow cytometry (CFC, as well as tetramer assays. Results Precision was measured in cryopreserved PBMC with a low, medium, or high response level to a CMV pp65 peptide or peptide mixture. Intra-assay precision was assessed using 6 replicates per assay; inter-assay precision was assessed by performing 8 assays on different days; and inter-operator precision was assessed using 3 different operators working on the same day. Percent CV values ranged from 4% to 133% depending upon the assay and response level. Linearity was measured by diluting PBMC from a high responder into PBMC from a non-responder, and yielded R2 values from 0.85 to 0.99 depending upon the assay and antigen. Conclusion These data provide target values for precision and linearity of single-cell assays for those wishing to validate these assays in their own laboratories. They also allow for comparison of the precision and linearity of ELISPOT, CFC, and tetramer across a range of response levels. There was a trend toward tetramer assays showing the highest precision, followed closely by CFC, and then ELISPOT; while all three assays had similar linearity. These findings are contingent upon the use of optimized protocols for each assay.

  10. CD107a Expression and IFN-γ Production as Markers for Evaluation of Cytotoxic CD3+ CD8+ T Cell Response to CMV Antigen in Women with Recurrent Spontaneous Abortion

    Directory of Open Access Journals (Sweden)

    Batoul Tarokhian

    2014-01-01

    Full Text Available Background: Some evidence has shown a relationship between primary human cytomegalovirus (CMV infection and pregnancy loss. The impact of CMV infection reactivation during pregnancy on adverse pregnancy outcomes is not completely understood. It is proposed that altered immune response, and therefore, recurrence or reactivation of latent CMV infection may relate to recurrent spontaneous abortion (RSA; however, few data are available in this regard. To find out about any cell mediated defect and reactivation of latent CMV infection in women with RPL, cellular immunity to the virus has been evaluated by specific cytotoxic T lymphocyte (CTL response to CMV. Materials and Methods: In a case control study, CTL CD107a expression and intercellular IFN-γ production in response to CMV pp65 antigen and staphylococcus enterotoxin B (SEB in women with RSA were assessed by flow cytometric analysis. Forty-four cases with history of recurrent pregnancy and forty-four controls with history of successful pregnancies were included. The FACSCaliber flow cytometer were used for analysis. Results: No significant difference was observed between CD107a expression and IFN-γ production in response to CMV PP65 antigen in RPL patients and control group. However, the cytotoxic response to SEB antigen in patients with RPL was significantly lower than control group (p=0.042. Conclusion: The results of this study show that impaired CD107a expression and IFN-γ production as CTL response to CMV does not appear to be a major contributing and immune incompetence factor in patients with RPL, but cytotoxic T cell response defect to other antigens requires to be assessed further in these patients.

  11. Diagnóstico rápido de citomegalovirus (CMV en pacientes inmunocomprometidos mediante anticuerpos monoclonales que reconocen proteinas precoces virales Rapid diagnosis of cytomegalovirus infection in immunocompromised patients by using monoclonal antibodies against early viral antigens

    Directory of Open Access Journals (Sweden)

    Maritza Alvarez

    1989-06-01

    Full Text Available Se aplicó la técnica de detección de antigenos precoces fluorescentes (DAPF usando el anticuerpo monoclonal E-13 McAb, mediante el cual se lograron detectar 15 casos positivos a CMV de 75 muestras de orina o sangre ("buffy coat" tomadas de 52 pacientes inmunocomprometidos ingresados en el Instituto de Nefrología de ciudad Habana. Aplicando las técnicas clásicas de aislamiento en fibroblastos humanos diploides (MRC-5, se lograron aislar 12 cepas de CMV de casos previamente positivos por DAPF; lográndose además un aislamiento en una muestra reportada negativa por fluorescencia. Se observó una coincidencia de un 80% entre ambas técnicas. Se detectó la presencia de anticuerpos IgG contra CMV en todos los casos estudiados, utilizando para ello la técnica ELISA.A technique was applied to detect early fluorescent antigens (DEFA of cytomegalovirus (CMV using the E13 monoclonal antibodies in 52 immunocompromised patients hospitalized in the Nephrology Institute of Havana. Of the 75 urine or blood (buffy coat samples taken, 15 were found positive to CMV. Using classical diploide human fibroblast isolation technique, 12 CMV strains were isloation of previously detected positive samples by DEFA. In addition, CMV was isolated from one sample reported to be negative by DEFA. A coincidence of 80% was found between both techniques. With the ELISA test, all the sample studied have IgG antibodies to CMV.

  12. Sensitivity of multiplex real-time PCR reactions, using the LightCycler and the ABI PRISM 7700 Sequence Detection System, is dependent on the concentration of the DNA polymerase.

    Science.gov (United States)

    Exner, M M; Lewinski, M A

    2002-10-01

    The introduction of multiplex PCR techniques to clinical laboratories has provided a means to streamline assays and to produce multiple results with minimal effort. While this methodology is very beneficial, care must be taken to ensure that reactions are properly optimized to allow for maximum sensitivity. This study was conducted to determine whether the sensitivity of multiplex-real-time PCR assays could be improved by increasing the concentration of DNA polymerase within a reaction. Multiplex reactions were designed to simultaneously detect the human HLA-DQ gene and a sequence from the UL83 region of the CMV genome. Two real-time PCR systems, one utilizing AmpliTaq Gold DNA polymerase and the ABI 7700 Sequence Detection System, and one utilizing FastStart Taq DNA polymerase and the Roche LightCycler were tested. The results indicated that increasing the AmpliTaq Gold concentration from 0.050 to 0.10 U/microl and the FastStart Taq concentration from 0.1875 to 0.375 U/microl increased detection sensitivity from 5,000 to 50 CMV copies per PCR reaction. In separate experiments, commercially prepared mastermixes were utilized for both real-time PCR platforms as per the manufacturer's suggestions or with the addition of supplemental DNA polymerase. In assays designed to detect 4 CMV genome copies per reaction, the addition of 2.5 U of AmpliTaq Gold to TaqMan Universal Mastermix increased the detection rate from 21 to 67%, and the addition of 5 U of FastStart Taq to FastStart DNA Master Hybridization Probes mastermix increased the detection rate from 17 to 56%. These results indicate that increasing the DNA polymerase concentration in multiplex real-time PCR reactions may be a simple way to optimize assay sensitivity.

  13. PCR, exit stage left ...

    CERN Multimedia

    2004-01-01

    The Prevessin Control Room during LEP's start up in 1989. The Prévessin Control Room (PCR) was recently engulfed in a wave of nostalgia. The PCR, scene of some of the greatest moments in CERN's history, is being dismantled to prepare for a complete overhaul. In February 2006, a new combined control centre for all the accelerators will open its doors on the same site, together with a new building currently under construction (see Bulletin issue 27/2004 of 28 June 2004). This marks the end of an important chapter in CERN's history. The Prévessin Control Room saw its first momentous event 28 years ago when the 400 GeV beam for the SPS was commissioned in the presence of Project Leader John Adams. It was also here that the first proton-antiproton collisions were observed, in 1981. Eight years later, in 1989, operators and directors alike jumped for joy at the announcement of the first electron-positron collisions at the start up of LEP, the biggest accelerator in the world. Today the 80 terminals and PCs have b...

  14. Comparing the methods of PCR with fluorogenic quantitative PCR in the detection of HCMV%PCR法与荧光定量PCR法在人巨细胞病毒检测中的作用比较

    Institute of Scientific and Technical Information of China (English)

    吴旗; 邹俊煊; 虞涛; 游上游; 张楚瑜

    2006-01-01

    目的比较常规PCR法与荧光定量PCR(FQ-PCR)法在人巨细胞病毒(HCMV)检测中的作用,寻找更适合于HCMV检测的方法.方法构建携带CMV-DNA的标准质粒,利用常规PCR技术及FQ-PCR技术分别对不同稀释度的标准质粒进行检测.结果RQ-PCR法对HCMV标准质粒检测的灵敏度及线性范围均显著高于常规PCR法.结论FQ-PCR法比常规PCR法更适合HCMV病毒的检测.

  15. Toracotomia em eqüinos sob anestesia com ventilação mecânica controlada Equine thoracotomy under anesthesia with controlled mechanical ventilation (CMV

    Directory of Open Access Journals (Sweden)

    Raquel Yvonne Arantes Baccarin

    1998-06-01

    Full Text Available Diversos procedimentos cirúrgicos e diagnósticos podem ser realizados através da cirurgia torácica em eqüinos. Para tanto faz-se necessária a utilização da ventilação controlada mecânica (VCM e a compreensão dos efeitos respiratórios deste tipo de ventilação durante a toracotomia. Foram utilizados seis eqüinos hígidos, sob anestesia com ventilação controlada, para a realização de acesso cirúrgico ao hemitórax direito com o objetivo de se avaliar, através da mensuração dos parâmetros fisiológicos, ventigrafia, pH e gases sangüíneos, a eficácia da ventilação controlada em otimizar a ventilação e a oxigenação e em prevenir a atelectasia pulmonar durante manipulações intratorácicas. Verificou-se diminuição dos valores da PaCO2 e aumento dos valores de pH durante a ventilação controlada, mesmo com o pneumotórax instalado. A pressão interpleural também apresentou aumento de seus valores durante a VCM. Concluiu-se que o uso da VCM, durante a prática da toracotomia, permite a padronização da freqüência respiratória com melhora da ventilação, e subseqüente normalização das alterações da pressão parcial de dióxido de carbono e pH, bem como mantém visualmente adequada a expansão pulmonar. A VCM promove aumento da pressão interpleural, enquanto o tórax permanece fechado.Several surgical and diagnostic procedures can be performed on thoracic surgery in the equine. To carry out these procedures controlled mechanical ventilation (CMV is necessary, as well as full understanding of the possible side effects of this type of ventilation. Six healthy equine were used under anesthesia wiith CMV to do right hemithorax approach. The goals were to evaluate efficiency of CMV in the improvement of ventilation and oxigenation as well as in the prevention of pulmonar atelectasis during intrathoracic manipulation. For such evaluation of physiologic parameters measurement, ventigraphy and blood gas analysis

  16. Pre-irradiation with low-dose 12C6+beam significantly enhances the efficacy of AdCMV-p53 gene therapy in human non-small lung cancer

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The combination of ionizing radiation and gene therapy has been investigated. However, there are very few reports about the combination of heavy-ion irradiation and gene therapy. To determine if the pre-exposure to low-dose heavy ion beam enhances the suppression of AdCMV-p53 on non-small lung cancer (NSLC), the cells pre-irradiated or non-irradiated were infected with 20, 40 MOI of AdCMV-p53. Survival fraction and the relative biology effect (RBE) were determined by clonogenic assay. The results showed that the proportions of p53 positive cells in 12C6+ beam induced AdCMV-p53 infected cells were more than 90%, which were signifi-cantly more than those in γ-ray induced AdCMV-p53 infected cells. The pre-exposure to low-dose 12C6+ beam significantly prevented the G0/G1 arrest and activated G2/M checkpoints. The pre-exposure to 12C6+ beam significantly improved cell to apoptosis. RBEs for the 12C6+ + AdCMV-p53 infection groups were 30%-60%, 20%-130% and 30%-70% more than those for the 12C6+-irradiated only, AdCMV-p53 infected only, and γ-irradiation induced AdCMVp53 infected groups, respectively. The data suggested that the pre-exposure to low-dose 12C6+ beam significantly promotes exogenous p53 expression in NSLC, and the suppression of AdCMV-p53 gene therapy on NSLC.

  17. Pre-irradiation with low-dose 12C6+ beam significantly enhances the efficacy of AdCMV-p53 gene therapy in human non-small lung cancer

    Science.gov (United States)

    Liu, Bing; Zhang, Hong; Li, Wenjian; Li, Qiang; Zhou, Guangming; Xie, Yi; Hao, Jifang; Min, Fengling; Zhou, Qingming; Duan, Xin

    2007-04-01

    The combination of ionizing radiation and gene therapy has been investigated. However, there are very few reports about the combination of heavy-ion irradiation and gene therapy. To determine if the pre-exposure to low-dose heavy ion beam enhances the suppression of AdCMV-p53 on non-small lung cancer (NSLC), the cells pre-irradiated or non-irradiated were infected with 20, 40 MOI of AdCMV-p53. Survival fraction and the relative biology effect (RBE) were determined by clonogenic assay. The results showed that the proportions of p53 positive cells in 12C6+ beam induced AdCMV-p53 infected cells were more than 90%, which were significantly more than those in γ-ray induced AdCMV-p53 infected cells. The pre-exposure to low-dose 12C6+ beam significantly prevented the G0/G1 arrest and activated G2/M checkpoints. The pre-exposure to 12C6+ beam significantly improved cell to apoptosis. RBEs for the 12C6+ + AdCMV-p53 infection groups were 30% 60%, 20% 130% and 30% 70% more than those for the 12C6+-irradiated only, AdCMV-p53 infected only, and γ-irradiation induced AdCMVp53 infected groups, respectively. The data suggested that the pre-exposure to low-dose 12C6+ beam significantly promotes exogenous p53 expression in NSLC, and the suppression of AdCMV-p53 gene therapy on NSLC.

  18. Pre-irradiation with low-dose 12C6+ beam significantly enhances the efficacy of AdCMV-p53 gene therapy in human non-small lung cancer

    Institute of Scientific and Technical Information of China (English)

    LIU Bing; DUAN Xin; ZHANG Hong; LI WenJian; LI Qiang; ZHOU GuangMing; XIE Yi; HAO JiFang; MIN FengLing; ZHOU QingMing

    2007-01-01

    The combination of ionizing radiation and gene therapy has been investigated. However, there are very few reports about the combination of heavy-ion irradiation and gene therapy. To determine if the pre-exposure to low-dose heavy ion beam enhances the suppression of AdCMV-p53 on non-small lung cancer (NSLC), the cells pre-irradiated or non-irradiated were infected with 20, 40 MOl of AdCMV-p53. Survival fraction and the relative biology effect (RBE) were determined by clonogenic assay. The results showed that the proportions of p53 positive cells in 12C6+ beam induced AdCMV-p53 infected cells were more than 90%, which were significantly more than those in γ-ray induced AdCMV-p53 infected cells. The pre-exposure to low-dose 12C6+ beam significantly prevented the G0/G1 arrest and activated G2/M checkpoints. The pre-exposure to 12C6+ beam significantly improved cell to apoptosis. RBEs for the 12C6+ + AdCMV-p53 infection groups were 30%-60%, 20% -130% and 30%-70% more than those for the 12C6+-irradiated only, AdCMV-p53 infected only, and γ-irradiation induced AdCMVp53 infected groups, respectively. The data suggested that the pre-exposure to low-dose 12C6+ beam significantly promotes exogenous p53 expression in NSLC, and the suppression of AdCMV-p53 gene therapy on NSLC.

  19. Recombination with coat protein transgene in a complemen-tation system based on Cucumber mosaic virus (CMV)

    Institute of Scientific and Technical Information of China (English)

    LEI; Wanli

    2001-01-01

    plant cell suspension cultures, in Plant Tissue Culture Manual (ed. Lindsey, K.), Dordrecht: Kluwer Academic Publishers, 1991, A3: 1-21.[12]Damm, B., Willmitzer, L., Arabidopsis protoplast transformation and re-generation, in Plant Tissue Culture Manual (ed. Lindsey, K.), Dordrecht: Kluwer Academic Publishers, 1991, A7: 1-20.[13]Saunders, J. A., Rhodes, S. C., Kaper, J. M., Effects of electroporation pulse wave on the incorporation of viral RNA into tobacco protoplasts, BioTechniques, 1989, 7: 1124-1131.[14]Laemmli, U. K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 1970, 227: 680-685.[15]Sambrook, J., Fritsch, E. F., Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., New York: Cold Spring Har-bor Laboratory Press, 1989, 18.60-18.75.[16]Ding, S. W., Rathjen, J. P., Li, W. X. et al., Efficient infection from cDNA clones of cucumber mosaic cucumovirus RNAs in a new plasmid vector, J. Gen. Virol., 1995, 76: 459-464.[17]Chomczynski, P., Sacchi, N., Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem., 1987, 162: 156-159.[18]Owen, J., Shintaku, M., Aeschleman, P., Nucleotide sequence and evolutionary relationships of cucumber mosaic virus (CMV) strains: CMV RNA3, J. Gen. Virol., 1990, 71: 2243-2249.[19]Roossinck, M. J., Zhang, L., Hellwald, K. H., Rearrangements in the 5′ nontranslated region and phylogenetic analysis of cucumber mosaic virus RNA3 indicate radial evolution of three subgroups, J. Virol., 1999, 73: 6752-6758.[20]Mori, M., Mise, K., Kobayashi, K., Infectivity of plasmids containing brome mosaic virus cDNA linked to the cauliflower mosaic virus 35S RNA promoter, J. Gen. Virol., 1991, 72: 243-246.[21]Aaziz, R., Tepfer, M., Recombination in RNA viruses and in virus-resistant transgenic plants, J. Gen. Virol., 1999, 80: 1339-1346.[22]Rubio, T., Borja, M., Scholthof, H. B. et al., Recombination with

  20. [Usefulness of a real-time quantitative polymerase-chain reaction (PCR) assay for the diagnosis of congenital and postnatal cytomegalovirus infection].

    Science.gov (United States)

    Reina, J; Weber, I; Riera, E; Busquets, M; Morales, C

    2014-05-01

    Cytomegalovirus (CMV) is the main virus causing congenital and postnatal infections in the pediatric population. The aim of this study is to evaluate the usefulness of a quantitative real-time PCR in the diagnosis of these infections using urine as a single sample. We studied all the urine samples of newborns (PCR (PCRc), and quantitative real-time PCR (PCRq). We analyzed 332 urine samples (270 to rule out congenital infection and 62 postnatal infections). Of the first, 22 were positive in the PCRq, 19 in the PCRc, and 17 in the culture. PCRq had a sensitivity of 100%, on comparing the culture with the rest of the techniques. Using the PCRq as a reference method, culture had a sensitivity of 77.2%, and PCRc 86.3%. In cases of postnatal infection, PCRq detected 16 positive urines, the PCRq 12, and the cell culture 10. The urines showed viral loads ranging from 2,178 to 116,641 copies/ml. The genomic amplification technique PCRq in real time was more sensitive than the other techniques evaluated. This technique should be considered as a reference (gold standard), leaving the cell culture as a second diagnostic level. The low cost and the automation of PCRq would enable the screening for CMV infection in large neonatal and postnatal populations. Copyright © 2013 Asociación Española de Pediatría. Published by Elsevier Espana. All rights reserved.

  1. The Presence of HLA-E-Restricted, CMV-Specific CD8+ T Cells in the Blood of Lung Transplant Recipients Correlates with Chronic Allograft Rejection.

    Directory of Open Access Journals (Sweden)

    Lucy C Sullivan

    Full Text Available The human cytomegalovirus (CMV immune evasion protein, UL40, shares an identical peptide sequence with that found in the leader sequence of many human leukocyte antigen (HLA-C alleles and when complexed with HLA-E, can modulate NK cell functions via interactions with the CD94-NKG2 receptors. However the UL40-derived sequence can also be immunogenic, eliciting robust CD8+ T cell responses. In the setting of solid organ transplantation these T cells may not only be involved in antiviral immunity but also can potentially contribute to allograft rejection when the UL40 epitope is also present in allograft-encoded HLA. Here we assessed 15 bilateral lung transplant recipients for the presence of HLA-E-restricted UL40 specific T cells by tetramer staining of peripheral blood mononuclear cells (PBMC. UL40-specific T cells were observed in 7 patients post-transplant however the magnitude of the response varied significantly between patients. Moreover, unlike healthy CMV seropositive individuals, longitudinal analyses revealed that proportions of such T cells fluctuated markedly. Nine patients experienced low-grade acute cellular rejection, of which 6 also demonstrated UL40-specific T cells. Furthermore, the presence of UL40-specific CD8+ T cells in the blood was significantly associated with allograft dysfunction, which manifested as Bronchiolitis Obliterans Syndrome (BOS. Therefore, this study suggests that minor histocompatibility antigens presented by HLA-E can represent an additional risk factor following lung transplantation.

  2. Fast detection and characterization of organic and inorganic gunshot residues on the hands of suspects by CMV-GC-MS and LIBS.

    Science.gov (United States)

    Tarifa, Anamary; Almirall, José R

    2015-05-01

    A rapid method for the characterization of both organic and inorganic components of gunshot residues (GSR) is proposed as an alternative tool to facilitate the identification of a suspected shooter. In this study, two fast screening methods were developed and optimized for the detection of organic compounds and inorganic components indicative of GSR presence on the hands of shooters and non-shooters. The proposed methods consist of headspace extraction of volatile organic compounds using a capillary microextraction of volatiles (CMV) device previously reported as a high-efficiency sampler followed by detection by GC-MS. This novel sampling technique has the potential to yield fast results (cotton swabs (previously analyzed for VOCs) and an additional 30 swab samples followed by spiking 1μL of the extract solution onto a Teflon disk and then analyzed by LIBS. Advantages of LIBS include fast analysis (~12s per sample) and high selectivity and sensitivity, with expected LODs 0.1-18ng for each of the target elements after sampling. The analytical performance of the LIBS method is also compared to previously reported methods (inductively coupled plasma-optical emission spectroscopy). The combination of fast CMV sampling, unambiguous organic compound identification with GC-MS and fast LIBS analysis provides the basis for a new comprehensive screening method for GSR.

  3. De novo PIK3R1 gain-of-function with recurrent sinopulmonary infections, long-lasting chronic CMV-lymphadenitis and microcephaly.

    Science.gov (United States)

    Kuhlen, Michaela; Hönscheid, Andrea; Loizou, Loizos; Nabhani, Schafiq; Fischer, Ute; Stepensky, Polina; Schaper, Jörg; Klapper, Wolfram; Siepermann, Meinolf; Schuster, Friedhelm; Meisel, Roland; Borkhardt, Arndt

    2016-01-01

    PIK3R1 (phosphoinositide-3-kinase, regulatory subunit 1) gain-of-function has recently been described in patients with recurrent sinopulmonary infections, chronic CMV-/EBV-infections, lymphoproliferation, and hypogammaglobulinemia. Here we report a 15-year-old boy with treatment refractory CMV lymphadenitis, severe combined immunodeficiency, microcephaly and a severe developmental defect of Th17 cells. To avoid poor outcome, hematopoietic stem cell transplantation (HSCT) was performed. Subsequently, whole exome sequencing revealed a de novo heterozygous G-to-C mutation (chr5: 5:67,589,663: G>C) at the splice donor site of the PIK3R1 gene. Our data suggest that PIK3R1 gain-of-function leads to developmental defects in helper and regulatory T-cell subsets, the latter expanding the immunological features of PIK3R1 gain-of-function. T-cell subsets play a critical role in the regulation of immune response against infectious agents and of autoimmunity and thus may be particularly accountable for the clinical phenotype of affected patients. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Infección Activa por el Citomegalovirus (CMV) en el trasplante alogénico de precursores hematopoyéticos:Investigación de nuevos factores biológicos para la estimación del riesgo y análisis integral de los mecanismos inmunitarios que la previenen y controlan

    OpenAIRE

    Giménez Quiles, Estela Berenice

    2016-01-01

    Citomegalovirus (CMV) es causa frecuente de morbididad y mortalidad en el marco del trasplante alogénico de precursores hematopoyéticos (alo-TPH). CMV puede ocasionar enfermedad orgánica o tisular (neumonía intersticial o enfermedad gastrointestinal, con mayor frecuencia), en virtud de su “citopatogenicidad” . CMV también puede causar morbididad a través de mecanismos indirectos, relacionados con su capacidad inmunosupresora y pro-inflamatoria; en ese contexto, CMV parece incrementar el ries...

  5. Infección Activa por el Citomegalovirus (CMV) en el trasplante alogénico de precursores hematopoyéticos:Investigación de nuevos factores biológicos para la estimación del riesgo y análisis integral de los mecanismos inmunitarios que la previenen y controlan

    OpenAIRE

    Giménez Quiles, Estela Berenice

    2016-01-01

    Citomegalovirus (CMV) es causa frecuente de morbididad y mortalidad en el marco del trasplante alogénico de precursores hematopoyéticos (alo-TPH). CMV puede ocasionar enfermedad orgánica o tisular (neumonía intersticial o enfermedad gastrointestinal, con mayor frecuencia), en virtud de su “citopatogenicidad” . CMV también puede causar morbididad a través de mecanismos indirectos, relacionados con su capacidad inmunosupresora y pro-inflamatoria; en ese contexto, CMV parece incrementar el ries...

  6. Digital PCR: A brief history

    OpenAIRE

    2014-01-01

    Digital PCR for quantification of a target of interest has been independently developed several times, being described in 1990 and 1991 using the term “limiting dilution PCR” and in 1999 using the term “digital PCR”. It came into use in the decade following its first development but its use was cut short by the description of real-time PCR in 1996. However digital PCR has now had a renaissance due to the recent development of new instruments and chemistry which have made it a much simpler and...

  7. Neurocryptococcosis: diagnosis by PCR method.

    Science.gov (United States)

    Paschoal, Regina Célia; Hirata, Mário Hiroyuki; Hirata, Rosário Crespo; Melhem, Márcia de Souza Carvalho; Dias, Amanda Latercia Tranches; Paula, Claudete Rodrigues

    2004-01-01

    Cryptococcus neoformans detection was optimized using PCR technique with the objective of application in the clinical laboratory diagnosis. The amplification area was ITS and 5,6S which encodes the ribosomal RNA (rRNA). A total of 72 cerebrospinal fluid (CSF) samples were used, obtained from cases with and without AIDS. The patients had cryptococcal meningitis (n = 56) and meningitis caused by other agents (n = 16). The results demonstrated that PCR test had the highest sensitivity rates, superior to culture (85.7%) and to India ink test (76.8%). PCR was found to be sensitive in detecting 1 cell/mL and highly specific since it did not amplify other fungal DNA. The comparative analysis of the methods showed that PCR is more sensitive and specific and is applicable as an important laboratorial resource for neurocryptococcosis diagnosis.

  8. A neutralizing anti-gH/gL monoclonal antibody is protective in the guinea pig model of congenital CMV infection.

    Directory of Open Access Journals (Sweden)

    Marcy R Auerbach

    2014-04-01

    Full Text Available Human cytomegalovirus (HCMV is the most common cause of congenital virus infection. Congenital HCMV infection occurs in 0.2-1% of all births, and causes birth defects and developmental abnormalities, including sensorineural hearing loss and developmental delay. Several key studies have established the guinea pig as a tractable model for the study of congenital HCMV infection and have shown that polyclonal antibodies can be protective. In this study, we demonstrate that an anti-guinea pig CMV (GPCMV glycoprotein H/glycoprotein L neutralizing monoclonal antibody protects against fetal infection and loss in the guinea pig. Furthermore, we have delineated the kinetics of GPCMV congenital infection, from maternal infection (salivary glands, seroconversion, placenta to fetal infection (fetus and amniotic fluid. Our studies support the hypothesis that a neutralizing monoclonal antibody targeting an envelope GPCMV glycoprotein can protect the fetus from infection and may shed light on the therapeutic intervention of HCMV congenital infection in humans.

  9. Primer design versus PCR bias in methylation independent PCR amplifications.

    Science.gov (United States)

    Wojdacz, Tomasz K; Borgbo, Tanni; Hansen, Lise Lotte

    2009-05-16

    Many protocols in methylation studies utilize one primer set to generate a PCR product from bisulfite modified template regardless of its methylation status (methylation independent amplification MIP). However, proportional amplification of methylated and unmethylated alleles is hard to achieve due to PCR bias favoring amplification of unmethylated relatively GC poor sequence. Two primer design systems have been proposed to overcome PCR bias in methylation independent amplifications. The first advises against including any CpG dinucleoteides into the primer sequence (CpG-free primers) and the second, recently published by us, is based on inclusion of a limited number of CpG sites into the primer sequence. Here we used the Methylation Sensitive High Resolution Melting (MS-HRM) technology to investigate the ability of primers designed according to both of the above mentioned primer design systems to proportionally amplify methylated and unmethylated templates. Ten "CpG-free" primer pairs and twenty primers containing limited number of CpGs were tested. In reconstruction experiments the "CpG-free" primers showed primer specific sensitivity and allowed us to detect methylation levels in the range from 5 to 50%. Whereas while using primers containing limited number of CpG sites we were able to consistently detect 1-0.1% methylation levels and effectively control PCR amplification bias. In conclusion, the primers with limited number of CpG sites are able to effectively reverse PCR bias and therefore detect methylated templates with significantly higher sensitivity than CpG free primers.

  10. M-CSF核内稳定表达细胞系的构建和鉴定%Construction of nuclear localization vector pCMV/M-CSF and establishment of its stably expressing cell line

    Institute of Scientific and Technical Information of China (English)

    涂剑; 滕淑静; 张晓红; 赵雪琴; 唐圣松

    2009-01-01

    目的:构建真核细胞pCMV/nuc/M-CSF载体,建立M-CSF核内稳定表达细胞系,为进一步研究M-CSF的核内作用奠定基础.方法:采用PCR扩增人M-CSF活性片段,将M-CSF片段插入真核表达载体pCMV/myc/nuc,强制性把M-CSF引入细胞核内,通过PCR、测序鉴定筛选阳性重组体pCMV/M-CSF,脂质体介导转染HeLa细胞,经G418筛选后,用RT-PCR、间接免疫荧光鉴定其在HeLa细胞中的表达及定位分布.结果:琼脂糖电泳结果显示插入片段为1 400 bp左右,与预期M-CSF分子大小相当;DNA测序分析表明插入质粒的M-CSF无读码框移位,并与来源序列一致.RT-PCR和间接免疫荧光检测表明,转染pCMV/M-CSF的HeLa细胞能稳定表达M-CSF mRNA与M-CSF蛋白,且表达的M-CSF定位于HeLa细胞的细胞核.结论:成功构建了真核细胞pCMV/nuc/M-CSF载体,建立了M-CSF核内稳定表达细胞系.

  11. Diagnóstico de infección congénita por citomegalovirus mediante PCR en gotas de sangre seca en el papel de filtro de la tamización neonatal

    OpenAIRE

    2015-01-01

    Objetivo: Determinar la frecuencia de la infección congénita por citomegalovirus en el grupo de recién nacidos prematuros y/o con restricción de crecimiento intrauterino y establecer asociación entre el diagnóstico de la infección congénita por CMV por PCR en sangre seca y la caracterización de los datos del libro de partos y la base de datos del RUAF. Materiales y Métodos: Es un estudio retrospectivo anónimo no ligado, desarrollado en el hospital la Victoria sede Instituto ...

  12. Methylation-Specific PCR Unraveled

    Directory of Open Access Journals (Sweden)

    Sarah Derks

    2004-01-01

    Full Text Available Methylation‐specific PCR (MSP is a simple, quick and cost‐effective method to analyze the DNA methylation status of virtually any group of CpG sites within a CpG island. The technique comprises two parts: (1 sodium bisulfite conversion of unmethylated cytosine's to uracil under conditions whereby methylated cytosines remains unchanged and (2 detection of the bisulfite induced sequence differences by PCR using specific primer sets for both unmethylated and methylated DNA. This review discusses the critical parameters of MSP and presents an overview of the available MSP variants and the (clinical applications.

  13. PCR

    African Journals Online (AJOL)

    ELO

    2012-01-05

    mail: ... Plates transferred to the laboratory were incubated at 25°C for 48 h for growing the .... Use of a probiotic to control lactococcosis and streptococcosis in ... Streptococcus difficile: two new streptococcal species causing a.

  14. 非对称PCR-FP技术同步检测HSV-1/-2、CMV和EBV%Simultaneous detection of four major human herpesviruses by asymmetric PCR-fluorescence polarization assay

    Institute of Scientific and Technical Information of China (English)

    张菊; 吴中亮; 李丁; 王香玲; 梁平; 郭宴海; 李宏伟; 颜真

    2009-01-01

    目的 应用非对称PCR-荧光偏振(FP)技术建立一种同步检测全血中4种主要疱疹病毒(单纯疱疹病毒1/2型,巨细胞病毒、EB病毒)的新方法.方法 以人疱疹病毒属通用引物行非对称PCR,扩增从179例样本中抽提的DNA.PCR扩增产物与特异性单纯疱疹病毒1/2型(HSV-1/2)、巨细胞病毒(CMV)和EB病毒(EBV)寡核苷酸探针混合物温育杂交,荧光偏振检测技术检测杂交液荧光偏振值,据荧光偏振值判断病毒感染类型,并以DNA序列测定结果为参照.结果 与DNA序列测定的阳性检测符合率为100%,但DNA序列测定检测均未检出多重混合感染.非对称PCR-FP方法对HSV-1/-2检测的灵敏度达到1.0×10~3拷贝/ml,时EBV和cMV检测的灵敏度达到2.0×10~3拷贝/ml.结论 本法对于4种主要疱疹病毒感染的筛查及预防、预后判断具重要价值.%Objective To establish a novel method to detect four major human herpesvirus simultaneously by fluorescence polarization (FP) assay based on asymmetric PCR. Methods A consensus primer system in human herpesvirus was used in an asymmetric PCR. The probes of herpes simplex virus types 1 and 2 (HSV-1/-2) ,cytomegalovirus (CMV) ,and Epstein-Barr virus (EBV) labeled with different fluorophores were hybridized respectively with target amplicons. The infection was determined by the increased FP value. The DNA extracted from 179 samples was subjected to FP and sequence assay to evaluate the feasibility of this method. Results The mini-mum detection level of FP assay was 10 genome copies for HSV -1/ -2 , 20 genome copies for both EBV and CMV. FP assay could detect co-infections more effectively than sequence assay. Conclusions A practical method was developed for the simultaneous detection of the four major human herpesviruses by FP assay based on an asymmetric PCR.

  15. Anti-CMV-IgG Positivity of Donors Is Beneficial for alloHSCT Recipients with Respect to the Better Short-Term Immunological Recovery and High Level of CD4+CD25high Lymphocytes

    Directory of Open Access Journals (Sweden)

    Emilia Jaskula

    2015-03-01

    Full Text Available Hematopoietic stem cell transplantation from anti-cytomegalovirus immunoglobulin G (anti-CMV-IgG positive donors facilitated immunological recovery post-transplant, which may indicate that chronic CMV infection has an effect on the immune system. This can be seen in the recipients after reconstitution with donor lymphocytes. We evaluated the composition of lymphocytes at hematologic recovery in 99 patients with hematologic malignancies post hematopoietic stem cell transplantation (HSCT. Anti-CMV-IgG seropositivity of the donor was associated with higher proportions of CD4+ (227.963 ± 304.858 × 106 vs. 102.050 ± 17.247 × 106 cells/L, p = 0.009 and CD4+CD25high (3.456 ± 0.436 × 106 vs. 1.589 ± 0.218 × 106 cells/L, p = 0.003 lymphocytes in the blood at hematologic recovery. The latter parameter exerted a diverse influence on the risk of acute graft-versus-host disease (GvHD if low (1.483 ± 0.360 × 106 vs. 3.778 ± 0.484 × 106 cells/L, p < 0.001 and de novo chronic GvHD (cGvHD if high (3.778 ± 0.780 × 106 vs. 2.042 ± 0.261 × 106 cells/L, p = 0.041. Higher values of CD4+ lymphocytes in patients who received transplants from anti-CMV-IgG-positive donors translated into a reduced demand for IgG support (23/63 vs. 19/33, p = 0.048, and these patients also exhibited reduced susceptibility to cytomegalovirus (CMV, Epstein–Barr virus (EBV and/or human herpes 6 virus (HHV6 infection/reactivation (12/50 vs. 21/47, p = 0.032. Finally, high levels (³0.4% of CD4+CD25high lymphocytes were significantly associated with better post-transplant survival (56% vs. 38%, four-year survival, p = 0.040. Donors who experience CMV infection/reactivation provide the recipients with lymphocytes, which readily reinforce the recovery of the transplanted patients’ immune system.

  16. Alanine scanning of cucumber mosaic virus (CMV 2b protein identifies different positions for cell-to-cell movement and gene silencing suppressor activity.

    Directory of Open Access Journals (Sweden)

    Katalin Nemes

    Full Text Available The multifunctional 2b protein of CMV has a role in the long distance and local movement of the virus, in symptom formation, in evasion of defense mediated by salicylic acid as well as in suppression of RNA silencing. The role of conserved amino acid sequence domains were analyzed previously in the protein function, but comprehensive analysis of this protein was not carried out until recently. We have analyzed all over the 2b protein by alanine scanning mutagenesis changing three consecutive amino acids (aa to alanine. We have identified eight aa triplets as key determinants of the 2b protein function in virus infection. Four of them (KKQ/22-24/AAA, QNR/31-33/AAA, RER/34-36/AAA, SPS/40-42/AAA overlap with previously determined regions indispensable in gene silencing suppressor function. We have identified two additional triplets necessary for the suppressor function of the 2b protein (LPF/55-57/AAA, NVE/10-12/AAA, and two other positions were required for cell-to-cell movement of the virus (MEL/1-3/AAA, RHV/70-72/AAA, which are not essential for suppressor activity.

  17. Molecular characterization of Cucumber mosaic virus infecting Gladiolus, revealing its phylogeny distinct from the Indian isolate and alike the Fny strain of CMV.

    Science.gov (United States)

    Dubey, Vimal Kumar; Aminuddin; Singh, Vijai Pal

    2010-08-01

    The majority of Gladiolus plants growing in the botanical garden at NBRI, Lucknow, India and adjoining areas exhibited symptoms of mosaic, color breaking, stunting of spikes and reduction in flower size. The occurrence of Cucumber mosaic virus (CMV) was suspected in symptomatic Gladiolus plants. Cucumber mosaic virus, the type species of the genus Cucumovirus of the family Bromoviridae, is an important plant virus worldwide, which infects many plants and causes quantity and quality losses. For virus characterization, total RNA was isolated from leaves of infected plants and used in reverse transcriptase polymerase chain reaction with a primer set designed in the Cucumber mosaic virus coat protein region. Viral amplicons of the expected 657 bp size were obtained from infected plants. No viral amplicon was obtained from healthy control plants. Viral amplicons were cloned and sequenced (DQ295914). Molecular characterization was performed and phylogenetic relationship determined by the comparison of coat protein gene nucleotide and amino acid sequences with other Cucumber mosaic virus isolates reported from India and worldwide. The nucleotide and amino acid percentage comparison and phylogenetic tree results revealed that Cucumber mosaic virus infecting Gladiolus show resemblance with the Fny strain, which is not common in the Asian continent.

  18. PCR detection of multiple human herpesvirus DNA in saliva from HIV-infected individuals in Teresina, State of Piauí, Brazil Detecção por PCR do DNA de vários herpesvírus humanos na saliva de indivíduos infectados pelo HIV em Teresina, Estado do Piauí, Brasil

    Directory of Open Access Journals (Sweden)

    Kátia Silene Sousa Carvalho

    2010-12-01

    Full Text Available INTRODUCTION: Human herpesviruses are frequently associated with orofacial diseases in humans (HSV-1, EBV, CMV and HHV-8, some can also cause systemic disease (CMV and HHV-8. The transmission of these viruses occurs by contact with infected secretions, especially saliva. Human immunodeficiency virus infection is associated with an increased risk of HHVs and related diseases. METHODS: This work aimed to detect HSV-1, EBV, CMV and HHV-8 DNA in saliva of HIV-infected patients from Teresina, northeast Brazil, by PCR and compare these findings with age and sex matched HIV-seronegative individuals. RESULTS: No difference in prevalence was verified between HHV detection in the saliva of HIV-seropositive individuals and controls. The individual frequencies of these viruses in these two populations were different. HIV seropositivity correlated positively with the presence of CMV (OR: 18.2, p= 0.00032 and EBV (OR: 3.44, p= 0.0081. No association between CD4 counts and the prevalence of HHVs in the saliva was observed; however, a strong association was determined between seropositivity and the presence of multiple HHV DNAs in saliva (OR: 4.83, p = 0.0028. CONCLUSIONS: These findings suggest the asymptomatic salivary shedding of HHVs is a common event between HIV-seropositive and seronegative individuals from Teresina, Piauí, Brazil, and, especially for HIV-seropositive patients, saliva is a risk factor for the acquisition/transmission of multiple HHVs.INTRODUÇÃO: Alguns herpesvírus humanos são frequentemente associados a doenças orofaciais em humanos. A transmissão destes vírus ocorre através do contato com secreções contaminadas, especialmente a saliva. A infecção pelo vírus da imunodeficiência humana é considerada um fator de risco para a aquisição de HHVs e doenças correlatas. MÉTODOS: Este trabalho teve como objetivo detectar por PCR o DNA de HSV-1, EBV, CMV e HHV-8 na saliva de pacientes infectados com HIV em Teresina, nordeste do

  19. CpG-island fragments from the HNRPA2B1/CBX3 genomic locus reduce silencing and enhance transgene expression from the hCMV promoter/enhancer in mammalian cells

    Directory of Open Access Journals (Sweden)

    Irvine Alistair

    2005-06-01

    Full Text Available Abstract Background The hCMV promoter is very commonly used for high level expression of transgenes in mammalian cells, but its utility is hindered by transcriptional silencing. Large genomic fragments incorporating the CpG island region of the HNRPA2B1 locus are resistant to transcriptional silencing. Results In this report we describe studies on the use of a novel series of vectors combining the HNRPA2B1 CpG island with the hCMV promoter for expression of transgenes in CHO-K1 cells. We show that the CpG island gives at least twenty-fold increases in the levels of EGFP and EPO observed in pools of transfectants, and that transgene expression levels remain high in such pools for more than 100 generations. These novel vectors also allow facile isolation of clonal CHO-K1 cell lines showing stable, high-level transgene expression. Conclusion Vectors incorporating the hnRPA2B1 CpG island give major benefits in transgene expression from the hCMV promoter, including substantial improvements in the level and stability of expression. The utility of these vectors for the improved production of recombinant proteins in CHO cells has been demonstrated.

  20. Perfil clínico da Enterocolite por Citomegalovírus (CMV na síndrome da imunodeficiência adquirida (Aids Clinical profile of Cytomegalovirus Colitis in AIDS

    Directory of Open Access Journals (Sweden)

    D.B. de Lima

    2000-03-01

    Full Text Available Múltiplos agentes estão envolvidos na etiopatogenia da diarréia em Aids. O exame de fezes e a colonoscopia são elementos decisivos para o diagnóstico específico. A enterocolite por CMV pode cursar com febre, emagrecimento, diarréia intermitente e hematoquesia. Outros agentes causadores de diarréia podem ter o mesmo espectro de apresentação. OBJETIVO: Definir o perfil clínico da enterocolite por CMV em pacientes com Aids, comparando os parâmetros clínicos, endoscópicos e de tempo de sobrevida entre dois grupos com diarréia crônica, grupo A com CMV e grupo B sem CMV. MÉTODOS: Foram acompanhados 48 pacientes com Aids e diarréia de duração maior que 30 dias, sendo 27 do grupo A e 21 do grupo B. Os parâmetros analisados foram idade, situação de risco, duração da diarréia, hematoquesia, intervalo de tempo entre diagnóstico da infecção por HIV e início de diarréia, achados endoscópicos e sobrevida. Foram realizados exames parasitológicos, culturas e colonoscopia com biópsias. Foi utilizado o teste "t-student" para amostras não pareadas e o teste Qui-Quadrado com correção de Yates para variáveis não paramétricas. Foram construídas curvas de sobrevida pelo método descrito por Kaplan-Meier e aplicado o teste de Mantel - Haenszel. Foi assumido como nível de significância estatística o valor de P menor que 0.05. RESULTADOS: O padrão endoscópico da infecção por CMV correspondeu a ulcerações associadas a hemorragia de submucosa 14 (51.8% P P > 0.001. Os outros parâmetros estudados não mostraram significância estatística. CONCLUSÕES: A presença de enterocolite por CMV em Aids é marcador de mau prognóstico e menor sobrevida. Existe um padrão endoscópico sugestivo da infecção por CMV.OBJECTIVE: To determine the clinical profile of CMV colitis in AIDS patients, comparing clinical, endoscopic parameters and survival time between 2 groups of AIDS patients having chronic diarrhea. Group A being CMV

  1. Two-temperature PCR for Microfluidics

    KAUST Repository

    Kodzius, Rimantas

    2010-05-01

    Since its invention in 1983, polymerase chain reaction (PCR) has been the method of choice for DNA amplification. Successful PCR depends on the optimization of several parameters, which is a cumbersome task due to the many variables (conditions and compon

  2. Real-time PCR in Food Science: PCR Diagnostics.

    Science.gov (United States)

    Rodriguez-Lazaro, David; Cook, Nigel; Hernandez, Marta

    2013-01-01

    A principal consumer demand is a guarantee of the safety and quality of food. The presence of foodborne pathogens and their potential hazard, the use of genetically modified organisms (GMOs) in food production, and the correct labelling in foods suitable for vegetarians are among the subjects where society demands total transparency. The application of controls within the quality assessment programmes of the food industry is a way to satisfy these demands, and is necessary to ensure efficient analytical methodologies are possessed and correctly applied by the Food Sector. The use of real-time PCR has become a promising alternative approach in food diagnostics. It possesses a number of advantages over conventional culturing approaches, including rapidity, excellent analytical sensitivity and selectivity, and potential for quantification. However, the use of expensive equipment and reagents, the need for qualified personnel, and the lack of standardized protocols are impairing its practical implementation for food monitoring and control.

  3. Real-Time PCR (qPCR) Primer Design Using Free Online Software

    Science.gov (United States)

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

    Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

  4. Real-Time PCR (qPCR) Primer Design Using Free Online Software

    Science.gov (United States)

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

    Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

  5. Anchored PCR (A-PCR):A new method for chromosome walking

    Institute of Scientific and Technical Information of China (English)

    CHEN Bojun; SUN Chao; WANG Yong; HU Yuanlei; LIN Zhongping

    2004-01-01

    @@ PCR-based techniques are most popular methods for isolation of DNA sequences flanking a known region.Such techniques published to date mainly include three types: inverse PCR (IPCR)[1-3], ligation-mediated PCR (LM-PCR)[4-9] and randomly primed PCR (RP-PCR)[10-12].IPCR was the first method developed for this kind of purpose. However, it is now rarely used because of the difficulty in finding suitable restriction sites in the target region or poor circularization of the template molecule.LM-PCR and RP-PCR are more frequently used nowadays, yet they also have some limitations. For example,LM-PCR depends on restriction sites within a reasonable distance in the flanking regions, while the amplified products of RP-PCR are generally small (<1 kb). Moreover, both methods often result in excessive amplification of non-specific molecules, which greatly reduces their efficiencies in obtaining sequences of interest. To resolve these problems, some new strategies have emerged in the past few years, such as Vectorette-PCR[6], biotin-capture PCR[7], TAIL-PCR[l2] and T-linker PCR[9]. These improved methods are more efficient than their old versions;however, most of them are still limited by restriction digestion or ligation. Although the intervening steps are avoided in TAIL-PCR, the amplified fragments are often small because of the use of random primers.

  6. Study of the bacterial diversity of foods: PCR-DGGE versus LH-PCR.

    Science.gov (United States)

    Garofalo, Cristiana; Bancalari, Elena; Milanović, Vesna; Cardinali, Federica; Osimani, Andrea; Sardaro, Maria Luisa Savo; Bottari, Benedetta; Bernini, Valentina; Aquilanti, Lucia; Clementi, Francesca; Neviani, Erasmo; Gatti, Monica

    2017-02-02

    The present study compared two culture-independent methods, polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and length-heterogeneity polymerase chain reaction (LH-PCR), for their ability to reveal food bacterial microbiota. Total microbial DNA and RNA were extracted directly from fourteen fermented and unfermented foods, and domain A of the variable regions V1 and V2 of the 16S rRNA gene was analyzed through LH-PCR and PCR-DGGE. Finally, the outline of these analyses was compared with bacterial viable counts obtained after bacterial growth on suitable selective media. For the majority of the samples, RNA-based PCR-DGGE revealed species that the DNA-based PCR-DGGE was not able to highlight. When analyzing either DNA or RNA, LH-PCR identified several lactic acid bacteria (LAB) and coagulase negative cocci (CCN) species that were not identified by PCR-DGGE. This phenomenon was particularly evident in food samples with viable loadsPCR was able to detect a higher number of peaks in the analyzed food matrices relative to species identified by PCR-DGGE. In light of these findings, it may be suggested that LH-PCR shows greater sensitivity than PCR-DGGE. However, PCR-DGGE detected some other species (LAB included) that were not detected by LH-PCR. Therefore, certain LH-PCR peaks not attributed to known species within the LH-PCR database could be solved by comparing them with species identified by PCR-DGGE. Overall, this study also showed that LH-PCR is a promising method for use in the food microbiology field, indicating the necessity to expand the LH-PCR database, which is based, up to now, mainly on LAB isolates from dairy products. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Application of Reverse Transcription-PCR and Real-Time PCR in Nanotoxicity Research

    Science.gov (United States)

    Mo, Yiqun; Wan, Rong; Zhang, Qunwei

    2016-01-01

    Reverse transcription-polymerase chain reaction (RT-PCR) is a relatively simple and inexpensive technique to determine the expression level of target genes and is widely used in biomedical science research including nanotoxicology studies for semiquantitative analysis. Real-time PCR allows for the detection of PCR amplification in the exponential growth phase of the reaction and is much more quantitative than traditional RT-PCR. Although a number of kits and reagents for RT-PCR and real-time PCR are commercially available, the basic principles are the same. Here, we describe the procedures for total RNA isolation by using TRI Reagent, for reverse transcription (RT) by M-MLV reverse transcriptase, and for PCR by GoTaq® DNA Polymerase. And real-time PCR will be performed on an iQ5 multicolor real-time PCR detection system by using iQ™ SYBR Green Supermix. PMID:22975959

  8. The polymerase chain reaction (PCR): general methods.

    Science.gov (United States)

    Waters, Daniel L E; Shapter, Frances M

    2014-01-01

    The polymerase chain reaction (PCR) converts very low quantities of DNA into very high quantities and is the foundation of many specialized techniques of molecular biology. PCR utilizes components of the cellular machinery of mitotic cell division in vitro which respond predictably to user inputs. This chapter introduces the principles of PCR and discusses practical considerations from target sequence definition through to optimization and application.

  9. Restriction endonucleases digesting DNA in PCR buffer

    Institute of Scientific and Technical Information of China (English)

    LIU Xue-dong; ZHENG Dong; ZHOU Yan-na; MAO Wei-wei; MA Jian-zhang

    2005-01-01

    Six commonly used restriction endonucleases (Res) (Acc I, Ban II, EcoR I, Hind III, Sac I, Sca I) were tested for their ability to directly digest DNA completely in the Polymerase Chain Reaction (PCR) buffers. The results showed that: with the requirement for additional magnesium supplemented as activator, Res, except EcoR I appeared star activity, completely digested unmethylated lambda DNA after overnight incubation in PCR buffer and functioned as equally well as in recommended Restriction Enzyme Buffer provided with each enzyme; all Res tested completely digested PCR products in PCR buffer, it implied digestion of PCR products may often be performed directly in the PCR tube without the requirement for any precipitation or purification steps; and the concentration of MgCl2 from 2.5 mmol·L-1 to 10 mmol·L-1 did not significantly affect activity of Res in PCR buffer. This simplified method for RE digestion of PCR products could have applications in restriction fragment length polymorphism (RFLP) analysis and single-stranded conformational polymorphism (SSCP) analysis of large PCR products. However, usage of this procedure for cloning applications needs further data.

  10. Real-time PCR in microfluidic devices

    Science.gov (United States)

    Becker, Holger; Hlawatsch, Nadine; Klemm, Richard; Moche, Christian; Hansen-Hagge, Thomas; Gärtner, Claudia

    2014-03-01

    A central method in a standard biochemical laboratory is represented by the polymerase chain reaction (PCR), therefore many attempts have been performed so far to implement this technique in lab-on-a-chip (LOC) devices. PCR is an ideal candidate for miniaturization because of a reduction of assay time and decreased costs for expensive bio-chemicals. In case of the "classical" PCR, detection is done by identification of DNA fragments electrophoretically separated in agarose gels. This method is meanwhile frequently replaced by the so-called Real-Time-PCR because here the exponential increase of amplificates can be observed directly by measurement of DNA interacting fluorescent dyes. Two main methods for on-chip PCRs are available: traditional "batch" PCR in chambers on a chip using thermal cycling, requiring about 30 minutes for a typical PCR protocol and continuous-flow PCR, where the liquid is guided over stationary temperature zones. In the latter case, the PCR protocol can be as fast as 5 minutes. In the presented work, a proof of concept is demonstrated for a real-time-detection of PCR products in microfluidic systems.

  11. Pitfalls in PCR troubleshooting: Expect the unexpected?

    Science.gov (United States)

    Schrick, Livia; Nitsche, Andreas

    2016-01-01

    PCR is a well-understood and established laboratory technique often used in molecular diagnostics. Huge experience has been accumulated over the last years regarding the design of PCR assays and their set-up, including in-depth troubleshooting to obtain the optimal PCR assay for each purpose. Here we report a PCR troubleshooting that came up with a surprising result never observed before. With this report we hope to sensitize the reader to this peculiar problem and to save troubleshooting efforts in similar situations, especially in time-critical and ambitious diagnostic settings.

  12. Detection of Eperythrozoon wenyoni by PCR assay

    Institute of Scientific and Technical Information of China (English)

    Jian WANG; Yutao ZHU; Jianhua QIN; Fumei ZHANG; Yuelan ZHAO

    2009-01-01

    The objective of this research was to develop a detection method for Eperythrozoon wenyoni infection using polymerase chain reaction (PCR) assay technique. A pair of primers was designed and synthesized according to the conservative sequence 16S rRNA. The PCR assay was performed with the primers. A 985-bp fragment was amplified by using PCR. The amplified fragments with the expected size were identified by EcoR I restriction digestion. The crossing-reaction, specific-reaction and duplicate-reaction indicated that the PCR method is a specific, sensitive, fast and effective method for diagnosing E. Wenyoni infection at group level.

  13. Pitfalls in PCR troubleshooting: Expect the unexpected?

    Directory of Open Access Journals (Sweden)

    Livia Schrick

    2016-01-01

    Full Text Available PCR is a well-understood and established laboratory technique often used in molecular diagnostics. Huge experience has been accumulated over the last years regarding the design of PCR assays and their set-up, including in-depth troubleshooting to obtain the optimal PCR assay for each purpose. Here we report a PCR troubleshooting that came up with a surprising result never observed before. With this report we hope to sensitize the reader to this peculiar problem and to save troubleshooting efforts in similar situations, especially in time-critical and ambitious diagnostic settings.

  14. Absolute quantification by droplet digital PCR versus analog real-time PCR

    Science.gov (United States)

    Hindson, Christopher M; Chevillet, John R; Briggs, Hilary A; Gallichotte, Emily N; Ruf, Ingrid K; Hindson, Benjamin J; Vessella, Robert L; Tewari, Muneesh

    2014-01-01

    Nanoliter-sized droplet technology paired with digital PCR (ddPCR) holds promise for highly precise, absolute nucleic acid quantification. Our comparison of microRNA quantification by ddPCR and real-time PCR revealed greater precision (coefficients of variation decreased by 37–86%) and improved day-to-day reproducibility (by a factor of seven) of ddPCR but with comparable sensitivity. When we applied ddPCR to serum microRNA biomarker analysis, this translated to superior diagnostic performance for identifying individuals with cancer. PMID:23995387

  15. Evaluation of AMPLICOR Neisseria gonorrhoeae PCR using cppB nested PCR and 16S rRNA PCR.

    Science.gov (United States)

    Farrell, D J

    1999-02-01

    Certain strains of Neisseria subflava and Neisseria cinerea are known to produce false-positive results with the AMPLICOR Neisseria gonorrhoeae PCR (Roche Diagnostic Systems, Branchburg, N.J.). The analytical sensitivity and analytical specificity of three PCR tests were assessed with 3 geographically diverse N. gonorrhoeae strains and 30 non-N. gonorrhoeae Neisseria spp. The sensitivities of the in-house nested cppB gene and the 16S rRNA PCR methods were greater than that of the AMPLICOR N. gonorrhoeae PCR with purified DNA from all 3 N. gonorrhoeae strains. Six of 14 clinical strains of N. subflava (1 from a vaginal swab, 5 from respiratory sites) produced false-positive AMPLICOR N. gonorrhoeae PCR results and were negative by the two other PCR methods. When applied to 207 clinical specimens selected from a population with a high prevalence ( approximately 9%) of infection, the results for 15 of 96 (15.6%) AMPLICOR-positive specimens and 14 of 17 (82.3%) AMPLICOR-equivocal specimens were not confirmed by the more sensitive nested cppB PCR method. Only 2 of 94 (2.1%) of AMPLICOR N. gonorrhoeae PCR-negative specimens from the same population tested positive by the nested cppB method. These results suggest that for this population the AMPLICOR N. gonorrhoeae PCR test is suitable as a screening test only and all positive results should be confirmed by a PCR method that is more specific and at least as sensitive. This study also illustrates that caution should be used when introducing commercially available nucleic acid amplification-based diagnostic tests into the regimens of tests used for populations not previously tested with these products.

  16. Digital PCR dynamic range is approaching that of real-time quantitative PCR.

    Science.gov (United States)

    Jones, Gerwyn M; Busby, Eloise; Garson, Jeremy A; Grant, Paul R; Nastouli, Eleni; Devonshire, Alison S; Whale, Alexandra S

    2016-12-01

    Digital PCR (dPCR) has been reported to be more precise and sensitive than real-time quantitative PCR (qPCR) in a variety of models and applications. However, in the majority of commercially available dPCR platforms, the dynamic range is dependent on the number of partitions analysed and so is typically limited to four orders of magnitude; reduced compared with the typical seven orders achievable by qPCR. Using two different biological models (HIV DNA analysis and KRAS genotyping), we have demonstrated that the RainDrop Digital PCR System (RainDance Technologies) is capable of performing accurate and precise quantification over six orders of magnitude thereby approaching that achievable by qPCR.

  17. Digital PCR for detection of citrus pathogens

    Science.gov (United States)

    Citrus trees are often infected with multiple pathogens of economic importance, especially those with insect or mite vectors. Real-time/quantitative PCR (qPCR) has been used for high-throughput detection and relative quantification of pathogens; however, target reference or standards are required. I...

  18. Testing for Genetically Modified Foods Using PCR

    Science.gov (United States)

    Taylor, Ann; Sajan, Samin

    2005-01-01

    The polymerase chain reaction (PCR) is a Nobel Prize-winning technique that amplifies a specific segment of DNA and is commonly used to test for the presence of genetic modifications. Students use PCR to test corn meal and corn-muffin mixes for the presence of a promoter commonly used in genetically modified foods, the cauliflower mosaic virus 35S…

  19. Validation of RNAi by real time PCR

    DEFF Research Database (Denmark)

    Josefsen, Knud; Lee, Ying Chiu

    2011-01-01

    Real time PCR is the analytic tool of choice for quantification of gene expression, while RNAi is concerned with downregulation of gene expression. Together, they constitute a powerful approach in any loss of function studies of selective genes. We illustrate here the use of real time PCR to veri...

  20. Testing for Genetically Modified Foods Using PCR

    Science.gov (United States)

    Taylor, Ann; Sajan, Samin

    2005-01-01

    The polymerase chain reaction (PCR) is a Nobel Prize-winning technique that amplifies a specific segment of DNA and is commonly used to test for the presence of genetic modifications. Students use PCR to test corn meal and corn-muffin mixes for the presence of a promoter commonly used in genetically modified foods, the cauliflower mosaic virus 35S…

  1. Construction of aptamer-based surface plasma resonance biosensor microarray for the rapid detection of toxoplasma godii and cytomegalovirus IgG antibodies%快速检测TOX、CMV IgG抗体的适配子型SPR传感器微阵列的初步构建

    Institute of Scientific and Technical Information of China (English)

    刘星; 秦莲花; 罗阳; 苗杰; 王丰; 黄庆; 黄君富; 胡忠义; 府伟灵

    2012-01-01

    Objective To construct the aptamer-based surface plasma resonance ( SPR) microarray for rapid assay of toxoplasma gondii (TOX) and cytomegalovirus (CMV) IgG antibodies. Methods Aptamers of the TOX gondii and CMV IgG antibodies were screened with the SELEX technique and integrated into the real-time online analyzing system on the SPR biosensor. Hybridization of TOX and CMV IgG antibodies in solution was detected according to the probe molecules fixed on the surface of biosensor. The stability and linear detection range of this assay were further investigated. Results The novel rapid method could assay the TOX and CMV IgG antibodies with a good stability and a linear assay range of 20 to 300 μmol/L. Conclusion The stability of the rapid assay we established is good. Combined SPR biosensor and aptamer techniques have a broad prospect in clinical assay of TOX and CMV IgG antibody level.%目的 初步构建弓形虫(toxoplas ma gondii,TOX)、巨细胞病毒(cytomegalovirus,CMV)IgG抗体的适配子型SPR传感器微阵列的快速检测方法.方法 采用SELEX技术筛选TOX、CMV IgG的适配子,并将其整合于SPR生物传感器实时在线分析系统,通过在传感器表面固定探针分子,对溶液中的TOX、CMV IgG进行杂交检测,并进一步研究该检测方法的稳定性与线性检范围.结果 该新型快速检测方法能够实现对TOX、CMV IgG的实时检测,检测系统稳定性良好,八通道间检测时互不影响,线性检测范围为20~300 μmol/L.结论 该实验建立的快速检测方法,具有稳定性好等优点,SPR传感器技术结合适配子技术在临床诊断工作中有着广阔的应用前景.

  2. Real-time PCR and PCR-tandem Mass Spectrometry for Biodetection

    Science.gov (United States)

    2005-10-01

    Real - time PCR and PCR- tandem mass spectrometry for biodetection Alvin Fox, University of South Carolina, School of Medicine Report Documentation...TYPE N/A 3. DATES COVERED - 4. TITLE AND SUBTITLE Real - time PCR and PCRtandem mass spectrometry for biodetection 5a. CONTRACT NUMBER 5b...interspace region Bacillus subtilis W23 standard Blank Barn dust House dust Cycle Real - time PCR (16s rRNA) - environmental samples Real - time

  3. Addressing PCR Biases in Environmental Microbiology Studies

    Science.gov (United States)

    Sipos, Rita; Székely, Anna; Révész, Sára; Márialigeti, Károly

    Each step of a molecular environmental microbiology study is prone to errors, though the qualitative and quantitative biases of PCR amplification could result in the most serious biases. One has to be aware of this fact, and well-characterized PCR biases have to be avoided by using target-optimized PCR protocols. The most important tasks are primer and thermal profile optimization. We have shown that primer mismatches, even in the case of universal primers, can cause almost complete missing of common taxa from clone libraries, for example. Similarly high annealing temperatures can drastically distort community composition of the sample in the PCR product. Strategies of primer selection and PCR thermal profile design are discussed in detail.

  4. COLD-PCR: Applications and Advantages.

    Science.gov (United States)

    Zuo, Zhuang; Jabbar, Kausar J

    2016-01-01

    Co-amplification at lower denaturation temperature-based polymerase chain reaction (COLD-PCR) is a single-step amplification method that results in the enhancement of both known and unknown minority alleles during PCR, irrespective of mutation type and position. This method is based on exploitation of the critical temperature, Tc, at which mutation-containing DNA is preferentially melted over wild type. COLD-PCR can be a good strategy for mutation detection in specimens with high nonneoplastic cell content, small specimens in which neoplastic cells are difficult to micro-dissect and therefore enrich, and whenever a mutation is suspected to be present but is undetectable using conventional PCR and sequencing methods. We describe in this chapter our COLD-PCR-based pyrosequencing method for KRAS mutation detection in various clinical samples using DNA extracted from either fresh or fixed paraffin-embedded tissue specimens.

  5. Mathematical analysis of the real time array PCR (RTA PCR) process

    NARCIS (Netherlands)

    Dijksman, Johan Frederik; Pierik, A.

    2012-01-01

    Real time array PCR (RTA PCR) is a recently developed biochemical technique that measures amplification curves (like with quantitative real time Polymerase Chain Reaction (qRT PCR)) of a multitude of different templates in a sample. It combines two different methods in order to profit from the

  6. Pre-PCR processing: strategies to generate PCR-compatible samples.

    Science.gov (United States)

    Rådström, Peter; Knutsson, Rickard; Wolffs, Petra; Lövenklev, Maria; Löfström, Charlotta

    2004-02-01

    Polymerase chain reaction (PCR) is recognized as a rapid, sensitive, and specific molecular diagnostic tool for the analysis of nucleic acids. However, the sensitivity and kinetics of diagnostic PCR may be dramatically reduced when applied directly to biological samples, such as blood and feces, owing to PCR-inhibitory components. As a result, pre-PCR processing procedures have been developed to remove or reduce the effects of PCR inhibitors. Pre-PCR processing comprises all steps prior to the detection of PCR products, that is, sampling, sample preparation, and deoxyribonucleic acid (DNA) amplification. The aim of pre-PCR processing is to convert a complex biological sample with its target nucleic acids/cells into PCR-amplifiable samples by combining sample preparation and amplification conditions. Several different pre-PCR processing strategies are used: (1) optimization of the DNA amplification conditions by the use of alternative DNA polymerases and/or amplification facilitators, (2) optimization of the sample preparation method, (3) optimization of the sampling method, and (4) combinations of the different strategies. This review describes different pre-PCR processing strategies to circumvent PCR inhibition to allow accurate and precise DNA amplification.

  7. Mathematical analysis of the real time array PCR (RTA PCR) process

    NARCIS (Netherlands)

    Dijksman, J.F.; Pierik, A.

    2012-01-01

    Real time array PCR (RTA PCR) is a recently developed biochemical technique that measures amplification curves (like with quantitative real time Polymerase Chain Reaction (qRT PCR)) of a multitude of different templates in a sample. It combines two different methods in order to profit from the adva

  8. 成人巨细胞病毒性肝炎临床表现及病理特点分析%Analysis on Clinical Manifestations and Pathological Features of CMV Hepatitis in Adult Patients

    Institute of Scientific and Technical Information of China (English)

    臧红; 朱冰; 游绍莉; 荣义辉; 张爱民; 刘婉姝; 辛绍杰

    2012-01-01

    目的 探讨成人巨细胞病毒性肝炎患者的临床表现及病理特点,以提高临床诊治水平.方法 对116例成人巨细胞病毒性肝炎患者临床表现、实验室检查及其中30例肝组织病理资料进行回顾性分析.结果 成人巨细胞病毒性肝炎患者平均年龄(38.2±13.3)岁,发病前为健康者110例,治愈好转率89.66%.主要为急性起病(86.21%),症状以发热(42.24%)、乏力(62.93%)、纳差(54.31%)为主,体征主要表现为巩膜黄染(40.52%)、脾大(18.10%)、肝大(14.66%)、淋巴结肿大(13.79%).肝功能指标以ALT、AST、GGT及LDH异常为主,黄疸型肝炎TBIL及TBA明显升高.病理特点主要表现为肝细胞变性、点灶状坏死,色素颗粒沉着,可见吞噬色素的Kupffer细胞,汇管区轻微扩大,偶见巨细胞包涵体.结论 成人巨细胞病毒性肝炎多为急性,预后较好,少数慢性化,严重可发生肝衰竭.其临床表现多样,结合病理特点可提高临床诊治水平.%Objective To explore the clinical manifestations and pathological characteristics of cytomegalovirus (CMV) hepatitis in adult patients so as to improve the level of diagnosis and treatment. Methods The characteristics of 116 adult patients with CMV hepatitis were retrospectively analyzed, including clinical features, serological examinations and 30 patients' liver - tissue pathological data. Results The average age of adult patients with CMV hepatitis was (38.2 ?3.3) years. 110 patients were previously healthy. The total improvement rate was 89.66%. 86.21 % of the cases were acute hepatitis. The most frequent symptoms were fever (42.24%), fatigue (62.93%) and anorexia (54.31%). The main signs were icteric sclera (40.52%), splenomegaly (18. 10%), hepatomegaly (14.66%), and lymphadenectasis (13.79%). The liver function tests showed that ALT, AST, GGT and LDH were abnormal and TBIL and TBA were significantly elevated in the patients with chol-estatic hepatitis. The pathological

  9. 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

  10. Material Biocompatibility for PCR Microfluidic Chips

    KAUST Repository

    Kodzius, Rimantas

    2010-04-23

    As part of the current miniaturization trend, biological reactions and processes are being adapted to microfluidics devices. PCR is the primary method employed in DNA amplification, its miniaturization is central to efforts to develop portable devices for diagnostics and testing purposes. A problem is the PCR-inhibitory effect due to interaction between PCR reagents and the surrounding environment, which effect is increased in high-surface-are-to-volume ration microfluidics. In this study, we evaluated the biocompatibility of various common materials employed in the fabrication of microfluidic chips, including silicon, several kinds of silicon oxide, glasses, plastics, wax, and adhesives. Two-temperature PCR was performed with these materials to determine their PCR-inhibitory effect. In most of the cases, addition of bovine serum albumin effectively improved the reaction yield. We also studied the individual PCR components from the standpoint of adsorption. Most of the materials did not inhibit the DNA, whereas they did show noticeable interaction with the DNA polymerase. Our test, instead of using microfluidic devices, can be easily conducted in common PCR tubes using a standard bench thermocycler. Our data supports an overview of the means by which the materials most bio-friendly to microfluidics can be selected.

  11. Quantitative DNA Analysis Using Droplet Digital PCR.

    Science.gov (United States)

    Vossen, Rolf H A M; White, Stefan J

    2017-01-01

    Droplet digital PCR (ddPCR) is based on the isolated amplification of thousands of individual DNA molecules simultaneously, with each molecule compartmentalized in a droplet. The presence of amplified product in each droplet is indicated by a fluorescent signal, and the proportion of positive droplets allows the precise quantification of a given sequence. In this chapter we briefly outline the basis of ddPCR, and describe two different applications using the Bio-Rad QX200 system: genotyping copy number variation and quantification of Illumina sequencing libraries.

  12. Real time PCR. Application in dengue studies

    Directory of Open Access Journals (Sweden)

    Jeanette Prada-Arismendy

    2011-06-01

    Full Text Available PCR (polymerase chain reaction is a routinely used tool in every diagnostic and research laboratory. This technique has been used in detection of mutations and pathogens, forensic investigation, and even is the base tool for human genome sequencing. A modification of PCR technique, real time PCR, allows the quantification of nucleic acids with higher sensibility, specificity and reproducibility. This article is intended to clarify the foundations of real-time PCR, using an application model for virology. In the actual work, it was quantified the viral load of dengue virus serotype 2 produced from infected murine macrophages; the obtained results in this work established that murine strain BALB/c presents a greater susceptibility to dengue virus infection, which establishes BALB/c murine strain as a best model of study for investigation of dengue virus infection physiopathology.

  13. Fundamentals of multiplexing with digital PCR.

    Science.gov (United States)

    Whale, Alexandra S; Huggett, Jim F; Tzonev, Svilen

    2016-12-01

    Over the past decade numerous publications have demonstrated how digital PCR (dPCR) enables precise and sensitive quantification of nucleic acids in a wide range of applications in both healthcare and environmental analysis. This has occurred in parallel with the advances in partitioning fluidics that enable a reaction to be subdivided into an increasing number of partitions. As the majority of dPCR systems are based on detection in two discrete optical channels, most research to date has focused on quantification of one or two targets within a single reaction. Here we describe 'higher order multiplexing' that is the unique ability of dPCR to precisely measure more than two targets in the same reaction. Using examples, we describe the different types of duplex and multiplex reactions that can be achieved. We also describe essential experimental considerations to ensure accurate quantification of multiple targets.

  14. Early kinetics of plasma cytomegalovirus DNA load in allogeneic stem cell transplant recipients in the era of highly sensitive real-time PCR assays: does it have any clinical value?

    Science.gov (United States)

    Giménez, Estela; Muñoz-Cobo, Beatriz; Solano, Carlos; Amat, Paula; Navarro, David

    2014-02-01

    We report that in a population of allogeneic stem cell transplant recipients, determination of the viral doubling time (dt) of the cytomegalovirus (CMV) DNA plasma load predicted the eventual need for inception of preemptive antiviral therapy, whereas the level of the initial plasma CMV DNA load did not. The data thus indicated that determination of the dt of CMV DNA may be useful in the therapeutic management of CMV infection in this clinical setting.

  15. The potential advantages of digital PCR for clinical virology diagnostics.

    Science.gov (United States)

    Hall Sedlak, Ruth; Jerome, Keith R

    2014-05-01

    Digital PCR (dPCR), a new nucleic acid amplification technology, offers several potential advantages over real-time or quantitative PCR (qPCR), the current workhorse of clinical molecular virology diagnostics. Several studies have demonstrated dPCR assays for human cytomegalovirus or HIV, which give more precise and reproducible results than qPCR assays without sacrificing sensitivity. Here we review the literature comparing dPCR and qPCR performance in viral molecular diagnostic assays and offer perspective on the future of dPCR in clinical virology diagnostics.

  16. Transgene detection by digital droplet PCR.

    Directory of Open Access Journals (Sweden)

    Dirk A Moser

    Full Text Available Somatic gene therapy is a promising tool for the treatment of severe diseases. Because of its abuse potential for performance enhancement in sports, the World Anti-Doping Agency (WADA included the term 'gene doping' in the official list of banned substances and methods in 2004. Several nested PCR or qPCR-based strategies have been proposed that aim at detecting long-term presence of transgene in blood, but these strategies are hampered by technical limitations. We developed a digital droplet PCR (ddPCR protocol for Insulin-Like Growth Factor 1 (IGF1 detection and demonstrated its applicability monitoring 6 mice injected into skeletal muscle with AAV9-IGF1 elements and 2 controls over a 33-day period. A duplex ddPCR protocol for simultaneous detection of Insulin-Like Growth Factor 1 (IGF1 and Erythropoietin (EPO transgenic elements was created. A new DNA extraction procedure with target-orientated usage of restriction enzymes including on-column DNA-digestion was established. In vivo data revealed that IGF1 transgenic elements could be reliably detected for a 33-day period in DNA extracted from whole blood. In vitro data indicated feasibility of IGF1 and EPO detection by duplex ddPCR with high reliability and sensitivity. On-column DNA-digestion allowed for significantly improved target detection in downstream PCR-based approaches. As ddPCR provides absolute quantification, it ensures excellent day-to-day reproducibility. Therefore, we expect this technique to be used in diagnosing and monitoring of viral and bacterial infection, in detecting mutated DNA sequences as well as profiling for the presence of foreign genetic material in elite athletes in the future.

  17. Universally Primed PCR (UP-PCR) and its applications for taxonomy in Trichoderma

    Institute of Scientific and Technical Information of China (English)

    Mette Lübeck

    2004-01-01

    @@ Universally Primed PCR (UP-PCR) is a PCR fingerprinting method that has demonstrated its applicability in different aspects of mycology. These applications constitute analysis of genome structures, identification of species, analysis of population and species diversity, revealing of genetic relatedness at infra-and inter-species level, and identification of UP-PCR markers at different taxonomic levels (strain, group and/or species) . A further development of the UP-PCR technique is an UP-PCR product cross hybridisation assay that facilitates investigation of sequence similarity (homology) of UP-PCR products and grouping of strains into UP-PCR hybridisation groups. This separates the strains into entities with high genetic similarity (DNA homology) . UP-PCR has been used as an aid in taxonomy and species delineation, and to monitor biocontrol strains following their release into the environment by fingerprint characterisation of pure cultures and through direct detection in soil by amplification of UP-PCR-derived SCAR markers. The technique has been applied to Trichoderma strains in particularly with the aims of strain recognition and classification.

  18. A naked-eye colorimetric "PCR developer"

    Science.gov (United States)

    Valentini, Paola; Pompa, Pier Paolo

    2016-04-01

    Despite several advances in molecular biology and diagnostics, Polymerase Chain Reaction (PCR) is currently the gold standard for nucleic acids amplification and detection, due to its versatility, low-cost and universality, with estimated market of several billion dollars/year. Nevertheless, PCR still relies on the laborious, time-consuming, and multi-step gel electrophoresis-based detection, which includes gel casting, electrophoretic run, gel staining, and gel visualization. In this work, we propose a "PCR developer", namely a universal one-step, one-tube method, based on controlled aggregation of gold nanoparticles (AuNPs), to detect PCR products by naked eye in few minutes, with no need for any instrumentation. We demonstrated the specificity and sensitivity of the PCR developer on different model targets, suitable for a qualitative detection in real-world diagnostics (i.e., gene rearrangements, genetically modified organisms, and pathogens). The PCR developer proved to be highly specific and ultra-sensitive, discriminating down to few copies of HIV viral DNA, diluted in an excess of interfering human genomic DNA, which is a clinically relevant viral load. Hence, it could be a valuable tool for both academic research and clinical applications.

  19. Diagnostic Performance of a Multiplex PCR assay for meningitis in an HIV-infected population in Uganda

    Science.gov (United States)

    Rhein, Joshua; Bahr, Nathan C; Hemmert, Andrew C; Cloud, Joann L; Bellamkonda, Satya; Oswald, Cody; Lo, Eric; Nabeta, Henry; Kiggundu, Reuben; Akampurira, Andrew; Musubire, Abdu; Williams, Darlisha; Meya, David B; Boulware, David R

    2015-01-01

    Meningitis remains a worldwide problem, and rapid diagnosis is essential to optimize survival. We evaluated the utility of a multiplex PCR test in differentiating possible etiologies of meningitis. Cerebrospinal fluid (CSF) from 69 HIV-infected Ugandan adults with meningitis was collected at diagnosis (n=51) and among persons with cryptococcal meningitis during therapeutic lumbar punctures (n=68). Cryopreserved CSF specimens were analyzed with BioFire FilmArray® Meningitis/Encephalitis panel, which targets 17 pathogens. The panel detected Cryptococcus in the CSF of patients diagnosed with a first-episode of cryptococcal meningitis by fungal culture with 100% sensitivity and specificity, and differentiated between fungal relapse and paradoxical immune reconstitution inflammatory syndrome in recurrent episodes. A negative FilmArray result was predictive of CSF sterility on follow-up lumbar punctures for cryptococcal meningitis. EBV was frequently detected in this immunosuppressed population (n=45). Other pathogens detected included: CMV (n=2), VZV (n=2), HHV-6 (n=1), and Streptococcus pneumoniae (n=1). The FilmArray Meningitis/Encephalitis panel offers a promising platform for rapid meningitis diagnosis. PMID:26711635

  20. Determination of PCR efficiency in chelex-100 purified clinical samples and comparison of real-time quantitative PCR and conventional PCR for detection of Chlamydia pneumoniae

    Directory of Open Access Journals (Sweden)

    Jensen Jørgen

    2002-07-01

    Full Text Available Abstract Background Chlamydia pneumoniae infection has been detected by serological methods, but PCR is gaining more interest. A number of different PCR assays have been developed and some are used in combination with serology for diagnosis. Real-time PCR could be an attractive new PCR method; therefore it must be evaluated and compared to conventional PCR methods. Results We compared the performance of a newly developed real-time PCR with a conventional PCR method for detection of C. pneumoniae. The PCR methods were tested on reference samples containing C. pneumoniae DNA and on 136 nasopharyngeal samples from patients with a chronic cough. We found the same detection limit for the two methods and that clinical performance was equal for the real-time PCR and for the conventional PCR method, although only three samples tested positive. To investigate whether the low prevalence of C. pneumoniae among patients with a chronic cough was caused by suboptimal PCR efficiency in the samples, PCR efficiency was determined based on the real-time PCR. Seventeen of twenty randomly selected clinical samples had a similar PCR efficiency to samples containing pure genomic C. pneumoniae DNA. Conclusions These results indicate that the performance of real-time PCR is comparable to that of conventional PCR, but that needs to be confirmed further. Real-time PCR can be used to investigate the PCR efficiency which gives a rough estimate of how well the real-time PCR assay works in a specific sample type. Suboptimal PCR efficiency of PCR is not a likely explanation for the low positivity rate of C. pneumoniae in patients with a chronic cough.

  1. Specific PCR and real-time PCR assays for detection and quantitation of 'Candidatus Phytoplasma phoenicium'.

    Science.gov (United States)

    Jawhari, Maan; Abrahamian, Peter; Sater, Ali Abdel; Sobh, Hana; Tawidian, Patil; Abou-Jawdah, Yusuf

    2015-02-01

    Almond witches' broom (AlmWB) is a fast-spreading lethal disease of almond, peach and nectarine associated with 'Candidatus Phytoplasma phoenicium'. The development of PCR and quantitative real-time PCR (qPCR) assays for the sensitive and specific detection of the phytoplasma is of prime importance for early detection of 'Ca. P. phoenicium' and for epidemiological studies. The developed qPCR assay herein uses a TaqMan(®) probe labeled with Black Hole Quencher Plus. The specificity of the PCR and that of the qPCR detection protocols were tested on 17 phytoplasma isolates belonging to 11 phytoplasma 16S rRNA groups, on samples of almond, peach, nectarine, native plants and insects infected or uninfected with the phytoplasma. The developed assays showed high specificity against 'Ca. P. phoenicium' and no cross-reactivity against any other phytoplasma, plant or insect tested. The sensitivity of the developed PCR and qPCR assays was similar to the conventional nested PCR protocol using universal primers. The qPCR assay was further validated by quantitating AlmWB phytoplasma in different hosts, plant parts and potential insect vectors. The highest titers of 'Ca. P. phoenicium' were detected in the phloem tissues of stems and roots of almond and nectarine trees, where they averaged from 10(5) to 10(6) genomic units per nanogram of host DNA (GU/ng of DNA). The newly developed PCR and qPCR protocols are reliable, specific and sensitive methods that are easily applicable to high-throughput diagnosis of AlmWB in plants and insects and can be used for surveys of potential vectors and alternative hosts.

  2. Signal and noise in bridging PCR

    Directory of Open Access Journals (Sweden)

    Thaler David S

    2002-07-01

    Full Text Available Abstract Background In a variant of the standard PCR reaction termed bridging, or jumping, PCR the primer-bound sequences are originally on separate template molecules. Bridging can occur if, and only if, the templates contain a region of sequence similarity. A 3' end of synthesis in one round of synthesis that terminates in this region of similarity can prime on the other. In principle, Bridging PCR (BPCR can detect a subpopulation of one template that terminates synthesis in the region of sequence shared by the other template. This study considers the sensitivity and noise of BPCR as a quantitative assay for backbone interruptions. Bridging synthesis is also important to some methods for computing with DNA. Results In this study, BPCR was tested over a 328 base pair segment of the E. coli lac operon and a signal to noise ratio (S/N of approximately 10 was obtained under normal PCR conditions with Taq polymerase. With special precautions in the case of Taq or by using the Stoffel fragment the S/N was improved to 100, i.e. 1 part of cut input DNA yielded the same output as 100 parts of intact input DNA. Conclusions In the E. coli lac operator region studied here, depending on details of protocol, between 3 and 30% per kilobase of final PCR product resulted from bridging. Other systems are expected to differ in the proportion of product that is bridged consequent to PCR protocol and the sequence analyzed. In many cases physical bridging during PCR will have no informational consequence because the bridged templates are of identical sequence, but in a number of special cases bridging creates, or, destroys, information.

  3. A method for amplification of unknown flanking sequences based on touchdown PCR and suppression-PCR.

    Science.gov (United States)

    Gao, Song; He, Dan; Li, Guangquan; Zhang, Yanhua; Lv, Huiying; Wang, Li

    2016-09-15

    Thermal asymmetric staggered PCR is the most widely used technique to obtain the flanking sequences. However, it has some limitations, including a low rate of positivity, and complex operation. In this study, a improved method of it was made based on suppression-PCR and touchdown PCR. The PCR fragment obtained by the amplification was used directly for sequencing after gel purification. Using this improved method, the positive rate of amplified flanking sequences of the ATMT mutants reached 99%. In addition, the time from DNA extraction to flanking sequence analysis was shortened to 2 days with about 6 dollars each sample.

  4. Study on Control Efficiency of Junkeduke on Tobacco Mosaic Virus(TMV) and Cucumber Mosaic Virus(CMV)%菌克毒克防治烟草花叶病效果研究

    Institute of Scientific and Technical Information of China (English)

    何杰忠

    2014-01-01

    研究菌克毒克对烟草花叶病的防治效果,结果表明:菌克毒克(8%宁南霉素水剂内销品)对烟草花叶病有较好的防治效果,平均防治效果达66.7%,可促进烟株的生长,经济效益显著。%Effect of Junkeduke on tobacco mosaic virus(TMV)and cucumber mosaic virus(CMV)were studied . The results showed that Junkeduke(8%Ningnanmycin domestic product)had better control efficincy,the average control efficiency reached 66.7%.It could promote the growth of tobacco plants,economic benefits was significant.

  5. Evaluation of Altona Diagnostics RealStar Zika Virus Reverse Transcription-PCR Test Kit for Zika Virus PCR Testing.

    Science.gov (United States)

    L'Huillier, Arnaud G; Lombos, Ernesto; Tang, Elaine; Perusini, Stephen; Eshaghi, Alireza; Nagra, Sandeep; Frantz, Christine; Olsha, Romy; Kristjanson, Erik; Dimitrova, Kristina; Safronetz, David; Drebot, Mike; Gubbay, Jonathan B

    2017-05-01

    With the emerging Zika virus (ZIKV) epidemic, accessible real-time reverse transcription-PCR (rRT-PCR) assays are needed to streamline testing. The commercial Altona Diagnostics RealStar ZIKV rRT-PCR test kit (Altona PCR) has been approved for emergency use authorization by the U.S. FDA. Our aim was to verify the Altona PCR by comparing it to the CDC-designed dual-target ZIKV rRT-PCR reference assay (reference PCR) and describe the demographics of patients tested for ZIKV by rRT-PCR in Ontario, Canada. A large set of clinical specimens was tested for ZIKV by the Altona PCR and the reference PCR. Positive or equivocal specimens underwent PCR and Sanger sequencing targeting the ZIKV NS5 gene. A total of 671 serum specimens were tested by the reference PCR: 58 (8.6%) were positive, 193 (28.8%) were equivocal, and 420 (62.6%) were negative. Ninety percent of the reference PCR-positive patients were tested in the first 5 days after symptom onset. The Altona PCR was performed on 284/671 specimens tested by the reference PCR. The Altona PCR was positive for 53/58 (91%) reference PCR-positive specimens and 16/193 (8%) reference PCR-equivocal specimens; the ZIKV NS5 PCR was positive for all 68 Altona PCR-positive specimens and negative for all 181 Altona PCR-negative specimens that underwent the NS5 PCR. The Altona PCR has very good sensitivity (91%) and specificity (97%) compared to the reference PCR. The Altona PCR can be used for ZIKV diagnostic testing and has less extensive verification requirements than a laboratory-developed test. Copyright © 2017 American Society for Microbiology.

  6. PCR+ In Diesel Fuels and Emissions Research

    Energy Technology Data Exchange (ETDEWEB)

    McAdams, H.T.

    2002-04-15

    In past work for the U.S. Department of Energy (DOE) and Oak Ridge National Laboratory (ORNL), PCR+ was developed as an alternative methodology for building statistical models. PCR+ is an extension of Principal Components Regression (PCR), in which the eigenvectors resulting from Principal Components Analysis (PCA) are used as predictor variables in regression analysis. The work was motivated by the observation that most heavy-duty diesel (HDD) engine research was conducted with test fuels that had been ''concocted'' in the laboratory to vary selected fuel properties in isolation from each other. This approach departs markedly from the real world, where the reformulation of diesel fuels for almost any purpose leads to changes in a number of interrelated properties. In this work, we present new information regarding the problems encountered in the conventional approach to model-building and how the PCR+ method can be used to improve research on the relationship between fuel characteristics and engine emissions. We also discuss how PCR+ can be applied to a variety of other research problems related to diesel fuels.

  7. PCR+ In Diesel Fuels and Emissions Research

    Energy Technology Data Exchange (ETDEWEB)

    McAdams, H.T.

    2002-04-15

    In past work for the U.S. Department of Energy (DOE) and Oak Ridge National Laboratory (ORNL), PCR+ was developed as an alternative methodology for building statistical models. PCR+ is an extension of Principal Components Regression (PCR), in which the eigenvectors resulting from Principal Components Analysis (PCA) are used as predictor variables in regression analysis. The work was motivated by the observation that most heavy-duty diesel (HDD) engine research was conducted with test fuels that had been ''concocted'' in the laboratory to vary selected fuel properties in isolation from each other. This approach departs markedly from the real world, where the reformulation of diesel fuels for almost any purpose leads to changes in a number of interrelated properties. In this work, we present new information regarding the problems encountered in the conventional approach to model-building and how the PCR+ method can be used to improve research on the relationship between fuel characteristics and engine emissions. We also discuss how PCR+ can be applied to a variety of other research problems related to diesel fuels.

  8. Direct Chromatin PCR (DC-PCR): Hypotonic Conditions Allow Differentiation of Chromatin States during Thermal Cycling

    Science.gov (United States)

    Vatolin, Sergei; Khan, Shahper N.; Reu, Frederic J.

    2012-01-01

    Current methods to study chromatin configuration are not well suited for high throughput drug screening since they require large cell numbers and multiple experimental steps that include centrifugation for isolation of nuclei or DNA. Here we show that site specific chromatin analysis can be achieved in one step by simply performing direct chromatin PCR (DC-PCR) on cells. The basic underlying observation was that standard hypotonic PCR buffers prevent global cellular chromatin solubilization during thermal cycling while more loosely organized chromatin can be amplified. Despite repeated heating to >90°C, 41 of 61 tested 5′ sequences of silenced genes (CDKN2A, PU.1, IRF4, FOSB, CD34) were not amplifiable while 47 could be amplified from expressing cells. Two gene regions (IRF4, FOSB) even required pre-heating of cells in isotonic media to allow this differentiation; otherwise none of 19 assayed sequences yielded PCR products. Cells with baseline expression or epigenetic reactivation gave similar DC-PCR results. Silencing during differentiation of CD34 positive cord blood cells closed respective chromatin while treatment of myeloma cells with an IRF4 transcriptional inhibitor opened a site to DC-PCR that was occupied by RNA polymerase II and NFκB as determined by ChIP. Translation into real-time PCR can not be achieved with commercial real-time PCR buffers which potently open chromatin, but even with simple ethidium bromide addition to standard PCR mastermix we were able to identify hits in small molecules screens that suppressed IRF4 expression or reactivated CDKN2A in myeloma cells using densitometry or visual inspection of PCR plates under UV light. While need in drug development inspired this work, application to genome-wide analysis appears feasible using phi29 for selective amplification of open cellular chromatin followed by library construction from supernatants since such supernatants yielded similar results as gene specific DC-PCR. PMID:22984542

  9. Direct chromatin PCR (DC-PCR: hypotonic conditions allow differentiation of chromatin states during thermal cycling.

    Directory of Open Access Journals (Sweden)

    Sergei Vatolin

    Full Text Available Current methods to study chromatin configuration are not well suited for high throughput drug screening since they require large cell numbers and multiple experimental steps that include centrifugation for isolation of nuclei or DNA. Here we show that site specific chromatin analysis can be achieved in one step by simply performing direct chromatin PCR (DC-PCR on cells. The basic underlying observation was that standard hypotonic PCR buffers prevent global cellular chromatin solubilization during thermal cycling while more loosely organized chromatin can be amplified. Despite repeated heating to >90 °C, 41 of 61 tested 5' sequences of silenced genes (CDKN2A, PU.1, IRF4, FOSB, CD34 were not amplifiable while 47 could be amplified from expressing cells. Two gene regions (IRF4, FOSB even required pre-heating of cells in isotonic media to allow this differentiation; otherwise none of 19 assayed sequences yielded PCR products. Cells with baseline expression or epigenetic reactivation gave similar DC-PCR results. Silencing during differentiation of CD34 positive cord blood cells closed respective chromatin while treatment of myeloma cells with an IRF4 transcriptional inhibitor opened a site to DC-PCR that was occupied by RNA polymerase II and NFκB as determined by ChIP. Translation into real-time PCR can not be achieved with commercial real-time PCR buffers which potently open chromatin, but even with simple ethidium bromide addition to standard PCR mastermix we were able to identify hits in small molecules screens that suppressed IRF4 expression or reactivated CDKN2A in myeloma cells using densitometry or visual inspection of PCR plates under UV light. While need in drug development inspired this work, application to genome-wide analysis appears feasible using phi29 for selective amplification of open cellular chromatin followed by library construction from supernatants since such supernatants yielded similar results as gene specific DC-PCR.

  10. Diagnosis of Cutaneous Leishmaniasis by Multiplex PCR

    Directory of Open Access Journals (Sweden)

    M Heiat

    2010-07-01

    Full Text Available Introduction: Annually, more than 14 million people are reported to be infected with Leishmaniasis all over the world. In Iran, this disease is seen in the form of cutaneous and visceral leishmaniasis, of which the cutaneous form is more wide spread. In recent years, cutaneous leishmaniaisis is diagnosed by PCR utilizing specific primers in order to amplify different parasite genes including ribosomal RNA genes, kinetoplast DNA or tandem repeating sequences. The aim of this research was to detect early stage cutaneous leishmaniasis using Multiplex-PCR technique. Methods: In this study, 67 samples were prepared from patients with cutaneous leishmaniasis. DNA was extracted with phenolchloroform. Each specimen was analyzed using two different pairs of PCR primers. The sensitivity of each PCR was optimized on pure Leishmania DNA prior to use for diagnosis. Two standard parasites L. major and L. tropica were used as positive control. Results: DNA amplification fragments were two 115 bp and 683 bp for AB and UL primers, respectively. The sensitivity of two primers was not equal for detection of L. major and L. tropica. The sensivity of PCR with AB primer was 35 cells, while that for UL primer was 40 cells. Conclusion: The results of this study indicate that PCR is a sensitive diagnostic assay for cutaneous leishmaniasis and could be employed as the new standard for routine diagnosis when species identification is not required. However, the ability to identify species is especially important in prognosis of the disease and in deciding appropriate therapy, especially in regions where more than one type of species and disease are seen by clinicians.

  11. pcrEfficiency: a Web tool for PCR amplification efficiency prediction

    Directory of Open Access Journals (Sweden)

    Mallona Izaskun

    2011-10-01

    Full Text Available Abstract Background Relative calculation of differential gene expression in quantitative PCR reactions requires comparison between amplification experiments that include reference genes and genes under study. Ignoring the differences between their efficiencies may lead to miscalculation of gene expression even with the same starting amount of template. Although there are several tools performing PCR primer design, there is no tool available that predicts PCR efficiency for a given amplicon and primer pair. Results We have used a statistical approach based on 90 primer pair combinations amplifying templates from bacteria, yeast, plants and humans, ranging in size between 74 and 907 bp to identify the parameters that affect PCR efficiency. We developed a generalized additive model fitting the data and constructed an open source Web interface that allows the obtention of oligonucleotides optimized for PCR with predicted amplification efficiencies starting from a given sequence. Conclusions pcrEfficiency provides an easy-to-use web interface allowing the prediction of PCR efficiencies prior to web lab experiments thus easing quantitative real-time PCR set-up. A web-based service as well the source code are provided freely at http://srvgen.upct.es/efficiency.html under the GPL v2 license.

  12. Molecular Subtyping of Salmonella Typhimurium with Multiplex Oligonucleotide Ligation-PCR (MOL-PCR).

    Science.gov (United States)

    Wuyts, Véronique; Mattheus, Wesley; Roosens, Nancy H C; Marchal, Kathleen; Bertrand, Sophie; De Keersmaecker, Sigrid C J

    2017-01-01

    A multiplex oligonucleotide ligation-PCR (MOL-PCR) assay is a valuable high-throughput technique for the detection of bacteria and viruses, for characterization of pathogens and for diagnosis of genetic diseases, as it allows one to combine different types of molecular markers in a high-throughput multiplex assay. A MOL-PCR assay starts with a multiplex oligonucleotide ligation reaction for detection of the molecular marker, followed by a singleplex PCR for signal amplification and analysis of the MOL-PCR products on a Luminex platform. This last step occurs through a liquid bead suspension array in which the MOL-PCR products are hybridized to MagPlex-TAG beads.In this chapter, we describe the complete procedure for a MOL-PCR assay for subtyping of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) and its monophasic variant S. 1,4[5],12:i:- from DNA isolation through heat lysis up to data interpretation through a Gödel Prime Product. The subtyping assay consists of 50 discriminative molecular markers and two internal positive control markers divided over three MOL-PCR assays.

  13. Mathematical analysis of the real time array PCR (RTA PCR) process

    NARCIS (Netherlands)

    Dijksman J.F.; Pierik, A.

    2012-01-01

    Real Time Array PCR is a recently developed biochemical technique that measures amplification curves (like quantitative real time Polymerase Chain Reaction (qPCR)) of a multitude of different templates ina sample. It combines two different techniques to profit from theadvantages of both techniques,

  14. Mathematical analysis of the real time array PCR (RTA PCR) process

    NARCIS (Netherlands)

    Dijksman J.F.; Pierik, A.

    2012-01-01

    Real Time Array PCR is a recently developed biochemical technique that measures amplification curves (like quantitative real time Polymerase Chain Reaction (qPCR)) of a multitude of different templates ina sample. It combines two different techniques to profit from theadvantages of both techniques,

  15. Propidium monoazide reverse transcription PCR and RT-qPCR for detecting infectious enterovirus and norovirus

    Science.gov (United States)

    Presently there is no established cell line or small animal model that allows for the detection of infectious human norovirus. Current methods based on RT-PCR and RT-qPCR detect both infectious and non-infectious virus and thus the conclusions that may be drawn regarding the publ...

  16. PCR clonality detection in Hodgkin lymphoma.

    NARCIS (Netherlands)

    Hebeda, K.M.; Altena, M.C. van; Rombout, P.D.M.; Krieken, J.H.J.M. van; Groenen, P.J.T.A.

    2009-01-01

    B-cell clonality detection in whole tissue is considered indicative of B-cell non-Hodgkin lymphoma (NHL). We tested frozen tissue of 24 classical Hodgkin lymphomas (cHL) with a varying tumor cell load with the multiplex polymerase chain reaction (PCR) primer sets for IGH and IGK gene rearrangement (

  17. The Power of Real-Time PCR

    Science.gov (United States)

    Valasek, Mark A.; Repa, Joyce J.

    2005-01-01

    In recent years, real-time polymerase chain reaction (PCR) has emerged as a robust and widely used methodology for biological investigation because it can detect and quantify very small amounts of specific nucleic acid sequences. As a research tool, a major application of this technology is the rapid and accurate assessment of changes in gene…

  18. Aspergillus PCR: one step closer to standardization.

    NARCIS (Netherlands)

    White, P.L.; Bretagne, S.; Klingspor, L.; Melchers, W.J.G.; McCulloch, E.; Schulz, B.; Finnstrom, N.; Mengoli, C.; Barnes, R.A.; Donnelly, J.P.; Loeffler, J.

    2010-01-01

    PCR has been used as an aid in the diagnosis of invasive aspergillosis for almost 2 decades. A lack of standardization has limited both its acceptance as a diagnostic tool and multicenter clinical evaluations, preventing its inclusion in disease-defining criteria. In 2006, the European Aspergillus

  19. Inverse PCR for Point Mutation Introduction.

    Science.gov (United States)

    Silva, Diogo; Santos, Gustavo; Barroca, Mário; Collins, Tony

    2017-01-01

    Inverse PCR is a powerful tool for the rapid introduction of desired mutations at desired positions in a circular double-stranded DNA sequence. Here, custom-designed mutant primers oriented in the inverse direction are used to amplify the entire circular template with incorporation of the required mutation(s). By careful primer design it can be used to perform such diverse modifications as the introduction of point mutations and multiple mutations, the insertion of new sequences, and even sequence deletions. Three primer formats are commonly used; nonoverlapping, partially overlapping and fully overlapping primers, and here we describe the use of nonoverlapping primers for introduction of a point mutation. Use of such a primer setup in the PCR reaction, with one of the primers containing the desired mismatch mutation, results in the amplification of a linear, double-stranded, mutated product. Methylated template DNA is removed from the nonmethylated PCR product by DpnI digestion and the PCR product is then phosphorylated by polynucleotide kinase treatment before being recircularized by ligation, and transformed to E. coli. This relatively simple site-directed mutagenesis procedure is of major importance in biology and biotechnology today where it is commonly employed for the study and engineering of DNA, RNA, and proteins.

  20. Aspergillus PCR: one step closer to standardization.

    NARCIS (Netherlands)

    White, P.L.; Bretagne, S.; Klingspor, L.; Melchers, W.J.G.; McCulloch, E.; Schulz, B.; Finnstrom, N.; Mengoli, C.; Barnes, R.A.; Donnelly, J.P.; Loeffler, J.

    2010-01-01

    PCR has been used as an aid in the diagnosis of invasive aspergillosis for almost 2 decades. A lack of standardization has limited both its acceptance as a diagnostic tool and multicenter clinical evaluations, preventing its inclusion in disease-defining criteria. In 2006, the European Aspergillus P

  1. Real-time PCR: Advanced technologies and applications

    Science.gov (United States)

    This book brings together contributions from 20 experts in the field of PCR, providing a broad perspective of the applications of quantitative real-time PCR (qPCR). The editors state in the preface that the aim is to provide detailed insight into underlying principles and methods of qPCR to provide ...

  2. Real Time PCR: Principles and Application

    Directory of Open Access Journals (Sweden)

    Safie Amini

    2005-09-01

    Full Text Available The polymerase chain reaction (PCR has been used as the new golden standard for detecting a wide variety of templates across a range of scientific specialties and also as an essential tool in research laboratories. PCR has completely revolutionized the detection of RNA and DNA viruses(1. Real Time vs. Traditional PCRReal time chemistry allows the detection of PCR amplification during the early phase of the reaction. Measuring the kinetic of the reaction in the early phase of PCR provides a distinct advantage over traditional PCR detection. Traditional methods use agarose gel electrophoresis for detection of PCR amplification at the final phase or end point. End point detection is really time consuming; it takes several hours to have the result. On the other hand, results are based on size discrimination. Also, the result of end point is variable from sample to sample. While gels may not resolve this variability in yield, real time PCR is sensitive enough to detect this change.Some problems with end point detection are: poor precision, low sensitivity, short dynamic range (<2 log, low resolution, non-automated procedure, size-based discrimination only, and post PCR processing (carry-over contamination and results are not expressed as numbers(2.Detection of PCR Products in Real-timeReal-time PCR and RT-PCR allow accurate quantification of starting amounts of DNA, cDNA, and RNA targets. Fluorescence is measured during each cycle, which greatly increases the dynamic range of the reaction since the amount offluorescence is proportional to the amount of PCR product. PCR products can be detected using either fluorescent dyes that bind to double-stranded DNA or fluorescently labeled sequence-specific probes(3.SYBR Green ISYBR® Green I binds all double-stranded DNA molecules, emitting a fluorescent signal of a defined wavelength on binding. The excitation and emission maxima of SYBR Green I are at 494 nm and 521 nm, respectively, and are compatible for

  3. Clinical Identification of Common Species of Dermatophytes by PCR and PCR-RFLP

    Institute of Scientific and Technical Information of China (English)

    丁娟; 李家文; 刘志香; 谭志建

    2004-01-01

    To find a fast and efficient way of identifying seven common dermatophytes in clinical practice, we used the techniques of polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) targeting Topoisomerase Ⅱ gene. The DNA of 7 dermatophytes, along with Candida albicans, Aspergillus terreus and Aspergillus flavus were amplified by consensus primer dPsD1. They were then subjected to a second PCR with primers dPsD2 and species-specific primers PsT and PsME separately. 6 of the products generated by dPsD2 were digested with restriction enzyme Hinc Ⅱ. DNA fragments of 3390 bp and 2380 bp was amplified by using consensus primer dPsD1 and dPsD2 from the genomic DNA of each dermatophyte species separately. By combining the results of the two species-specific primer sets (PsT and PsME), all species of dermatophyte yielded unique sizes-set of PCR products expect for T. mentagrophytes and T. tonsurans.From the restriction profiles of Hinc Ⅱ , 6 of the 7 dermatophytoses were diagnosed to species level including T. mentagrophytes and T. tonsurans. By combining the results of the PCR and PCRRFLP, the 7 common dermatophytes can be identified to species level. It is conclude that the multiplex PCR and PCR-RFLP identification targeting the DNA topoisomerase Ⅱ gene is rapid and efficient.

  4. Quantitative PCR and Digital PCR for Detection of Ascaris lumbricoides Eggs in Reclaimed Water

    Directory of Open Access Journals (Sweden)

    Lucrecia Acosta Soto

    2017-01-01

    Full Text Available The reuse of reclaimed water from wastewater depuration is a widespread and necessary practice in many areas around the world and must be accompanied by adequate and continuous quality control. Ascaris lumbricoides is one of the soil-transmitted helminths (STH with risk for humans due to its high infectivity and an important determinant of transmission is the inadequacy of water supplies and sanitation. The World Health Organization (WHO recommends a limit equal to or lower than one parasitic helminth egg per liter, to reuse reclaimed water for unrestricted irrigation. We present two new protocols of DNA extraction from large volumes of reclaimed water. Quantitative PCR (qPCR and digital PCR (dPCR were able to detect low amounts of A. lumbricoides eggs. By using the first extraction protocol, which processes 500 mL of reclaimed water, qPCR can detect DNA concentrations as low as one A. lumbricoides egg equivalent, while dPCR can detect DNA concentrations as low as five A. lumbricoides egg equivalents. By using the second protocol, which processes 10 L of reclaimed water, qPCR was able to detect DNA concentrations equivalent to 20 A. lumbricoides eggs. This fact indicated the importance of developing new methodologies to detect helminth eggs with higher sensitivity and precision avoiding possible human infection risks.

  5. Digital PCR to assess gene-editing frequencies (GEF-dPCR) mediated by designer nucleases.

    Science.gov (United States)

    Mock, Ulrike; Hauber, Ilona; Fehse, Boris

    2016-03-01

    Genome editing using designer nucleases such as transcription activator-like effector nucleases (TALENs) or clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 nucleases is an emerging technology in basic and applied research. Whereas the application of editing tools, namely CRISPR-Cas9, has recently become very straightforward, quantification of resulting gene knockout rates still remains a bottleneck. This is particularly true if the product of a targeted gene is not easily detectable. To address this problem, we devised a novel gene-editing frequency digital PCR (GEF-dPCR) technique. GEF-dPCR exploits two differently labeled probes that are placed within one amplicon at the gene-editing target site to simultaneously detect wild-type and nonhomologous end-joining (NHEJ)-affected alleles. Taking advantage of the principle of dPCR, this enables concurrent quantification of edited and wild-type alleles in a given sample. We propose that our method is optimal for the monitoring of gene-edited cells in vivo, e.g., in clinical settings. Here we describe preparation, design of primers and probes, and setup and analysis of GEF-dPCR. The setup of GEF-dPCR requires up to 2 weeks (depending on the starting point); once the dPCR has been established, the protocol for sample analysis takes <1 d.

  6. 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.

  7. EXTRAÇÃO DE SOLO EXPOSTO E INFERÊNCIA DO TIPO DE SOLO A PARTIR DO ESTUDO DA DRENAGEM USANDO-SE TÉCNICAS DE SENSORIAMENTO REMOTO E GEOPROCESSAMENTO

    Directory of Open Access Journals (Sweden)

    Jedman Dantas Motta

    Full Text Available RESUMO Este trabalho procura obter uma região de solo exposto a partir da observação do atributo cor e inferir sobre determinado tipo de solo a partir de um mapa de drenagem obtido através de imagens temáticas TM-Landsat (banda 5 e composições coloridas 7R/2G/1B, 7R/5G/2B e 5R/4G/3B georefenciadas. A região considerada localiza-se próximo à cidade de Campina Grande, Estado da Paraíba, onde existem poucos trabalhos utilizando produtos Landsat, e quase nenhum trabalho cartográfico, devido à grande concentração de nuvens durante todo o ano, o que dificulta a obtenção dos dados por meio aéreo ou espacial.

  8. Análise de eventos hidrológicos extremos, usando-se a distribuição GEV e momentos LH Analysis of extreme hydrological events using GEV distribution and LH moments

    Directory of Open Access Journals (Sweden)

    Manoel M. F. de Queiroz

    2006-06-01

    Full Text Available A distribuição de probabilidade generalizada de valores extremos (GEV, tem facilitado muitas aplicações em hidrologia, utilizada na modelação de eventos extremos naturais. Estudos sobre o assunto mostram que estimadores de máxima verossimilhança dos parâmetros da GEV são instáveis em pequenas amostras, podendo fornecer valores absurdos do parâmetro de forma, quando então são recomendados estimadores de momentos LH, baseados na combinação linear de estatísticas de altas ordens, introduzidas para caracterizar a parte mais alta da distribuição e os valores extremos dos dados; contudo, não se dispõe de programas computacionais para PC, que modelem eventos extremos via momentos LH. Objetivou-se, com este trabalho, apresentar a modelação de eventos hidrológicos extremos através da distribuição GEV, utilizando-se momentos LH para estimar seus parâmetros e o teste estatístico proposto por Wang (1998 para verificação da qualidade dos ajustes desenvolvidos no ambiente Matlab. Como resultados, são apresentados as estimativas dos parâmetros da GEV, os valores das taxas de momentos LH: coeficientes de variação, assimetria e curtose, e os valores do teste de qualidade de ajuste, em aplicações com dados de vazão de rios do Paraná.The generalized extreme-value (GEV distribution has facilitated many applications in hydrology, used to model a wide variety of natural extreme events. Previous studies show that small-sample maximum-likelihood estimators parameters are unstable and demonstrates that absurd values of the GEV shape parameter can be generated. It is recommended that LH moments estimators, based on linear combinations of higher-order statistics, should be introduced for characterizing the upper part of distributions and larger events in data. However, there have been no computer packages for PC that model extreme events by LH moments. The objective of this paper was to present the modeling of hydrological extreme events by GEV distribution, using LH moments to estimate its parameters and the goodness-of-fit test proposed by Wang (1998 to evaluate the goodness-of-fit, both developed in Matlab. The results are presented for the estimation of the parameters of the GEV, the values coefficient of variation, skewness, kurtosis and goodness-of-fit test values, and fitting extreme flow observed in Paraná Rivers by GEV distribution.

  9. Umidade do solo no semiárido pernambucano usando-se reflectometria no domínio do tempo (TDR Soil moisture in Pernambuco semiarid using time domain reflectometry (TDR

    Directory of Open Access Journals (Sweden)

    Thais E. M. dos Santos

    2011-07-01

    Full Text Available Objetivou-se com o presente trabalho investigar a variabilidade temporal do conteúdo superficial da água no solo, através da reflectometria no domínio do tempo (TDR, em consequência das características de precipitação ocorridas no semiárido pernambucano, estudando esta dinâmica sob diferentes tipos de cobertura superficial do solo. O estudo foi realizado em uma encosta de uma bacia representativa, em um Argissolo Amarelo Eutrófico típico, onde foram instaladas quatro parcelas experimentais dotadas de duas sondas TDR, para investigação da umidade do solo e do sistema para monitoramento do escoamento superficial. Os tratamentos adotados foram: cobertura natural (CN, solo descoberto (SD, palma forrageira (P e barramentos, associados à cobertura morta (B+CM. A partir dos resultados obtidos durante o período de ocorrência de chuvas erosivas, a umidade do solo apresentou elevada variabilidade no tempo, estando relacionada aos diferentes tipos de cobertura e propriedades do solo. A cobertura morta mostrou ser a prática conservacionista mais adequada para manutenção da umidade do solo.Present study aimed to investigate the temporal variability of surface water content in soil by time domain reflectometry (TDR, as consequence of precipitation characteristics of Pernambuco semiarid, studying such dynamics under different types of the soil cover. The study was conducted in a slope of a representative catchment, in a Typic Hapludalf soil, where four experimental plots were installed with two TDR probes for soil moisture investigation as well as monitoring the runoff. Treatments were natural cover (CN, bare soil (SD, cactus (P and microdams associated with mulch (B + CM. From the results obtained during a period with erosive rainfall, it was found that soil moisture observed during the experimental period showed high variability in time, related to different types of coverage and soil properties. Mulching was the most appropriate conservation practice for maintaining soil moisture.

  10. Production of tomato seedlings using different substrates and trays in three protected environments Produção de mudas de tomate usando-se diferentes substratos e tipos de bandejas em três ambientes protegidos

    Directory of Open Access Journals (Sweden)

    Edilson Costa

    2012-10-01

    Full Text Available The seedlings production is an essential part for vegetables production. Thus, this study aimed to evaluate the environment, the substrates and the containers in the development of tomato seedlings, cv. Santa crus Kada Gigante, in Aquidauana -MS, Brazil region, from October to November, 2008. Polystyrene trays with 72; 128 and 200 cells, filled with four substrates (soil; Plantmax®; coconut fiber and vermiculite were tested in three protected environments (greenhouse; screened with Sombrite® and screened with Aluminet®. The experimental design was completely randomized, factorial scheme 3x4 (three trays x four substrates, with four replications, being analyzed individual variance analysis and joint analysis for the environments. The environment with screens (Sombrite® and Aluminet®, the trays with 72 cells and the vermiculite produced better results.A produção de mudas é uma etapa essencial na cadeia produtiva de hortaliças. Desta forma, este trabalho teve os objetivos de avaliar a ambiência, os substratos e os recipientes no desenvolvimento de mudas de tomateiro, cv. Santa Cruz Kada Gigante, na região de Aquidauana-MS, de outubro a novembro de 2008. Foram testadas bandejas de poliestireno com 72; 128 e 200 células, preenchidas com quatro substratos: solo; Plantmax®; fibra de coco fina, e vermiculita, em três ambientes protegidos: estufa, telado com Sombrite® e telado com Aluminet®. O experimento foi conduzido em delineamento inteiramente casualizado, em esquema fatorial 3 x 4 (três bandejas x quatro substratos, com quatro repetições, realizando-se de análises de variâncias individuais e análise conjunta dos ambientes. Os ambientes telados (Sombrite® e Aluminet®, a bandeja com 72 células e a vermiculita produziram as melhores mudas.

  11. Quantificação da área de infarto usando-se SPECT de perfusão miocárdica de alta resolução em ratos

    Directory of Open Access Journals (Sweden)

    Luciano Fonseca Lemos de Oliveira

    2013-07-01

    Full Text Available FUNDAMENTO: Técnicas de imageamento in vivo permitem avaliar sequencialmente a morfologia e a função dos órgãos em diversos modelos experimentais. Desenvolvemos um dispositivo de adaptação de uma gama-câmara clínica para obter imagens tomográficas por emissão de fótons singulares (SPECT de alta resolução, baseado em colimador pinhole. OBJETIVO: Determinar a acurácia desse sistema na quantificação da área de infarto miocárdico em ratos. MÉTODOS: Treze ratos Wistar machos (250 g foram submetidos a infarto do miocárdio por oclusão da artéria coronária esquerda. Após 4 semanas, foram adquiridas imagens tomográficas com o sistema desenvolvido, 1,5 hora após a injeção endovenosa de 555MBq de 99mTc-Sestamibi. Na reconstrução tomográfica, utilizamos software especialmente desenvolvido baseado no algoritmo de Máxima Verossimilhança. Comparamos as médias e analisamos a correlação entre a extensão dos defeitos perfusionais detectados pela cintilografia e a extensão da fibrose miocárdica avaliada pela histologia. RESULTADOS: As imagens apresentaram ótima relação órgão-alvo/fundo, com apropriada visualização das paredes e da cavidade do ventrículo esquerdo. Todos os animais exibindo áreas de infarto foram corretamente identificados pelas imagens de perfusão. Não houve diferença entre a área do infarto medida pelo SPECT (21,1 ± 21,2% e pela histologia (21,7 ± 22,0%; p = 0,45, obtendo forte correlação entre os valores da área de infarto mensurada pelos dois métodos (r = 0,99; p < 0,0001. CONCLUSÃO: O sistema desenvolvido apresentou resolução espacial adequada e elevada acurácia para detecção e quantificação das áreas de infarto miocárdico, sendo uma opção de baixo custo e grande versatilidade na obtenção de imagens em SPECT de alta resolução de órgãos de pequenos roedores.

  12. Prevalência e fatores associados à infecção pelo Mycobacterium tuberculosis entre agentes comunitários de saúde no Brasil, usando-se a prova tuberculínica

    Directory of Open Access Journals (Sweden)

    Wesley Pereira Rogerio

    2015-10-01

    Full Text Available Resumo Este artigo tem por objetivo determinar a prevalência e os fatores associados à infecção latente pelo Mycobacterium tuberculosis entre agentes comunitários de saúde (ACS, usando dois pontos de corte da prova tuberculínica 5mm e 10mm. Trata-se de estudo transversal, com dados coletados sobre sexo, idade, cicatriz de BCG, prova tuberculínica (PT anterior, tempo que trabalha na profissão de ACS, atuar em unidade básica de saúde (UBS, ter tido contato intradomiciliar com tuberculose (TB, usar álcool, ser fumante e apresentar comorbidades. Para controle de variáveis de confusão e estimativa da medida de efeito (OR, foi empregada a regressão logística. Aplicou-se PT, com leitura após 48-72 horas. As prevalências foram de 57,88% e 37,3%, respectivamente, para 5mm e 10mm. Manteve-se associada à positividade para o ponto de corte de 10mm a condição de trabalhar em UBS com Programa de Controle de Tuberculose (PCT implementado e já ter tido contato intradomiciliar com TB. Já para o ponto de corte de 5mm, trabalhar em UBS com PCT e implementado. São necessárias ações de conscientização nos municípios e fortalecimento das ações de educação permanente sobre a temática.

  13. Detection of Main Causal Viruses in Trichosanthes kirilowii by RT-PCR%利用RT-PCR技术检测吊瓜病毒种类的研究

    Institute of Scientific and Technical Information of China (English)

    徐京; 王珍华; 庞基良

    2011-01-01

    Zucchini yellow mosaic virus(ZYMV), Watermelon mosaic virus(WMV), Cucumber mosaic virus (CMV), Squash mosaic virus (SqMV) and Papaya ringspot virus (PRSV) are the most serious pathogens infecting cucurbitaceous crops. Five pairs of specific primers were designed based on the conserved sequences of the 5 viruses, and RT-PCR was applied to detect total RNA which was extracted from Trichosanthes kirilowii Maxim. leaves infected virus. The results showed that the causal virus of T. kirilowii in Changxing was PRSV with 100% incidence rate as no multiplex virus infection was detected.The RT-PCR results of different sampling parts of T. kirilowii suggested that the most suitable part for virus detection was old leave and the most virus-free part was young leave. The cause of more PRSV expressed in shoot tip than in young leaves was discussed, and it was possibly that the expression of virus was related to plant's metabolism intensity.%小西葫芦黄花叶病毒(Zucchini yellow mosaic virus,ZYMV)、西瓜花叶病毒(Watermelon mosaic virus,WMV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)、南瓜花叶病毒(Squash mosaic virus,SqMV)和番木瓜环斑病毒(Papayava ringspot virus,PRSV)是危害葫芦科作物最严重、最广泛的5种病毒.根据已知的5种病毒序列设计特异性引物,对感染病毒的吊瓜总RNA进行RT-PCR扩增.结果表明长兴吊瓜的病毒种类主要为PRSv,感染率为100%,无复合病毒感染.吊瓜植株不同取样部位(茎尖、卷须、嫩叶、茎、花、子房、老叶等)的RT-PCR表明老叶是较好的病毒检测取样部位,而嫩叶是吊瓜脱毒最佳的外植体取样部位.对茎尖的PRSV表达量高过幼叶的原因也进行了分析,认为可能是病毒的表达量与植株的代谢强度有关.

  14. Rapid PCR thermocycling using microscale thermal convection.

    Science.gov (United States)

    Muddu, Radha; Hassan, Yassin A; Ugaz, Victor M

    2011-03-05

    Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable concentration level. But the design of conventional PCR thermocycling hardware, predominantly based on massive metal heating blocks whose temperature is regulated by thermoelectric heaters, severely limits the achievable reaction speed(1). Considerable electrical power is also required to repeatedly heat and cool the reagent mixture, limiting the ability to deploy these instruments in a portable format. Thermal convection has emerged as a promising alternative thermocycling approach that has the potential to overcome these limitations(2-9). Convective flows are an everyday occurrence in a diverse array of settings ranging from the Earth's atmosphere, oceans, and interior, to decorative and colorful lava lamps. Fluid motion is initiated in the same way in each case: a buoyancy driven instability arises when a confined volume of fluid is subjected to a spatial temperature gradient. These same phenomena offer an attractive way to perform PCR thermocycling. By applying a static temperature gradient across an appropriately designed reactor geometry, a continuous circulatory flow can be established that will repeatedly transport PCR reagents through temperature zones associated with the denaturing, annealing, and extension stages of the reaction (Figure 1). Thermocycling can therefore be actuated in a pseudo-isothermal manner by simply holding two opposing surfaces at fixed temperatures, completely eliminating the need to repeatedly heat and cool the instrument. One of the main challenges facing design of convective thermocyclers is the need to precisely control the spatial velocity and temperature distributions within the reactor to ensure that the reagents sequentially occupy the correct temperature zones for a sufficient period of time(10,11). Here we describe results of our efforts to probe the full 3-D velocity and

  15. A quadruplex PCR (qxPCR) assay for adulteration in dairy products.

    Science.gov (United States)

    Agrimonti, Caterina; Pirondini, Andrea; Marmiroli, Marta; Marmiroli, Nelson

    2015-11-15

    This study describes the development of a quadruplex quantitative Real Time PCR (qxPCR) based on SYBR®GreenER chemistry, for rapid identification of DNA of cow, goat, sheep and buffalo in dairy products, and for quantification of cow DNA in these products. The platform was applied to: (i) mixes of milks at fixed percentages; (ii) cheeses prepared with the same mixes; (iii) commercial dairy products. The methodology enabled the detection of DNA from cow in mixes of milk and cheeses with a limit of detection (LOD) of 0.1%. When applied to commercial dairy products the qxPCR gave results comparable with each single-plex Real Time PCR. A good correlation (R(2)>0.9) between peaks' area of derivative of melting curves of amplicons and percentages of cow milk in milk mixes and cheeses, allows for an estimation of cow DNA in a dynamic range varying from 0.1-5% to 1-25%.

  16. Real‐time PCR (qPCR) primer design using free online software

    National Research Council Canada - National Science Library

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

    ...‐stranded DNA product, its success depends greatly on proper primer design. Many types of online primer design software are available, which can be used free of charge to design desirable SYBR® Green‐based qPCR primers...

  17. Establishment of a perinatal cytomegaiovirus (CMV) induced hepatobiliary system injury model in guinea pigs%豚鼠围产期巨细胞病毒感染肝胆损伤模型的建立

    Institute of Scientific and Technical Information of China (English)

    王玮; 郑珊

    2009-01-01

    Objective To establish a perinatal cytomegalovirus (CMV) induced hepatobiliary system injury model in guinea pigs. Methods Three experimental groups were designed as follows. (1) Prenatal group (Group P): Female guinea pigs on the 40th to 43rd gestational day were randomly allocated into 3 subgroups. Guinea pigs in group P1 and P2 accepted intraperitoneal injection of virus supernatant with the dose of 1 10 9 TCID per dam and saline respectively, while guinea pigs in group P3 served as blank control. Live-born pups were sacrificed within 24 hours, on day 10 or 20 after birth. Samples of livers, extrahepatic bile duct and blood were collected. Weight gain, clinical signs of hepa-tobiliary injury (I. E. , jaundice in non-fur-covered skin, acholic stools) and survival were recorded. (2) Neonatal group (Group N) : A subset of healthy pups accepted intraperitoneal injection of virus supernatant at the dose of 1 108 TCID per pup within 24 hours after birth. Samples were collected on day 10, 20, or 30 after birth. (3) Infantile Group (Group Ⅰ): Healthy pups were inoculated with a same dose of virus supernatant on day 10 after birth. Samples were harvested on day 10, 20 or 30 after inoculation. The level of total bilirubin (TB), direct bilirubin (DB), ALT and AST in blood samples was analyzed. Serial sections of the liver or extrahepatic bile duct fixed in formalin were stained with Hematoxylin and Eosin. Hybridization in situ was applied on frozen sections to detect the distribution of viral mRNA. Results Compared to those in the control group, TB, DB, ALT and AST levels in Pups infected prenatally were significantly higher within the first 10 days of life (P<0. 05), and de-creased to the normal level on 20th day of life. A few pups of P1 group got the signs of jaundice (ie. Acholic stools ), which were associated with increased TB and DB level. In the neonatal group, a higher AST level was observed on the 10th day post inoculation in pups but it returned to normal soon

  18. Chicken QTL mapping by multiplex PCR

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    To facilitate rapid determination of the chromosomal location of quantitative trait loci, the current approaches to gene mapping are improved using a multiplex PCR technique. The high-throughput linkage analysis method described here allows selection of 178 from 328 microsatellite markers through the multiplex PCR method combined with the semi-automatic fluorescence-labeled DNA analysis technology. Those polymorphism markers are distributed on 23 autosomes and one sex chromosome (chromosome Z), covering 3080cM genetic distance. The average marker density is 18cM, dispersed into 30 different sets. These selected polymorphism microsatellite markers segregate with the family members, following the Mendel's heritage laws, and are very useful for chicken linkage map analysis as well as for the research on some important economic quantitative characters of chicken.

  19. PCR-SSCP检测水稻SNPs

    Institute of Scientific and Technical Information of China (English)

    李飞; 郭凌; 余潮

    2006-01-01

    本文选择5个水稻SNPs位点为对象,研究凝胶组成和电泳条件等对PCR-SSCP检测SNPs的影响.发现凝胶浓度、交联度、甘油和电泳温度等均可明显影响DNA单链分子迁移率,且对不同DNA单链分子的影响程度不同.因此,可以通过调节凝胶浓度、交联度、甘油和电泳温度等提高PCR-SSCP检测水稻SNPs的分辨率及效果.在一定条件下,较低交联度和4°电泳条件检测SNPs的效果更好.

  20. Application of PCR techniques in toxicology

    Directory of Open Access Journals (Sweden)

    Maja Kazubek

    2010-10-01

    Full Text Available Molecular biology techniques have become widely used in toxicology, leading to the creation of a new science – molecular toxicology. The goal of molecular toxicology is to detect and study the changes induced by xenobiotics at the molecular level. The research scope of molecular toxicology includes examination of mutations in genomic DNA, differences in mRNA expression and study of genotype indicating individual sensitivity.The processes of activation and detoxification of xenobiotics, drugs and environmental carcinogens involve several enzymes (xenobiotic-metabolizing enzymes – XMEs. Most of the chemicals entering our bodies, regardless of whether they have medical, pathogenic or carcinogenic properties, require metabolic activation by phase I enzymes (cytochrome P-450. In the next process the phase I products are usually detoxified by phase II enzymes, mainly by epoxide hydrolase, glutathione transferase, N-acetyltransferase or sulfotransferase. PCR techniques allow precise study of the effects of xenobiotics on cells and tissues by examining the level of activation of genes coding for phase I and II enzymes, or by testing the activity of other elements of the transcriptome. Studies of sensitivity of individual cells or tissues based on examination of mutation or gene polymorphism presence are also relevant.This paper presents the possibility of using various PCR techniques in toxicology and especially in the study of genetically determined sensitivity to xenobiotics. It also covers the possibilities of applying qPCR and qRT-PCR methods in the search for exposure biomarkers with particular emphasis on individual cytochrome P450 isoforms. Furthermore, it provides information about the possibility of implementing the differential display technique in the identification of new genes activated by toxic agents.

  1. Miniature PCR based portable bioaerosol monitor development.

    Science.gov (United States)

    Agranovski, I E; Usachev, E V; Agranovski, E; Usacheva, O V

    2017-01-01

    A portable bioaerosol monitor is greatly demanded technology in many areas including air quality control, occupational exposure assessment and health risk evaluation, environmental studies and, especially, in defence and bio-terrorism applications. Our recent groundwork allowed us to formulate the concept of a portable bioaerosol monitor, which needs to be light, user friendly, reliable and capable of detecting airborne pathogens within 1-1·5 h on the spot. Conceptually, the event of a bioaerosol concentration burst is determined by triggers to commence the representative air sampling with sequential real-time polymerase chain reaction (PCR) confirmation of the targeted micro-organism present in the air. To minimize reagent consumption and idle running of the technology, an event of a bioaerosol burst is confirmed by three parameters: aerosol particle size, concentration and composition. Only particle sizes above 200 nm attract interest in the bioaerosol. Only an elevated aerosol concentration above the threshold (background aerosol concentration) is a signal to commence the analytical procedure. The combination of our previously developed personal bioaerosol sampler, aerosol particle counter based trigger and portable real-time PCR device formed the basis of the bioaerosol monitoring technology. The portable real-time PCR device was advanced to provide internally controlled detection, significantly reducing false-positive alarms. The technique is capable of detecting selected airborne micro-organisms on the spot within 30-80 min, depending on the genome organization of the particular strain. Due to recent outbreaks of infectious airborne diseases and the continuing threat of intentionally released bioaerosol attacks, investigations into the possibility of the early and reliable detection of pathogenic micro-organisms in the air is becoming increasingly important. The proposed technology consisting of a bioaerosol sampler, technology trigger and PCR device is

  2. Reproducibility of AMPLICOR enterovirus PCR test results.

    OpenAIRE

    1997-01-01

    The reproducibility of AMPLICOR enterovirus PCR test results was determined with clinical samples of cerebrospinal fluid, serum, urine, and throat and rectal swabs. Among 608 samples from which duplicate aliquots were run simultaneously, only seven pairs gave discordant results. Among 104 samples from which duplicate aliquots were run in separate assays, no discordance was seen. Overall, the reproducibility of test kit results was 99% (705 of 712).

  3. A PCR amplification method without DNA extraction.

    Science.gov (United States)

    Li, Hongwei; Xu, Haiyue; Zhao, Chunjiang; Sulaiman, Yiming; Wu, Changxin

    2011-02-01

    To develop a simple and inexpensive method for direct PCR amplification of animal DNA from tissues, we optimized different components and their concentration in lysis buffer systems. Finally, we acquired the optimized buffer system composed of 10 mmol tris(hydroxymethyl)aminomethane (Tris)-Cl (pH 8.0), 2 mmol ethylene diamine tetraacetic (EDTA) (pH 8.0), 0.2 mol NaCl and 200 μg/mL Proteinase K. Interestingly, the optimized buffer is also very effective when working with common human sample types, including blood, buccal cells and hair. The direct PCR method requires fewer reagents (Tris-Cl, EDTA, Protease K and NaCl) and less incubation time (only 35 min). The cost of treating every sample is less than $0.02, and all steps can be completed on a thermal cycler in a 96-well format. So, the proposed method will significantly improve high-throughput PCR-based molecular assays in animal systems and in common human sample types.

  4. Clostridium perfringens isolate typing by multiplex PCR

    Directory of Open Access Journals (Sweden)

    MR Ahsani

    2010-01-01

    Full Text Available Clostridium perfringens is an important pathogen that provokes numerous different diseases. This bacterium is classified into five different types, each of which capable of causing a different disease. There are various methods for the bacterial identification, many are labor-intensive, time-consuming, expensive and also present low sensitivity and specificity. The aim of this research was to identify the different types of C. perfringens using PCR molecular method. In this study, 130 sheep-dung samples were randomly collected from areas around the city of Kerman, southeastern Iran. After processing and culturing of samples, the produced colonies were morphologically studied, gram stain test was also carried out and the genera of these bacteria were identified through biochemical tests. DNA extracted from isolated bacteria for genotyping was tested by multiplex PCR with specific primers. Based on length of synthesized fragments by PCR, toxin types and bacterial strains were detected. C. perfringens isolated types were divided as follows: 17.39% type A, 21.74% type B, 34.78% type C and 26.09% type D. It should be emphasized that, up to the present moment, C. perfringens type A has not been reported in Iran.

  5. Comparison of Droplet Digital PCR and Quantitative PCR Assays for Quantitative Detection of Xanthomonas citri Subsp. citri.

    Directory of Open Access Journals (Sweden)

    Yun Zhao

    Full Text Available Droplet digital polymerase chain reaction (ddPCR is a novel molecular biology technique providing absolute quantification of target nucleic acids without the need for an external calibrator. Despite its emerging applications in medical diagnosis, there are few reports of its use for the detection of plant pathogens. This work was designed to assess the diagnosis potential of the ddPCR for absolute quantitative detection of Xanthomonas citri subsp. citri, a quarantine plant pathogenic bacterium that causes citrus bacterial canker in susceptible Citrus species. We transferred an established quantitative PCR (qPCR assay for citrus bacterial canker diagnosis directly to the ddPCR format and compared the performance of the two methods. The qPCR assay has a broader dynamic range compared to the ddPCR assay and the ddPCR assay has a significantly higher degree of sensitivity compared to the qPCR assay. The influence of PCR inhibitors can be reduced considerably in the ddPCR assay because the collection of end-point fluorescent signals and the counting of binomial events (positive or negative droplets are associated with a Poisson algorithm. The ddPCR assay also shows lower coefficient of variation compared to the qPCR assay especially in low target concentration. The linear association of the measurements by ddPCR and qPCR assays is strong (Pearson correlation = 0.8633; P<0.001. Receiver operating characteristic analysis indicates the ddPCR methodology is a more robust approach for diagnosis of citrus bacterial canker. In summary, the results demonstrated that the ddPCR assay has the potential for the quantitative detection of X. citri subsp. citri with high precision and accuracy as compared with the results from qPCR assay. Further studies are required to evaluate and validate the value of ddPCR technology in the diagnosis of plant disease and quarantine applications.

  6. Amp-PCR: combining a random unbiased Phi29-amplification with a specific real-time PCR, performed in one tube to increase PCR sensitivity.

    Directory of Open Access Journals (Sweden)

    Lena Erlandsson

    Full Text Available In clinical situations where a diagnostic real-time PCR assay is not sensitive enough, leading to low or falsely negative results, or where detection earlier in a disease progression would benefit the patient, an unbiased pre-amplification prior to the real-time PCR could be beneficial. In Amp-PCR, an unbiased random Phi29 pre-amplification is combined with a specific real-time PCR reaction. The two reactions are separated physically by a wax-layer (AmpliWax® and are run in sequel in the same sealed tube. Amp-PCR can increase the specific PCR signal at least 100×10(6-fold and make it possible to detect positive samples normally under the detection limit of the specific real-time PCR. The risk of contamination is eliminated and Amp-PCR could replace nested-PCR in situations where increased sensitivity is needed e.g. in routine PCR diagnostic analysis. We show Amp-PCR to work on clinical samples containing circular and linear viral dsDNA genomes, but can work well on DNA of any origin, both from non-cellular (virus and cellular sources (bacteria, archae, eukaryotes.

  7. Overcoming inhibition in real-time diagnostic PCR.

    Science.gov (United States)

    Hedman, Johannes; Rådström, Peter

    2013-01-01

    PCR is an important and powerful tool in several fields, including clinical diagnostics, food analysis, and forensic analysis. In theory, PCR enables the detection of one single cell or DNA molecule. However, the presence of PCR inhibitors in the sample affects the amplification efficiency of PCR, thus lowering the detection limit, as well as the precision of sequence-specific nucleic acid quantification in real-time PCR. In order to overcome the problems caused by PCR inhibitors, all the steps leading up to DNA amplification must be optimized for the sample type in question. Sampling and sample treatment are key steps, but most of the methods currently in use were developed for conventional diagnostic methods and not for PCR. Therefore, there is a need for fast, simple, and robust sample preparation methods that take advantage of the accuracy of PCR. In addition, the thermostable DNA polymerases and buffer systems used in PCR are affected differently by inhibitors. During recent years, real-time PCR has developed considerably and is now widely used as a diagnostic tool. This technique has greatly improved the degree of automation and reduced the analysis time, but has also introduced a new set of PCR inhibitors, namely those affecting the fluorescence signal. The purpose of this chapter is to view the complexity of PCR inhibition from different angles, presenting both molecular explanations and practical ways of dealing with the problem. Although diagnostic PCR brings together scientists from different diagnostic fields, end-users have not fully exploited the potential of learning from each other. Here, we have collected knowledge from archeological analysis, clinical diagnostics, environmental analysis, food analysis, and forensic analysis. The concept of integrating sampling, sample treatment, and the chemistry of PCR, i.e., pre-PCR processing, will be addressed as a general approach to overcoming real-time PCR inhibition and producing samples optimal for PCR

  8. 锚定PCR(Anchored PCR):一种新的染色体步行方法

    Institute of Scientific and Technical Information of China (English)

    陈柏君; 孙超; 王勇; 胡鸢雷; 林忠平

    2004-01-01

    基于PCR的技术是克隆已知DNA片段侧翼序列的最常用方法.到目前为止,这些方法大致可以分为3种类型:反向PCR(inverse PCR)、连接介导的PCR(1igation-mediated PCR)和随机引物PCR(randomly primed PCR).反向PCR是使用最早的方法,其原理是用限制性内切酶消化基因组总

  9. Lab-on-a-chip PCR: real time PCR in miniaturized format for HLA diagnostics

    Science.gov (United States)

    Gaertner, Claudia; Becker, Holger; Hlawatsch, Nadine; Klemm, Richard; Moche, Christian; Sewart, René; Frank, Rainer; Willems, Andreas

    2014-05-01

    In case of transplantation or the identification of special metabolic diseases like coeliac disease, HLA typing has to be done fast and reliably with easy-to-handle devices by using limited amount of sample. Against this background a lab-on-a-chip device was realized enabling a fast HLA typing via miniaturized Real-time PCR. Hereby, two main process steps were combined, namely the extraction of DNA from whole blood and the amplification of the target DNA by Real-time PCR giving rise-to a semi-quantitative analysis. For the implementation of both processes on chip, a sample preparation and a real-time module were used. Sample preparation was carried out by using magnetic beads that were stored directly on chip as dry powder, together with all lysis reagents. After purification of the DNA by applying a special buffer regime, the sample DNA was transferred into the PCR module for amplification and detection. Coping with a massively increased surface-to-volume ratio, which results in a higher amount of unspecific binding on the chip surface, special additives needed to be integrated to compensate for this effect. Finally the overall procedure showed a sensitivity comparable to standard Real-time PCR but reduced the duration of analysis to significantly less than one hour. The presented work demonstrates that the combination of lab-on-a-chip PCR with direct optical read-out in a real-time fashion is an extremely promising tool for molecular diagnostics.

  10. Species identification and quantification in meat and meat products using droplet digital PCR (ddPCR).

    Science.gov (United States)

    Floren, C; Wiedemann, I; Brenig, B; Schütz, E; Beck, J

    2015-04-15

    Species fraud and product mislabelling in processed food, albeit not being a direct health issue, often results in consumer distrust. Therefore methods for quantification of undeclared species are needed. Targeting mitochondrial DNA, e.g. CYTB gene, for species quantification is unsuitable, due to a fivefold inter-tissue variation in mtDNA content per cell resulting in either an under- (-70%) or overestimation (+160%) of species DNA contents. Here, we describe a reliable two-step droplet digital PCR (ddPCR) assay targeting the nuclear F2 gene for precise quantification of cattle, horse, and pig in processed meat products. The ddPCR assay is advantageous over qPCR showing a limit of quantification (LOQ) and detection (LOD) in different meat products of 0.01% and 0.001%, respectively. The specificity was verified in 14 different species. Hence, determining F2 in food by ddPCR can be recommended for quality assurance and control in production systems. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  11. Evaluation of PCR and PCR-RFLP protocols for identifying Shiga toxins.

    Science.gov (United States)

    Ziebell, Kim A; Read, Susan C; Johnson, Roger P; Gyles, Carlton L

    2002-06-01

    This study evaluated two generic polymerase chain reaction (PCR) protocols, and nine subtyping protocols and three PCR-restriction fragment length polymorphism (RFLP) protocols for detection of stx genes. The PCR protocols were evaluated by testing 12 reference isolates and 496 field strains of Shiga toxin-producing Escherichia coli (STEC). Both generic methods detected all stx genes. In tests with the reference isolates, all methods detected stx1 and stx2, seven subtyping methods detected stx2v(EH250), seven detected stx2e and only two detected stx2f. Four of the subtyping protocols identified stx genes in all of the field isolates. The PCR-RFLP protocols gave contradictory results for approximately 20% of the strains tested. The observed limitations of the protocols were shown to be due to nucleotide sequence variation in the region of the PCR primers. One subtyping protocol that detected the virulence-related genes, eae and ehxA, and all stx except for the stx2f gene, was modified by newly designed primers so that it identified all stx genes. This modified protocol provides comprehensive characterization of STEC in a single multiplex reaction.

  12. Viral diagnostics in the era of digital PCR

    Science.gov (United States)

    Sedlak, Ruth Hall; Jerome, Keith R.

    2012-01-01

    Unlike quantitative PCR (qPCR), digital PCR (dPCR) achieves sensitive and accurate absolute quantitation of a DNA sample without the need for a standard curve. A single PCR reaction is divided into many separate reactions that each have a positive or negative signal. By applying Poisson statistics, the number of DNA molecules in the original sample is directly calculated from the number of positive and negative reactions. The recent availability of multiple commercial dPCR platforms has led to increased interest in clinical diagnostic applications, such as low viral load detection and low abundance mutant detection, where dPCR could be superior to traditional qPCR.Here we review current literature that demonstrates dPCR’s potential utility in viral diagnostics, particularly through absolute quantification of target DNA sequences and rare mutant allele detection. PMID:23182074

  13. Modification of Cre Gene by PCR Method

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Cre/LoxP site-specified recombination system is mainly used for excision,inversion and integration of target gene.Therefore,this system can be used for plant marker free genetic transformation,site-specific transgene expression and so on.However,the application of this system was limited due to its low expression and excision efficiency.In this study,an intron,which can enhance gene expression in plants,was inserted into Cre by using PCR method.And a modified Cre gene,named Crein,was obtained.This gene was ...

  14. Analysis of extracellular RNA by digital PCR

    Directory of Open Access Journals (Sweden)

    Kenji eTakahashi

    2014-06-01

    Full Text Available The transfer of extracellular RNA is emerging as an important mechanism for intracellular communication. The ability for the transfer of functionally active RNA molecules from one cell to another within vesicles such as exosomes enables a cell to modulate cellular signaling and biological processes within recipient cells. The study of extracellular RNA requires sensitive methods for the detection of these molecules. In this methods article, we will describe protocols for the detection of such extracellular RNA using sensitive detection technologies such as digital PCR. These protocols should be valuable to researchers interested in the role and contribution of extracellular RNA to tumor cell biology.

  15. Real-Time PCR for Universal Phytoplasma Detection and Quantification

    DEFF Research Database (Denmark)

    Christensen, Nynne Meyn; Nyskjold, Henriette; Nicolaisen, Mogens

    2013-01-01

    Currently, the most efficient detection and precise quantification of phytoplasmas is by real-time PCR. Compared to nested PCR, this method is less sensitive to contamination and is less work intensive. Therefore, a universal real-time PCR method will be valuable in screening programs and in other...

  16. Evaluation of Aspergillus PCR Protocols for Testing Serum Specimens

    NARCIS (Netherlands)

    White, P.L.; Mengoli, C.; Bretagne, S.; Cuenca-Estrella, M.; Finnstrom, N.; Klingspor, L.; Melchers, W.J.G.; McCulloch, E.; Barnes, R.A.; Donnelly, J.P.; Loeffler, J.

    2011-01-01

    A panel of human serum samples spiked with various amounts of Aspergillus fumigatus genomic DNA was distributed to 23 centers within the European Aspergillus PCR Initiative to determine analytical performance of PCR. Information regarding specific methodological components and PCR performance was

  17. Multiplex polymerase chain reaction (PCR) on a SU-8 chip

    DEFF Research Database (Denmark)

    Christensen, Troels Balmer; Bang, Dang Duong; Wolff, Anders

    2008-01-01

    We present the detection of Campylobacter at species level using multiplex PCR in a micro fabricated PCR chip. The chip is based on the polymer SU-8 that allows integration with different microfluidic components, e.g., sample pre-treatment before PCR, and DNA detection simultaneously with or afte...

  18. Determining Fungi rRNA Copy Number by PCR

    Science.gov (United States)

    The goal of this project is to improve the quantification of indoor fungal pollutants via the specific application of quantitative PCR (qPCR). Improvement will be made in the controls used in current qPCR applications. This work focuses on the use of two separate controls within ...

  19. Evaluation of Aspergillus PCR Protocols for Testing Serum Specimens

    NARCIS (Netherlands)

    White, P.L.; Mengoli, C.; Bretagne, S.; Cuenca-Estrella, M.; Finnstrom, N.; Klingspor, L.; Melchers, W.J.G.; McCulloch, E.; Barnes, R.A.; Donnelly, J.P.; Loeffler, J.

    2011-01-01

    A panel of human serum samples spiked with various amounts of Aspergillus fumigatus genomic DNA was distributed to 23 centers within the European Aspergillus PCR Initiative to determine analytical performance of PCR. Information regarding specific methodological components and PCR performance was re

  20. Determining Fungi rRNA Copy Number by PCR

    Science.gov (United States)

    The goal of this project is to improve the quantification of indoor fungal pollutants via the specific application of quantitative PCR (qPCR). Improvement will be made in the controls used in current qPCR applications. This work focuses on the use of two separate controls within ...

  1. An efficient multistrategy DNA decontamination procedure of PCR reagents for hypersensitive PCR applications.

    Directory of Open Access Journals (Sweden)

    Sophie Champlot

    Full Text Available BACKGROUND: PCR amplification of minute quantities of degraded DNA for ancient DNA research, forensic analyses, wildlife studies and ultrasensitive diagnostics is often hampered by contamination problems. The extent of these problems is inversely related to DNA concentration and target fragment size and concern (i sample contamination, (ii laboratory surface contamination, (iii carry-over contamination, and (iv contamination of reagents. METHODOLOGY/PRINCIPAL FINDINGS: Here we performed a quantitative evaluation of current decontamination methods for these last three sources of contamination, and developed a new procedure to eliminate contaminating DNA contained in PCR reagents. We observed that most current decontamination methods are either not efficient enough to degrade short contaminating DNA molecules, rendered inefficient by the reagents themselves, or interfere with the PCR when used at doses high enough to eliminate these molecules. We also show that efficient reagent decontamination can be achieved by using a combination of treatments adapted to different reagent categories. Our procedure involves γ- and UV-irradiation and treatment with a mutant recombinant heat-labile double-strand specific DNase from the Antarctic shrimp Pandalus borealis. Optimal performance of these treatments is achieved in narrow experimental conditions that have been precisely analyzed and defined herein. CONCLUSIONS/SIGNIFICANCE: There is not a single decontamination method valid for all possible contamination sources occurring in PCR reagents and in the molecular biology laboratory and most common decontamination methods are not efficient enough to decontaminate short DNA fragments of low concentration. We developed a versatile multistrategy decontamination procedure for PCR reagents. We demonstrate that this procedure allows efficient reagent decontamination while preserving the efficiency of PCR amplification of minute quantities of DNA.

  2. Comparison of a Commercially Available Repetitive-Element PCR System (DiversiLab) with PCR Ribotyping for Typing of Clostridium difficile Strains ▿

    OpenAIRE

    Eckert, C; Van Broeck, J; Spigaglia, P.; Burghoffer, B.; Delmée, M; Mastrantonio, P; Barbut, F

    2011-01-01

    This study compared a repetitive-element PCR (rep-PCR) method (DiversiLab system) to PCR ribotyping. The discriminatory power of rep-PCR was 0.997. Among the PCR ribotype 027 isolates tested, different rep types could be distinguished. rep-PCR showed a higher discriminatory power than PCR ribotyping. Nevertheless, this method requires technical skill, and visual interpretation of rep-PCR fingerprint patterns may be difficult.

  3. Comparison of a Commercially Available Repetitive-Element PCR System (DiversiLab) with PCR Ribotyping for Typing of Clostridium difficile Strains ▿

    OpenAIRE

    Eckert, C.; Van Broeck, J.; Spigaglia, P.; Burghoffer, B; Delmée, M; Mastrantonio, P; Barbut, F

    2011-01-01

    This study compared a repetitive-element PCR (rep-PCR) method (DiversiLab system) to PCR ribotyping. The discriminatory power of rep-PCR was 0.997. Among the PCR ribotype 027 isolates tested, different rep types could be distinguished. rep-PCR showed a higher discriminatory power than PCR ribotyping. Nevertheless, this method requires technical skill, and visual interpretation of rep-PCR fingerprint patterns may be difficult.

  4. Identification of periodontopathogen microorganisms by PCR technique

    Directory of Open Access Journals (Sweden)

    Milićević Radovan

    2008-01-01

    Full Text Available INTRODUCTION Periodontitis is an inflammatory disease of the supporting tissues of teeth and is a major cause of tooth loss in adults. The onset and progression of periodontal disease is attributed to the presence of elevated levels of a consortium of pathogenic bacteria. Gram negative bacteria, mainly strict anaerobes, play the major role. OBJECTIVE The present study aimed to assess the presence of the main types of microorganisms involved in the aetiopathogenesis of periodontal disease: Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Eikenella corrodens, Treponema denticola, Tanerella forsythia and Prevotella intermedia in different samples collected from the oral cavity of 90 patients diagnosed with periodontitis. METHOD Bacterial DNA detection was performed in diverse biological materials, namely in dental plaque, gingival tissue and saliva, by means of multiplex PCR, a technique that allows simultaneous identification of two different bacterial genomes. RESULTS In the dental plaque of the periodontitis patients, Treponema denticola dominated. In the gingival tissue, Tannerella forsythia and Treponema denticola were the microbiota most frequently detected, whilst in saliva Treponema denticola and Eikenella corrodens were found with the highest percentage. CONCLUSION The identification of microorganisms by multiplex PCR is specific and sensitive. Rapid and precise assessment of different types of periodontopathogens is extremely important for early detection of the infection and consequently for the prevention and treatment of periodontal disease. In everyday clinical practice, for routine bacterial evaluation in patients with periodontal disease, the dental plaque is the most suitable biological material, because it is the richest in periodontal bacteria.

  5. In silico PCR primer designing and validation.

    Science.gov (United States)

    Kumar, Anil; Chordia, Nikita

    2015-01-01

    Polymerase chain reaction (PCR) is an enzymatic reaction whose efficiency and sensitivity largely depend on the efficiency of the primers that are used for the amplification of a concerned gene/DNA fragment. Selective amplification of nucleic acid molecules initially present in minute quantities provides a powerful tool for analyzing nucleic acids. In silico method helps in designing primers. There are various programs available for PCR primer design. Here we described designing of primers using web-based tools like "Primer3" and "Web Primer". For designing the primer, DNA template sequence is required that can be taken from any of the available sequence databases, e.g., RefSeq database. The in silico validation can be carried out using BLAST tool and Gene Runner software, which check their efficiency and specificity. Thereafter, the primers designed in silico can be validated in the wet lab. After that, these validated primers can be synthesized for use in the amplification of concerned gene/DNA fragment.

  6. A Guide to Using STITCHER for Overlapping Assembly PCR Applications.

    Science.gov (United States)

    O'Halloran, Damien M

    2017-01-01

    Overlapping PCR is commonly used in many molecular applications that include stitching PCR fragments together, generating fluorescent transcriptional and translational fusions, inserting mutations, making deletions, and PCR cloning. Overlapping PCR is also used for genotyping and in detection experiments using techniques such as loop-mediated isothermal amplification (LAMP). STITCHER is a web tool providing a central resource for researchers conducting all types of overlapping assembly PCR experiments with an intuitive interface for automated primer design that's fast, easy to use, and freely available online.

  7. cDNA amplification by SMART-PCR and suppression subtractive hybridization (SSH)-PCR.

    Science.gov (United States)

    Hillmann, Andrew; Dunne, Eimear; Kenny, Dermot

    2009-01-01

    The comparison of two RNA populations that differ from the effects of a single-independent variable, such as a drug treatment or a specific genetic defect, can identify differences in the abundance of specific transcripts that vary in a population-dependent manner. There are a variety of methods for identifying differentially expressed genes, including microarray, SAGE, qRT-PCR, and DDGE. This protocol describes a potentially less sensitive yet relatively easy and cost-effective alternative that does not require prior knowledge of the transcriptomes under investigation and is particularly applicable when minimal levels of starting material, RNA, are available. RNA input can often be a limiting factor when analyzing RNA from, for example, rigorously purified blood cells. This protocol describes the use of SMART-PCR to amplify cDNA from sub-microgram levels of RNA. The amplified cDNA populations under comparison are then subjected to suppression subtractive hybridization (SSH-PCR), a technique that couples subtractive hybridization with suppression PCR to selectively amplify fragments of differentially expressed genes. The final products are cDNA populations enriched for significantly over-represented transcripts in either of the two input RNA preparations. These cDNA populations may then be cloned to make subtracted cDNA libraries and/or used as probes to screen subtracted cDNA, global cDNA, or genomic DNA libraries.

  8. Simultaneous detection of ricin and abrin DNA by real-time PCR (qPCR).

    Science.gov (United States)

    Felder, Eva; Mossbrugger, Ilona; Lange, Mirko; Wölfel, Roman

    2012-09-01

    Ricin and abrin are two of the most potent plant toxins known and may be easily obtained in high yield from the seeds using rather simple technology. As a result, both toxins are potent and available toxins for criminal or terrorist acts. However, as the production of highly purified ricin or abrin requires sophisticated equipment and knowledge, it may be more likely that crude extracts would be used by non-governmental perpetrators. Remaining plant-specific nucleic acids in these extracts allow the application of a real-time PCR (qPCR) assay for the detection and identification of abrin or ricin genomic material. Therefore, we have developed a duplex real-time PCR assays for simultaneous detection of ricin and abrin DNA based on the OmniMix HS bead PCR reagent mixture. Novel primers and hybridization probes were designed for detection on a SmartCycler instrument by using 5'-nuclease technology. The assay was thoroughly optimized and validated in terms of analytical sensitivity. Evaluation of the assay sensitivity by probit analysis demonstrated a 95% probability of detection at 3 genomes per reaction for ricin DNA and 1.2 genomes per reaction for abrin DNA. The suitability of the assays was exemplified by detection of ricin and abrin contaminations in a food matrix.

  9. A survey of tools for the analysis of quantitative PCR (qPCR data

    Directory of Open Access Journals (Sweden)

    Stephan Pabinger

    2014-09-01

    Our comprehensive survey showed that most tools use their own file format and only a fraction of the currently existing tools support the standardized data exchange format RDML. To allow a more streamlined and comparable analysis of qPCR data, more vendors and tools need to adapt the standardized format to encourage the exchange of data between instrument software, analysis tools, and researchers.

  10. Simultaneous Detection of Ricin and Abrin DNA by Real-Time PCR (qPCR

    Directory of Open Access Journals (Sweden)

    Roman Wölfel

    2012-08-01

    Full Text Available Ricin and abrin are two of the most potent plant toxins known and may be easily obtained in high yield from the seeds using rather simple technology. As a result, both toxins are potent and available toxins for criminal or terrorist acts. However, as the production of highly purified ricin or abrin requires sophisticated equipment and knowledge, it may be more likely that crude extracts would be used by non-governmental perpetrators. Remaining plant-specific nucleic acids in these extracts allow the application of a real-time PCR (qPCR assay for the detection and identification of abrin or ricin genomic material. Therefore, we have developed a duplex real-time PCR assays for simultaneous detection of ricin and abrin DNA based on the OmniMix HS bead PCR reagent mixture. Novel primers and hybridization probes were designed for detection on a SmartCycler instrument by using 5′-nuclease technology. The assay was thoroughly optimized and validated in terms of analytical sensitivity. Evaluation of the assay sensitivity by probit analysis demonstrated a 95% probability of detection at 3 genomes per reaction for ricin DNA and 1.2 genomes per reaction for abrin DNA. The suitability of the assays was exemplified by detection of ricin and abrin contaminations in a food matrix.

  11. DNA polymerase preference determines PCR priming efficiency

    Science.gov (United States)

    2014-01-01

    Background Polymerase chain reaction (PCR) is one of the most important developments in modern biotechnology. However, PCR is known to introduce biases, especially during multiplex reactions. Recent studies have implicated the DNA polymerase as the primary source of bias, particularly initiation of polymerization on the template strand. In our study, amplification from a synthetic library containing a 12 nucleotide random portion was used to provide an in-depth characterization of DNA polymerase priming bias. The synthetic library was amplified with three commercially available DNA polymerases using an anchored primer with a random 3’ hexamer end. After normalization, the next generation sequencing (NGS) results of the amplified libraries were directly compared to the unamplified synthetic library. Results Here, high throughput sequencing was used to systematically demonstrate and characterize DNA polymerase priming bias. We demonstrate that certain sequence motifs are preferred over others as primers where the six nucleotide sequences at the 3’ end of the primer, as well as the sequences four base pairs downstream of the priming site, may influence priming efficiencies. DNA polymerases in the same family from two different commercial vendors prefer similar motifs, while another commercially available enzyme from a different DNA polymerase family prefers different motifs. Furthermore, the preferred priming motifs are GC-rich. The DNA polymerase preference for certain sequence motifs was verified by amplification from single-primer templates. We incorporated the observed DNA polymerase preference into a primer-design program that guides the placement of the primer to an optimal location on the template. Conclusions DNA polymerase priming bias was characterized using a synthetic library amplification system and NGS. The characterization of DNA polymerase priming bias was then utilized to guide the primer-design process and demonstrate varying amplification

  12. A RAPID PCR-QUALITY DNA EXTRACTION METHOD IN FISH

    Institute of Scientific and Technical Information of China (English)

    LI Zhong; LIANG Hong-Wei; ZOU Gui-Wei

    2012-01-01

    PCR has been a general preferred method for biological research in fish, and previous research have enabled us to extract and purify PCR-quality DNA templates in laboratories[1-4]. The same problem among these procedures is waiting for tissue digesting for a long time. The overabundance time spent on PCR-quality DNA extraction restricts the efficiency of PCR assay, especially in large-scale PCR amplification, such as SSR-based genetic-mapping construction [5,6], identification of germ plasm resource[7,8] and evolution research [9,10], etc. In this study, a stable and rapid PCR-quality DNA extraction method was explored, using a modified alkaline lysis protocol. Extracting DNA for PCR only takes approximately 25 minutes. This stable and rapid DNA extraction method could save much laboratory time and promotes.%PCR has been a general preferred method for biological research in fish,and previous research have enabled us to extract and purify PCR-quality DNA templates in laboratories [1-4].The same problem among these procedures is waiting for tissue digesting for a long time.The overabundance time spent on PCR-quality DNA extraction restricts the efficiency of PCR assay,especially in large-scale PCR amplification,such as SSR-based genetic-mapping construction [5,6],identification of germ plasm resource[7,8] and evolution research [9,10],etc.In this study,a stable and rapid PCR-quality DNA extraction method was explored,using a modified alkaline lysis protocol.Extracting DNA for PCR only takes approximately 25 minutes.This stable and rapid DNA extraction method could save much laboratory time and promotes.

  13. Comparison of droplet digital PCR to real-time PCR for quantification of hepatitis B virus DNA.

    Science.gov (United States)

    Tang, Hui; Cai, Qingchun; Li, Hu; Hu, Peng

    2016-06-16

    Quantitative real-time PCR (qPCR) has been widely implemented for clinical hepatitis B viral load testing, but a lack of standardization and relatively poor precision hinder its usefulness. Droplet digital PCR (ddPCR) is a promising tool that offers high precision and direct quantification. In this study, we compared the ddPCR QX100 platform by Bio-Rad with the CFX384 Touch Real-Time PCR Detection System (Bio-Rad, USA) to detect serial plasmid DNA dilutions of known concentrations as well as HBV DNA extracted from patient serum samples. Both methods showed a high degree of linearity and quantitative correlation. However, ddPCR assays generated more reproducible results and detected lower copy numbers than qPCR assays. Patient sample quantifications by ddPCR and qPCR were highly agreeable based on the Bland-Altman analysis. Collectively, our findings demonstrate that ddPCR offers improved analytical sensitivity and specificity for HBV measurements and is suitable for clinical HBV detection.

  14. The dissemination of CMV in urine of different group from Guangxi and the relationship between CMV infection and renal disease%人巨细胞病毒在广西不同人群中的感染情况及其与肾脏疾病关系的初步研究

    Institute of Scientific and Technical Information of China (English)

    陈智平; 覃锦耀; 王健; 阳晶; 阳艳华; 蔡豪斌; 何雨; 李山; 王丽兰; 劳小霞; 李泰阶; 胡春卉; 邓燕

    2010-01-01

    目的 调查广西壮族自治区孕妇、患病新生儿、肾病患者、肾移植患者及正常体检者尿中人巨细胞病毒阳性率,了解人类巨细胞病毒在广西不同人群中的分布情况,初步探讨人巨细胞病毒感染与肾脏疾病的关系.方法 用荧光定量PCR检测研究对象晨尿中人巨细胞病毒DNA.结果 孕妇、患病新生儿、肾病者、肾移植患者及正常体检者尿中人巨细胞病毒DNA阳性率依次为8.18%、3.45%、18.54%、25.42%、0.56%,其在各类肾病患者中的感染率均在10%左右,尤其在狼疮肾炎患者中的感染率更是达到27.78%.结论 人巨细胞病毒在黄疸和早产新生儿中的感染率均较高,需要加强母婴人巨细胞病毒感染的防控工作.人巨细胞病毒很可能在肾病患者发病时对患者肾脏造成损害,并且加重肾病患者的病情,成为肾衰的一个诱因.%Objective To detect Human Cytomegalovirus (HCMV) DNA in urine samples from the following groups:pregnant women,sick newborns,hospitalized nephropathy patients,renal transplant recipients and normal population.Preliminarily study the relationship of HCMVinfection and renal disease.Methods To detect HCMV DNA in morning urine samples by Real-time fluorescence quantitative PCR (FQ-PCR).Results The positive rates of HCMV DNA in the urine of pregnant women,sick newborns,hospitalized nephropathy patients,renal transplant recipients and normal population are 8.1 8%,3.45%,18.54%,25.42%,0.56%.Conclusion The infection rates of HCMV in the urine of pregnant women and sick newborns are very high in Guangxi,it should take serious measures to prevent and control the situation.HCMV probably participate in the iniury of kidney,and worsen the disease.It should be one of the causes of renal disease.

  15. Recovery of DNA of Giardia intestinalis cysts from surface water concentrates measured with PCR and real time PCR

    Directory of Open Access Journals (Sweden)

    Adamska M.

    2011-11-01

    Full Text Available The most important restriction for the detection in water samples is the low concentration of Giardia intestinalis cysts, additional difficulty is the presence of PCR inhibitors. We have carried out trials in order to assess the sensitivity of semi-nested PCR and TaqMan real time PCR on the basis of DNA extracted from G. intestinalis cysts coming from spiked environmental and distilled water samples, filtrated with the use of Filta-Max® equipment (1623 Method. Removal of inhibitors was carried out with addition of BSA in different concentrations. During the filtration and concentration of water samples, losses of cysts have been recorded. Moreover, addition of BSA to the PCR and real time PCR mix increases the sensitivity of reaction. The optimal concentration of BSA for semi-nested PCR was 15 and 20 ng/μl, whereas for real time PCR 5 ng/μl.

  16. Calibrated user-friendly reverse transcriptase-PCR assay

    DEFF Research Database (Denmark)

    Bor, M V; Sørensen, B S; Rammer, P

    1998-01-01

    We report a competitive reverse transcriptase-PCR (RT-PCR) assay and a calibrated user-friendly RT-PCR assay (CURT-PCR) for epidermal growth factor receptor (EGFR) mRNA. A calibrator was prepared from isolated rat liver RNA, and the amount of EGFR mRNA was determined by competitive RT-PCR. In CUR...... and to accept or reject the results according to existing rules for quality assurance....... and the corresponding RNA internal standard. Competitive RT-PCR and CURT-PCR were used for rat liver samples from 21 different animals. Comparable results were obtained by the two methods. The imprecision of the CURT-PCR method was 8% (n = 20), and the imprecision of the traditional competitive RT-PCR was 16% (n = 17......). We conclude that the CURT-PCR method developed is suitable for routine applications such as quantitation of EGFR expression in tumor biopsies. The imprecision is relatively low. Furthermore, the use of a calibration curve makes it possible to analyze a large number of samples in one analytical run...

  17. False negative results from using common PCR reagents

    Directory of Open Access Journals (Sweden)

    Atwood Allison A

    2011-10-01

    Full Text Available Abstract Background The sensitivity of the PCR reaction makes it ideal for use when identifying potentially novel viral infections in human disease. Unfortunately, this same sensitivity also leaves this popular technique open to potential contamination with previously amplified PCR products, or "carry-over" contamination. PCR product carry-over contamination can be prevented with uracil-DNA-glycosylase (UNG, and it is for this reason that it is commonly included in many commercial PCR master-mixes. While testing the sensitivity of PCR assays to detect murine DNA contamination in human tissue samples, we inadvertently discovered that the use of this common PCR reagent may lead to the production of false-negative PCR results. Findings We show here that contamination with minute quantities of UNG-digested PCR product or any negative control PCR reactions containing primer-dimers regardless of UNG presence can completely block amplification from as much as 60 ng of legitimate target DNA. Conclusions These findings could potentially explain discrepant results from laboratories attempting to amplify MLV-related viruses including XMRV from human samples, as none of the published reports used internal-tube controls for amplification. The potential for false negative results needs to be considered and carefully controlled in PCR experiments, especially when the target copy number may be low - just as the potential for false positive results already is.

  18. Detection of Human Papillomavirus DNA in Cervical Samples: Analysis of the New PGMY-PCR Compared To the Hybrid Capture II and MY-PCR Assays and a Two-Step Nested PCR Assay

    OpenAIRE

    Giovannelli, Lucia; Lama, Anna; Capra, Giuseppina; Giordano, Viviana; Aricò, Pietro; Ammatuna, Pietro

    2004-01-01

    The PGMY-PCR for human papillomavirus (HPV) was evaluated, in parallel with nested PCR (nPCR), in samples with noted Hybrid Capture II (HCII) and MY-PCR results. PGMY-PCR detected HPV DNA in 2.5% of HCII-negative-MY-PCR-negative samples and in 71.7% of HCII-positive-MY-PCR-negative samples; also, it detected the MY-PCR-negative-nPCR-negative types HPV-42, HPV-44, HPV-51, HPV-87, and HPV-89.

  19. IDH1 mutation detection by droplet digital PCR in glioma.

    Science.gov (United States)

    Wang, Jing; Zhao, Yi-ying; Li, Jian-feng; Guo, Cheng-cheng; Chen, Fu-rong; Su, Hong-kai; Zhao, Hua-fu; Long, Ya-kang; Shao, Jian-yong; To, Shing shun Tony; Chen, Zhong-ping

    2015-11-24

    Glioma is the most frequent central nervous system tumor in adults. The overall survival of glioma patients is disappointing, mostly due to the poor prognosis of glioblastoma (Grade IV glioma). Isocitrate dehydrogenase (IDH) is a key factor in metabolism and catalyzes the oxidative decarboxylation of isocitrate. Mutations in IDH genes are observed in over 70% of low-grade gliomas and some cases of glioblastoma. As the most frequent mutation, IDH1(R132H) has been served as a predictive marker of glioma patients. The recently developed droplet digital PCR (ddPCR) technique generates a large amount of nanoliter-sized droplets, each of which carries out a PCR reaction on one template. Therefore, ddPCR provides high precision and absolute quantification of the nucleic acid target, with wide applications for both research and clinical diagnosis. In the current study, we collected 62 glioma tissue samples (Grade II to IV) and detected IDH1 mutations by Sanger direct sequencing, ddPCR, and quantitative real-time PCR (qRT-PCR). With the results from Sanger direct sequencing as the standard, the characteristics of ddPCR were compared with qRT-PCR. The data indicated that ddPCR was much more sensitive and much easier to interpret than qRT-PCR. Thus, we demonstrated that ddPCR is a reliable and sensitive method for screening the IDH mutation. Therefore, ddPCR is able to applied clinically in predicting patient prognosis and selecting effective therapeutic strategies. Our data also supported that the prognosis of Grade II and III glioma was better in patients with an IDH mutation than in those without mutation.

  20. Comparison of a commercially available rep-PCR system (Diversilab(R)) with PCR-ribotyping for typing of Clostridium difficile strains.

    OpenAIRE

    Eckert, C.; van Broeck, Johan; Spigaglia, P.; Burghoffer, B; Delmée, Michel; Mastrantonio, P; Barbut, F

    2011-01-01

    This study compared a rep-PCR method (DiversiLab® system) to PCR-ribotyping. The discriminatory power of rep-PCR was 0.997. Among the PCR-ribotype 027, different rep-types could be distinguished. Rep-PCR showed a higher discriminatory power than PCR-ribotyping. Nevertheless, this method requires technical skill and visual interpretation of rep-PCR fingerprint patterns may be difficult.

  1. COMPARISON OF 16S rRNA-PCR-RFLP, LipL32-PCR AND OmpL1-PCR METHODS IN THE DIAGNOSIS OF LEPTOSPIROSIS

    Directory of Open Access Journals (Sweden)

    Tülin GÜVEN GÖKMEN

    Full Text Available SUMMARY Leptospirosis is still one of the most important health problems in developing countries located in humid tropical and subtropical regions. Human infections are generally caused by exposure to water, soil or food contaminated with the urine of infected wild and domestic animals such as rodents and dogs. The clinical course of leptospirosis is variable and may be difficult to distinguish from many other infectious diseases. The dark-field microscopy (DFM, serology and nucleic acid amplification techniques are used to diagnose leptospirosis, however, a distinctive standard reference method is still lacking. Therefore, in this study, we aimed to determine the presence of Leptospira spp., to differentiate the pathogenic L. interrogans and the non-pathogenic L. biflexa, and also to determine the sensitivity and specificity values of molecular methods as an alternative to conventional ones. A total of 133 serum samples, from 47 humans and 86 cattle were evaluated by two conventional tests: the Microagglutination Test (MAT and the DFM, as well as three molecular methods, the 16S rRNA-PCR followed by Restriction Fragment Lenght Polymorphism (RFLP of the amplification products 16S rRNA-PCR-RFLP, LipL32-PCR and OmpL1-PCR. In this study, for L. interrogans, the specificity and sensitivity rates of the 16S rRNA-PCR and the LipL32-PCR were considered similar (100% versus 98.25% and 100% versus 98.68%, respectively. The OmpL1-PCR was able to classify L. interrogans into two intergroups, but this PCR was less sensitive (87.01% than the other two PCR methods. The 16S rRNA-PCR-RFLP could detect L. biflexa DNA, but LipL32-PCR and OmpL1-PCR could not. The 16S rRNA-PCR-RFLP provided an early and accurate diagnosis and was able to distinguish pathogenic and non-pathogenic Leptospira species, hence it may be used as an alternative method to the conventional gold standard techniques for the rapid disgnosis of leptospirosis.

  2. COMPARISON OF 16S rRNA-PCR-RFLP, LipL32-PCR AND OmpL1-PCR METHODS IN THE DIAGNOSIS OF LEPTOSPIROSIS

    Science.gov (United States)

    GÖKMEN, Tülin GÜVEN; SOYAL, Ayben; KALAYCI, Yıldız; ÖNLEN, Cansu; KÖKSAL, Fatih

    2016-01-01

    SUMMARY Leptospirosis is still one of the most important health problems in developing countries located in humid tropical and subtropical regions. Human infections are generally caused by exposure to water, soil or food contaminated with the urine of infected wild and domestic animals such as rodents and dogs. The clinical course of leptospirosis is variable and may be difficult to distinguish from many other infectious diseases. The dark-field microscopy (DFM), serology and nucleic acid amplification techniques are used to diagnose leptospirosis, however, a distinctive standard reference method is still lacking. Therefore, in this study, we aimed to determine the presence of Leptospira spp., to differentiate the pathogenic L. interrogans and the non-pathogenic L. biflexa, and also to determine the sensitivity and specificity values of molecular methods as an alternative to conventional ones. A total of 133 serum samples, from 47 humans and 86 cattle were evaluated by two conventional tests: the Microagglutination Test (MAT) and the DFM, as well as three molecular methods, the 16S rRNA-PCR followed by Restriction Fragment Lenght Polymorphism (RFLP) of the amplification products 16S rRNA-PCR-RFLP, LipL32-PCR and OmpL1-PCR. In this study, for L. interrogans, the specificity and sensitivity rates of the 16S rRNA-PCR and the LipL32-PCR were considered similar (100% versus 98.25% and 100% versus 98.68%, respectively). The OmpL1-PCR was able to classify L. interrogans into two intergroups, but this PCR was less sensitive (87.01%) than the other two PCR methods. The 16S rRNA-PCR-RFLP could detect L. biflexa DNA, but LipL32-PCR and OmpL1-PCR could not. The 16S rRNA-PCR-RFLP provided an early and accurate diagnosis and was able to distinguish pathogenic and non-pathogenic Leptospira species, hence it may be used as an alternative method to the conventional gold standard techniques for the rapid disgnosis of leptospirosis. PMID:27680169

  3. Multiplex enrichment quantitative PCR (ME-qPCR): a high-throughput, highly sensitive detection method for GMO identification.

    Science.gov (United States)

    Fu, Wei; Zhu, Pengyu; Wei, Shuang; Zhixin, Du; Wang, Chenguang; Wu, Xiyang; Li, Feiwu; Zhu, Shuifang

    2017-04-01

    Among all of the high-throughput detection methods, PCR-based methodologies are regarded as the most cost-efficient and feasible methodologies compared with the next-generation sequencing or ChIP-based methods. However, the PCR-based methods can only achieve multiplex detection up to 15-plex due to limitations imposed by the multiplex primer interactions. The detection throughput cannot meet the demands of high-throughput detection, such as SNP or gene expression analysis. Therefore, in our study, we have developed a new high-throughput PCR-based detection method, multiplex enrichment quantitative PCR (ME-qPCR), which is a combination of qPCR and nested PCR. The GMO content detection results in our study showed that ME-qPCR could achieve high-throughput detection up to 26-plex. Compared to the original qPCR, the Ct values of ME-qPCR were lower for the same group, which showed that ME-qPCR sensitivity is higher than the original qPCR. The absolute limit of detection for ME-qPCR could achieve levels as low as a single copy of the plant genome. Moreover, the specificity results showed that no cross-amplification occurred for irrelevant GMO events. After evaluation of all of the parameters, a practical evaluation was performed with different foods. The more stable amplification results, compared to qPCR, showed that ME-qPCR was suitable for GMO detection in foods. In conclusion, ME-qPCR achieved sensitive, high-throughput GMO detection in complex substrates, such as crops or food samples. In the future, ME-qPCR-based GMO content identification may positively impact SNP analysis or multiplex gene expression of food or agricultural samples. Graphical abstract For the first-step amplification, four primers (A, B, C, and D) have been added into the reaction volume. In this manner, four kinds of amplicons have been generated. All of these four amplicons could be regarded as the target of second-step PCR. For the second-step amplification, three parallels have been taken for

  4. Blood grouping based on PCR methods and agarose gel electrophoresis.

    Science.gov (United States)

    Sell, Ana Maria; Visentainer, Jeane Eliete Laguila

    2015-01-01

    The study of erythrocyte antigens continues to be an intense field of research, particularly after the development of molecular testing methods. More than 300 specificities have been described by the International Society for Blood Transfusion as belonging to 33 blood group systems. The polymerase chain reaction (PCR) is a central tool for red blood cells (RBC) genotyping. PCR and agarose gel electrophoresis are low cost, easy, and versatile in vitro methods for amplifying defined target DNA (RBC polymorphic region). Multiplex-PCR, AS-PCR (Specific Allele Polymerase Chain Reaction), and RFLP-PCR (Restriction Fragment Length Polymorphism-Polymerase Chain Reaction) techniques are usually to identify RBC polymorphisms. Furthermore, it is an easy methodology to implement. This chapter describes the PCR methodology and agarose gel electrophoresis to identify the polymorphisms of the Kell, Duffy, Kidd, and MNS blood group systems.

  5. Microfluidics-Based PCR for Fusion Transcript Detection.

    Science.gov (United States)

    Chen, Hui

    2016-01-01

    The microfluidic technology allows the production of network of submillimeter-size fluidic channels and reservoirs in a variety of material systems. The microfluidic-based polymerase chain reaction (PCR) allows automated multiplexing of multiple samples and multiple assays simultaneously within a network of microfluidic channels and chambers that are co-ordinated in controlled fashion by the valves. The individual PCR reaction is performed in nanoliter volume, which allows testing on samples with limited DNA and RNA. The microfluidics devices are used in various types of PCR such as digital PCR and single molecular emulsion PCR for genotyping, gene expression, and miRNA expression. In this chapter, the use of a microfluidics-based PCR for simultaneous screening of 14 known fusion transcripts in patients with leukemia is described.

  6. Optimized MOL-PCR for Characterization of Microbial Pathogens.

    Science.gov (United States)

    Wuyts, Véronique; Roosens, Nancy H C; Bertrand, Sophie; Marchal, Kathleen; De Keersmaecker, Sigrid C J

    2016-01-06

    Characterization of microbial pathogens is necessary for surveillance, outbreak detection, and tracing of outbreak sources. This unit describes a multiplex oligonucleotide ligation-PCR (MOL-PCR) optimized for characterization of microbial pathogens. With MOL-PCR, different types of markers, like unique sequences, single-nucleotide polymorphisms (SNPs) and indels, can be simultaneously analyzed in one assay. This assay consists of a multiplex ligation for detection of the markers, a singleplex PCR for signal amplification, and hybridization to MagPlex-TAG beads for readout on a Luminex platform after fluorescent staining. The current protocol describes the MOL-PCR, as well as methods for DNA isolation, probe design, and data interpretation and it is based on an optimized MOL-PCR assay for subtyping of Salmonella Typhimurium.

  7. Standardization of diagnostic PCR for the detection of foodborne pathogens

    DEFF Research Database (Denmark)

    Malorny, B.; Tassios, P.T.; Radstrom, P.

    2003-01-01

    In vitro amplification of nucleic acids using the polymerase chain reaction (PCR) has become, since its discovery in the 1980s, a powerful diagnostic tool for the analysis of microbial infections as well as for the analysis of microorganisms in food samples. However, despite its potential, PCR has...... neither gained wide acceptance in routine diagnostics nor been widely incorporated in standardized methods. Lack of validation and standard protocols, as well as variable quality of reagents and equipment, influence the efficient dissemination of PCR methodology from expert research laboratories to end......-user laboratories. Moreover, the food industry understandably requires and expects officially approved standards. Recognizing this, in 1999, the European Commission approved the research project, FOOD-PCR (http://www.PCR.dk), which aims to validate and standardize the use of diagnostic PCR for the detection...

  8. Template-blocking PCR: an advanced PCR technique for genome walking.

    Science.gov (United States)

    Bae, Jung-Hoon; Sohn, Jung-Hoon

    2010-03-01

    This article describes the development of an improved method for the isolation of genomic fragments adjacent to a known DNA sequence based on a cassette ligation-mediated polymerase chain reaction (PCR) technique. To reduce the nonspecific amplification of PCR-based genome walking, the 3' ends of the restriction enzyme-digested genomic DNA fragments were blocked with dideoxynucleoside triphosphate (ddNTP) and ligated with properly designed cassettes. The modified genomic DNA fragments flanked with cassettes were used as a template for the amplification of a target gene with a gene-specific primer (GSP) and a cassette primer (CP). The ddNTP blocking of the genomic DNA ends significantly reduced the nonspecific amplification and resulted in a simple and rapid walking along the genome. The efficiency of the template-blocking PCR method was confirmed by a carefully designed control experiment. The method was successfully applied for the cloning of the PGK1 promoter from Pichia ciferrii and two novel cellulase genes from Penicillium sp.

  9. Doubling Throughput of a Real-Time PCR

    OpenAIRE

    Christian D. Ahrberg; Pavel Neužil

    2015-01-01

    The invention of polymerase chain reaction (PCR) in 1983 revolutionized many areas of science, due to its ability to multiply a number of copies of DNA sequences (known as amplicons). Here we report on a method to double the throughput of quantitative PCR which could be especially useful for PCR-based mass screening. We concurrently amplified two target genes using only single fluorescent dye. A FAM probe labelled olionucleotide was attached to a quencher for one amplicon while the second one...

  10. 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.

  11. A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood.

    Science.gov (United States)

    Wiencke, John K; Bracci, Paige M; Hsuang, George; Zheng, Shichun; Hansen, Helen; Wrensch, Margaret R; Rice, Terri; Eliot, Melissa; Kelsey, Karl T

    2014-10-01

    Quantitating the copy number of demethylated CpG promoter sites of the CD3Z gene can be used to estimate the numbers and proportions of T cells in human blood and tissue. Quantitative methylation specific PCR (qPCR) is useful for studying T cells but requires extensive calibration and is imprecise at low copy numbers. Here we compared the performance of a new digital PCR platform (droplet digital PCR or ddPCR) to qPCR using bisulfite converted DNA from 157 blood specimens obtained from ambulatory care controls and patients with primary glioma. We compared both ddPCR and qPCR with conventional flow cytometry (FACS) evaluation of CD3 positive T cells. Repeated measures on the same blood sample revealed ddPCR to be less variable than qPCR. Both qPCR and ddPCR correlated significantly with FACS evaluation of peripheral blood CD3 counts and CD3/total leukocyte values. However, statistical measures of agreement showed that linear concordance was stronger for ddPCR than for qPCR and the absolute values were closer to FACS for ddPCR. Both qPCR and ddPCR could distinguish clinically significant differences in T cell proportions and performed similarly to FACS. Given the higher precision, greater accuracy, and technical simplicity of ddPCR, this approach appears to be a superior DNA methylation based method than conventional qPCR for the assessment of T cells.

  12. A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood

    Science.gov (United States)

    Wiencke, John K; Bracci, Paige M; Hsuang, George; Zheng, Shichun; Hansen, Helen; Wrensch, Margaret R; Rice, Terri; Eliot, Melissa; Kelsey, Karl T

    2014-01-01

    Quantitating the copy number of demethylated CpG promoter sites of the CD3Z gene can be used to estimate the numbers and proportions of T cells in human blood and tissue. Quantitative methylation specific PCR (qPCR) is useful for studying T cells but requires extensive calibration and is imprecise at low copy numbers. Here we compared the performance of a new digital PCR platform (droplet digital PCR or ddPCR) to qPCR using bisulfite converted DNA from 157 blood specimens obtained from ambulatory care controls and patients with primary glioma. We compared both ddPCR and qPCR with conventional flow cytometry (FACS) evaluation of CD3 positive T cells. Repeated measures on the same blood sample revealed ddPCR to be less variable than qPCR. Both qPCR and ddPCR correlated significantly with FACS evaluation of peripheral blood CD3 counts and CD3/total leukocyte values. However, statistical measures of agreement showed that linear concordance was stronger for ddPCR than for qPCR and the absolute values were closer to FACS for ddPCR. Both qPCR and ddPCR could distinguish clinically significant differences in T cell proportions and performed similarly to FACS. Given the higher precision, greater accuracy, and technical simplicity of ddPCR, this approach appears to be a superior DNA methylation based method than conventional qPCR for the assessment of T cells. PMID:25437051

  13. A thermodynamic approach to PCR primer design.

    Science.gov (United States)

    Mann, Tobias; Humbert, Richard; Dorschner, Michael; Stamatoyannopoulos, John; Noble, William Stafford

    2009-07-01

    We developed a primer design method, Pythia, in which state of the art DNA binding affinity computations are directly integrated into the primer design process. We use chemical reaction equilibrium analysis to integrate multiple binding energy calculations into a conservative measure of polymerase chain reaction (PCR) efficiency, and a precomputed index on genomic sequences to evaluate primer specificity. We show that Pythia can design primers with success rates comparable with those of current methods, but yields much higher coverage in difficult genomic regions. For example, in RepeatMasked sequences in the human genome, Pythia achieved a median coverage of 89% as compared with a median coverage of 51% for Primer3. For parameter settings yielding sensitivities of 81%, our method has a recall of 97%, compared with the Primer3 recall of 48%. Because our primer design approach is based on the chemistry of DNA interactions, it has fewer and more physically meaningful parameters than current methods, and is therefore easier to adjust to specific experimental requirements. Our software is freely available at http://pythia.sourceforge.net.

  14. Variation in copy number of the 28S rDNA of Aspergillus fumigatus measured by droplet digital PCR and analog quantitative real-time PCR.

    Science.gov (United States)

    Alanio, Alexandre; Sturny-Leclère, Aude; Benabou, Marion; Guigue, Nicolas; Bretagne, Stéphane

    2016-08-01

    Droplet digital PCR (ddPCR) after DNA digestion yielded a 28S rDNA copy number of 61 to 86 copies/genome when testing 10 unrelated Aspergillus fumigatus isolates, higher than with quantitative PCR. Unfortunately, ddPCR after DNA digestion did not improve the sensitivity of our PCR assay when testing serum patients with invasive aspergillosis.

  15. Comparison of kDNA PCR-hybridization assay with three PCR methods for canines visceral Leishmaniasis diagnosis

    Energy Technology Data Exchange (ETDEWEB)

    Pilatti, Marcia M.; Andrade, Antero S.R. [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil)], e-mail: marciapilatti@yahoo.com.br, e-mail: antero@cdtn.br; Ferreira, Sidney A. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Parasitologia], e-mail: saninoalmeida@gmail.com

    2009-07-01

    The sensitivity of the kDNA PCR-Hybridization assay, which uses radioactive DNA probes (labeled with {sup 32}P), was compared with three conventional PCR methods used for canine visceral leishmaniasis diagnosis. All PCR methods had two steps: a first amplification followed by hybridization or by a new amplification (nested or semi nested). Two methods (kDNA PCR-Hybridization and kDNA snPCR) used primers addressed to kinetoplast minicircles and the other two methods to the coding (LnPCR) and intergenic noncoding regions (ITS-1 nPCR) of the ribosomal rRNA genes. The comparison was accomplished in two groups of 23 infected dogs using samples collected by the conjunctival swab procedure. In the Group 1 the DNA was extracted from cotton swabs by phenol-chloroform and in Group 2 by boiling. The most efficient PCR methods in the Group 1 were those based on kDNA targets. The kDNA PCR-Hybridization was able to detect parasites in 22/23 dogs (95.6%) and in 40/46 samples (86.9%). The kDNA snPCR was positive for 21/23 dogs (91.3%) and for 40/46 samples (86.9%). The positivities of the kDNA based methods were significantly higher than the positivities verified for the methods based on ribosomal rRNA genes (p<0.05). In the Group 2 the kDNA PCR- Hybridization showed a better performance detecting parasites in 18/23 dogs (78.3%) and in 31/46 samples (67.4%), significantly higher than the other three methods (p<0.05). The higher sensitivity of the minicircle kDNA based assays reported by others was confirmed in this study and kDNA PCR-Hybridization showed the best sensitivity among the assays evaluated. (author)

  16. 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.

  17. 高速PCR技术%Rapid PCR Technology

    Institute of Scientific and Technical Information of China (English)

    赵晓光; 薛燕

    2011-01-01

    PCR technology has been one of the basic and key technologies of Modern Molecular Biology.How to shorten PCR time is a question badly needed to be solved on the premise of PCR amplification effect.In the paper we describe the concept,principle,advantage and application of rapidPCR technology which can highly increase the PCR speed.As addressed in the paper,the Key of rapid PCR is not only to increase the heating and cooling rate of PCR machine,more important,it relays on reducing the temperature difference between the PCR module and sample solution,so that the holding time of denaturation,annealing,elongation could be shorten,and the whole PCR time could be shorten dramatically.While,rapid PCR could also lead to another benefit to improving the specificity of PCR%PCR技术是现代分子生物学最基础的技术之一,在实现扩增效果的同时如何缩短PCR的实验时间成为实验工作者亟待解决的问题。本文从高速PCR的概念入手,阐述了实现高速PCR仪器技术的原理、优势,及可能的应用。指出了高速PCR技术的核心在于,不仅要提高PCR仪器的升降温速率,更重要的是提高PCR样品温度与金属样品槽的一致性,从而达到缩短变性,退火,延伸等停留时间的目的,进而大大的缩短PCR时间。同时也指出,高速PCR对提高反应特异性带来的好处等

  18. Comparison of droplet digital PCR with quantitative real-time PCR for determination of zygosity in transgenic maize.

    Science.gov (United States)

    Xu, Xiaoli; Peng, Cheng; Wang, Xiaofu; Chen, Xiaoyun; Wang, Qiang; Xu, Junfeng

    2016-12-01

    This study evaluated the applicability of droplet digital PCR (ddPCR) as a tool for maize zygosity determination using quantitative real-time PCR (qPCR) as a reference technology. Quantitative real-time PCR is commonly used to determine transgene copy number or GMO zygosity characterization. However, its effectiveness is based on identical reaction efficiencies for the transgene and the endogenous reference gene. Additionally, a calibrator sample should be utilized for accuracy. Droplet digital PCR is a DNA molecule counting technique that directly counts the absolute number of target and reference DNA molecules in a sample, independent of assay efficiency or external calibrators. The zygosity of the transgene can be easily determined using the ratio of the quantity of the target gene to the reference single copy endogenous gene. In this study, both the qPCR and ddPCR methods were used to determine insect-resistant transgenic maize IE034 zygosity. Both methods performed well, but the ddPCR method was more convenient because of its absolute quantification property.

  19. A novel three primers PCR (TP-PCR) method to obtain recombinant DNA molecule independent of restriction enzyme

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    In this note, we report a novel and efficient three primers PCR (TP-PCR) method to rapidly generate recombinant DNA molecule at precise junction between two arbitrary DNA fragments. TP-PCR method is characterized by its reaction system with two templates and three primers, which can produce a recombinant DNA molecule in one PCR reaction. The main advantages of this method are the independence of sequences at the recombination site, the rapidness, and the easy establishment of adequate conditions. This method has been successfully applied to constructing a fusion protein gene, sck gene.

  20. Hypervariable spacer regions are good sites for developing specific PCR-RFLP markers and PCR primers for screening actinorhizal symbionts

    Indian Academy of Sciences (India)

    Rajani Varghese; Vineeta S Chauhan; Arvind K Misra

    2003-06-01

    While the ribosomal RNA like highly conserved genes are good molecular chronometers for establishing phylogenetic relationships, they can also be useful in securing the amplification of adjoining hyper-variable regions. These regions can then be used for developing specific PCR primers or PCR-RFL profiles to be used as molecular markers. We report here the use of ITS region of rrn operon of Frankia for developing PCR-RFL profiles capable of discriminating between closely related frankiae. We have also made use of the ITS1 region of the nuclear rrn operon of Alnus nepalensis (D Don) for designing a PCR primer for specific amplification of nuclear DNA of this tree.

  1. 一种高特异性的改良降落PCR%Modified Touchdown PCR as a High Efficient Approach to Improve the Specificity of PCR

    Institute of Scientific and Technical Information of China (English)

    张贵星; 袁保梅; 许培荣; 王建民; 薛乐勋

    2002-01-01

    To improve the specificity of PCR in detection of genes in genomic DNA, a modified touchdown PCR method was designed by using the common Taq DNA polymerase and the Pfu DNA polymerase with proofreading ability. Genomic DNA was extracted from Dunaliella bardawil, a unicellular and wall-less alga being able to propagate in solution of high concentration of NaCl. Two kinds of PCR using Taq and Pfu DNA polymerases were carried out, utilizing the genomic DNA mentioned above as a template, to amplify the upstream promoter sequence of the cbr (carotene biosynthesis-related) gene. Traditional PCR was used as control. Electrophoresis of the PCRs using Taq DNA polymerase demonstrated that TD-PCR produced a single band of 1 272 bp which was corresponding to the expected cbr promoter, in contrast, however, in the lane of common PCR, two extra nonspecific bands appeared as well as the expected 1 272 bp band. PCRs using the Pfu DNA polymerase displayed that common PCR program generated two bands of 1 272 bp and 500 bp, while the TD-PCR program brought about only the expected 1 272 bp band. PCRs using modified touchdown PCR program with both kinds of polymerase were of higher specificity than common PCR. The modified TD-PCR program can efficiently improve the specificity of PCR. Similar programs are expected to clone gene fragments that are difficult to be cloned by the common PCR method, or to raise the specificity of PCR in which the false positives are difficult to be eliminated.%为提高基因组DNA中的基因PCR检出的特异性,设计了一种改良的降落PCR程序,并分别用TaqDNA聚合酶及高保真Pfu DNA聚合酶进行实验.自盐藻Dunaliella bardawil中提取基因组DNA作为PCR模板,使用Taq DNA聚合酶及Pfu DNA聚合酶,运用普通PCR和降落PCR程序,扩增胡萝卜素生物合成相关基因(cbr)上游启动子序列,并电泳比较PCR扩增产物的特异性.结果显示,使用普通Taq酶PCR,普通PCR程序产生200bp,500bp和1272 bp长的三条

  2. Diagnostic multiplex PCR for toxin genotyping of Clostridium perfringens isolates.

    Science.gov (United States)

    Baums, Christoph G; Schotte, Ulrich; Amtsberg, Gunter; Goethe, Ralph

    2004-05-20

    In this study we provide a protocol for genotyping Clostridium perfringens with a new multiplex PCR. This PCR enables reliable and specific detection of the toxin genes cpa, cpb, etx, iap, cpe and cpb2 from heat lysed bacterial suspensions. The efficiency of the protocol was demonstrated by typing C. perfringens reference strains and isolates from veterinary bacteriological routine diagnostic specimens.

  3. Clinical consequences of PCR based diagnosis of intestinal parasitic infections

    NARCIS (Netherlands)

    Rijsman, Lucas H; Monkelbaan, Jan F; Kusters, Johannes G

    The implementation of Polymerase Chain Reaction (PCR) based diagnostics of intestinal protozoa have led to higher sensitivity and (subtype) specificity, more convenient sampling and the possibility for high-throughput screening. An increasing number of clinical laboratories use PCR for routine

  4. Two unusual occurrences of trichomoniasis: rapid species identification by PCR.

    Science.gov (United States)

    Bellanger, A P; Cabaret, O; Costa, J M; Foulet, F; Bretagne, S; Botterel, F

    2008-09-01

    PCR analysis in two unusual occurrences of trichomoniasis, trichomonal empyema due to Trichomonas tenax and Trichomonas vaginalis in an infant urine sample, allowed us to obtain rapid and accurate trichomonad species identification. The weak sensitivity of wet preparations and the low viability of the flagellates can be remedied by the PCR method.

  5. Inhibitory effect of common microfluidic materials on PCR outcome

    KAUST Repository

    Kodzius, Rimantas

    2013-10-10

    In this study, we established a simple method for evaluating the PCR compatibility of various common materials employed when fabricating microfluidic chips, including silicon, several kinds of silicon oxide, glasses, plastics, wax, and adhesives. Two-temperature PCR was performed with these materials to determine their PCR-inhibitory effect. In most cases, adding bovine serum albumin effectively improved the reaction yield. We also studied the individual PCR components from the standpoint of adsorption. Most of the materials did not inhibit the DNA, although they noticeably interacted with the polymerase. We provide a simple method of performing PCR-compatibility testing of materials using inexpensive instrumentation that is common in molecular biology laboratories. Furthermore, our method is direct, being performed under actual PCR conditions with high temperature. Our results provide an overview of materials that are PCR-friendly for fabricating microfluidic devices. The PCR reaction, without any additives, performed best with pyrex glass, and it performed worst with PMMA or acrylic glue materials.

  6. Sexing Bovine Embryos Using PCR Amplification of Bovine SRY Sequence

    Institute of Scientific and Technical Information of China (English)

    曾溢滔; 张美兰; 陈美珏; 周霞娣; 黄英; 任兆瑞; 黄淑帧; 胡明信; 吴学清; 高建明; 张斌; 徐慧如

    1994-01-01

    This study analyses the bovine SRY DNA sequence by direct sequencing procedure, followed by the designation of the PCR primers specific for bovine SRY. Using PCR amplification of bovine SRY gene, the embryo sex was determined. The results of the embryo sex identification were confirmed after the embryo transfer and pregnancies.

  7. Clinical consequences of PCR based diagnosis of intestinal parasitic infections

    NARCIS (Netherlands)

    Rijsman, Lucas H; Monkelbaan, Jan F; Kusters, Johannes G

    2016-01-01

    The implementation of Polymerase Chain Reaction (PCR) based diagnostics of intestinal protozoa have led to higher sensitivity and (subtype) specificity, more convenient sampling and the possibility for high-throughput screening. An increasing number of clinical laboratories use PCR for routine detec

  8. Evaluation of Aspergillus PCR protocols for testing serum specimens.

    Science.gov (United States)

    White, P Lewis; Mengoli, Carlo; Bretagne, Stéphane; Cuenca-Estrella, Manuel; Finnstrom, Niklas; Klingspor, Lena; Melchers, Willem J G; McCulloch, Elaine; Barnes, Rosemary A; Donnelly, J Peter; Loeffler, Juergen

    2011-11-01

    A panel of human serum samples spiked with various amounts of Aspergillus fumigatus genomic DNA was distributed to 23 centers within the European Aspergillus PCR Initiative to determine analytical performance of PCR. Information regarding specific methodological components and PCR performance was requested. The information provided was made anonymous, and meta-regression analysis was performed to determine any procedural factors that significantly altered PCR performance. Ninety-seven percent of protocols were able to detect a threshold of 10 genomes/ml on at least one occasion, with 83% of protocols reproducibly detecting this concentration. Sensitivity and specificity were 86.1% and 93.6%, respectively. Positive associations between sensitivity and the use of larger sample volumes, an internal control PCR, and PCR targeting the internal transcribed spacer (ITS) region were shown. Negative associations between sensitivity and the use of larger elution volumes (≥100 μl) and PCR targeting the mitochondrial genes were demonstrated. Most Aspergillus PCR protocols used to test serum generate satisfactory analytical performance. Testing serum requires less standardization, and the specific recommendations shown in this article will only improve performance.

  9. Clinical consequences of PCR based diagnosis of intestinal parasitic infections

    NARCIS (Netherlands)

    Rijsman, Lucas H; Monkelbaan, Jan F|info:eu-repo/dai/nl/344499383; Kusters, Johannes G

    2016-01-01

    The implementation of Polymerase Chain Reaction (PCR) based diagnostics of intestinal protozoa have led to higher sensitivity and (subtype) specificity, more convenient sampling and the possibility for high-throughput screening. An increasing number of clinical laboratories use PCR for routine detec

  10. Comparison between ICT and PCR for diagnosis of Chlamydia trachomatis.

    Science.gov (United States)

    Khan, E R; Hossain, M A; Paul, S K; Mahmud, C; Hasan, M M; Rahman, M M; Nahar, K; Kubayashi, N

    2012-04-01

    Chlamydia trachomatis is an obligate intracellular gram-negative bacterium which is the most prevalent cause of bacterial sexually transmitted infections (STI). The present study was carried to diagnose genital Chlamydia trachomatis infection among women of reproductive age, attending Mymensingh Medical College Hospital, during July 2009 to June 2010 by Immunochromatographic test (ICT) and Polymerase chain reaction (PCR). A total of 70 females were included in this study. Out of 70 cases 56 were symptomatic and 14 asymptomatic. Endocervical swabs were collected from each of the cases and examined by Immunochromatographic test (ICT) for antigen detection and Polymerase chain reaction (PCR) for detection of endogenous plasmid-based nucleic acid. A total 29(41.4%) of the cases were found positive for C. trachomatis either by ICT or PCR. Of the 56 symptomatic cases, 19(33.9%) were found ICT positive and 17(30.4%) were PCR positive. Among 14 asymptomatic females, 2(14.3%) were ICT positive and none were PCR positive. Though PCR is highly sensitive but a total of twelve cases were found ICT positive but PCR negative. It may be due to presence of plasmid deficient strain of C trachomatis which could be amplified by ompA based (Chromosomal gene) multiplex PCR.

  11. mRNA and DNA PCR tests in cutaneous tuberculosis

    Directory of Open Access Journals (Sweden)

    Chandanmal Suthar

    2013-01-01

    Full Text Available Background: The microbiologic diagnosis of cutaneous tuberculosis is difficult because most lesions harbor only a small number of mycobacteria that cannot usually be detected by staining for the organism or by culture. Nucleic acid amplification tests based on the polymerase chain reaction (PCR are potentially useful in this situation. Aims: To evaluate the utility of mRNA PCR and DNA PCR in the diagnosis of cutaneous tuberculosis. Methods: Biopsies from 28 cases of cutaneous tuberculosis and 19 controls with other diseases were subjected to microbiologic tests including direct smears for mycobacteria, culture and both mRNA PCR and DNA PCR. The laboratory was blinded to the clinical diagnosis. Results: None of the patients or controls showed a positive reaction on mRNA PCR test. Seven of 28 cases and 5 out of 19 controls showed a positive result on DNA PCR test yielding a sensitivity of 25% and a specificity of 73.7%. Conclusion: The results of PCR tests in cutaneous tuberculosis should be interpreted in the light of clinical and histopathological findings.

  12. Multiplex PCR identification of Taenia spp. in rodents and carnivores.

    Science.gov (United States)

    Al-Sabi, Mohammad N S; Kapel, Christian M O

    2011-11-01

    The genus Taenia includes several species of veterinary and public health importance, but diagnosis of the etiological agent in definitive and intermediate hosts often relies on labor intensive and few specific morphometric criteria, especially in immature worms and underdeveloped metacestodes. In the present study, a multiplex PCR, based on five primers targeting the 18S rDNA and ITS2 sequences, produced a species-specific banding patterns for a range of Taenia spp. Species typing by the multiplex PCR was compared to morphological identification and sequencing of cox1 and/or 12S rDNA genes. As compared to sequencing, the multiplex PCR identified 31 of 32 Taenia metacestodes from rodents, whereas only 14 cysts were specifically identified by morphology. Likewise, the multiplex PCR identified 108 of 130 adult worms, while only 57 were identified to species by morphology. The tested multiplex PCR system may potentially be used for studies of Taenia spp. transmitted between rodents and carnivores.

  13. Engineered DNA Polymerase Improves PCR Results for Plastid DNA

    Directory of Open Access Journals (Sweden)

    Melanie Schori

    2013-02-01

    Full Text Available Premise of the study: Secondary metabolites often inhibit PCR and sequencing reactions in extractions from plant material, especially from silica-dried and herbarium material. A DNA polymerase that is tolerant to inhibitors improves PCR results. Methods and Results: A novel DNA amplification system, including a DNA polymerase engineered via directed evolution for improved tolerance to common plant-derived PCR inhibitors, was evaluated and PCR parameters optimized for three species. An additional 31 species were then tested with the engineered enzyme and optimized protocol, as well as with regular Taq polymerase. Conclusions: PCR products and high-quality sequence data were obtained for 96% of samples for rbcL and 79% for matK, compared to 29% and 21% with regular Taq polymerase.

  14. KRAS mutation analysis by PCR: a comparison of two methods.

    Directory of Open Access Journals (Sweden)

    Louise Bolton

    Full Text Available KRAS mutation assays are important companion diagnostic tests to guide anti-EGFR antibody treatment of metastatic colorectal cancer. Direct comparison of newer diagnostic methods with existing methods is an important part of validation of any new technique. In this this study, we have compared the Therascreen (Qiagen ARMS assay with Competitive Allele-Specific TaqMan PCR (castPCR, Life Technologies to determine equivalence for KRAS mutation analysis.DNA was extracted by Maxwell (Promega from 99 colorectal cancers. The ARMS-based Therascreen and a customized castPCR assay were performed according to the manufacturer's instructions. All assays were performed on either an Applied Biosystems 7500 Fast Dx or a ViiA7 real-time PCR machine (both from Life Technologies. The data were collected and discrepant results re-tested with newly extracted DNA from the same blocks in both assay types.Of the 99 tumors included, Therascreen showed 62 tumors to be wild-type (WT for KRAS, while 37 had KRAS mutations on initial testing. CastPCR showed 61 tumors to be wild-type (WT for KRAS, while 38 had KRAS mutations. Thirteen tumors showed BRAF mutation in castPCR and in one of these there was also a KRAS mutation. The custom castPCR plate included several other KRAS mutations and BRAF V600E, not included in Therascreen, explaining the higher number of mutations detected by castPCR. Re-testing of discrepant results was required in three tumors, all of which then achieved concordance for KRAS. CastPCR assay Ct values were on average 2 cycles lower than Therascreen.There was excellent correlation between the two methods. Although castPCR assay shows lower Ct values than Therascreen, this is unlikely to be clinically significant.

  15. PCR detection of Babesia ovata from questing ticks in Japan.

    Science.gov (United States)

    Sivakumar, Thillaiampalam; Tattiyapong, Muncharee; Okubo, Kazuhiro; Suganuma, Keisuke; Hayashida, Kyoko; Igarashi, Ikuo; Zakimi, Satoshi; Matsumoto, Kotaro; Inokuma, Hisashi; Yokoyama, Naoaki

    2014-04-01

    Babesia ovata is a tick-transmitted hemoprotozoan parasite of cattle. In the present study, we analyzed tick DNA samples (n=1459) prepared from questing ticks collected from various cattle pastures in Hokkaido (Shibecha, Taiki, Otofuke, Memuro, and Shin-Hidaka districts) and Okinawa (Yonaguni Island) prefectures of Japan for B. ovata. When all the tick DNA samples were screened by a previously described B. ovata-specific apical membrane antigen-1 (AMA-1) gene-based polymerase chain reaction (PCR) assay, none of the DNA samples was positive. Therefore, we developed a PCR assay based on the protozoan beta-tubulin (β-tubulin) gene to detect B. ovata from ticks in Japan. In the specificity test, the PCR assay amplified the expected 444-bp target gene fragment from B. ovata DNA. No PCR products were amplified from DNA samples from other blood pathogens, bovine blood, or ticks. In addition, the PCR assay detected 100 fg of B. ovata-genomic DNA extracted from an in vitro culture of the parasites. Subsequently, when all the tick DNA samples were screened by this new PCR assay, 18 were positive for B. ovata. Positive samples were found only in the Yonaguni and Memuro areas. In Okinawa, where all the ticks were identified as Haemaphysalis longicornis, 9.7% of the samples were PCR-positive, while a single tick (Ixodes ovatus) from Memuro was infected with B. ovata. When the nucleotide sequences of the PCR amplicons were phylogenetically analyzed, they formed a separate clade containing a previously described β-tubulin gene sequence from B. ovata (Miyake strain), confirming that the PCR assay had detected only B. ovata from the tick DNA samples. This is the first report that describes the PCR detection of B. ovata in ticks. The findings warrant transmission experiments to evaluate I. ovatus as a potential vector of B. ovata.

  16. Analysis on the Factors Affecting PCR-SSCP%影响PCR-SSCP的因素分析

    Institute of Scientific and Technical Information of China (English)

    姜运良; 李宁; 赵兴波; 吴常信

    2000-01-01

    PCR-SSCP是一种基于PCR的单链构象多态性(single-stranded conformation polymorphism)分析技术,是DNA已知突变的检测或未知变异分析中十分常用和实用的技术之一.本研究从非变性聚丙烯酰胺凝胶的配制、样品的变性处理、电泳条件、显色方法和结果分析等方面对影响PCR-SSCP实验室操作的因素进行了分析,并结合作者的经验给出了大小在100~200 bp 范围以内的DNA片段进行SSCP分析的操作程序:凝胶浓度为10 %~15 %,98℃变性5~10 min,4℃条件下5 W过夜电泳,银染法染色.

  17. Transient neonatal diabetes mellitus with macroglossia diagnosed by methylation specific PCR (MS-PCR

    Directory of Open Access Journals (Sweden)

    Hye Young Jin

    2010-03-01

    Full Text Available Transient neonatal diabetes mellitus (TNDM has been associated with paternal uniparental isodisomy of chromosome 6, paternally inherited duplication of 6q24, or a methylation defect at a CpG island of the ZAC or HYMAI gene. We experienced a case of TNDM in which the patient presented with hyperglycemia, macroglossia, and intrauterine growth retardation, caused by a paternally derived HYMAI. An 18-day-old female infant was admitted to the hospital because of macroglossia and recurrent hyperglycemia. In addition to the macroglossia, she also presented with large fontanelles, micrognathia, and prominent eyes. Serum glucose levels were 200&#8211;300 mg/dL and they improved spontaneously 2 days after admission. To identify the presence of a maternal methylated allele, bisulfite-treated genomic DNA from peripheral blood was prepared and digested with BssHII after polymerase chain reaction (PCR amplification with methylation-specific HYMAI primers. PCR and restriction fragment length polymorphism analysis showed that the patient had only the paternal origin of the HYMA1 gene. TNDM is associated with a methylation defect in chromosome 6, suggesting that an imprinted gene on chromosome 6 is responsible for this phenotype.

  18. How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments

    Directory of Open Access Journals (Sweden)

    David Svec

    2015-03-01

    Full Text Available We have examined the imprecision in the estimation of PCR efficiency by means of standard curves based on strategic experimental design with large number of technical replicates. In particular, how robust this estimation is in terms of a commonly varying factors: the instrument used, the number of technical replicates performed and the effect of the volume transferred throughout the dilution series. We used six different qPCR instruments, we performed 1–16 qPCR replicates per concentration and we tested 2–10 μl volume of analyte transferred, respectively. We find that the estimated PCR efficiency varies significantly across different instruments. Using a Monte Carlo approach, we find the uncertainty in the PCR efficiency estimation may be as large as 42.5% (95% CI if standard curve with only one qPCR replicate is used in 16 different plates. Based on our investigation we propose recommendations for the precise estimation of PCR efficiency: (1 one robust standard curve with at least 3–4 qPCR replicates at each concentration shall be generated, (2 the efficiency is instrument dependent, but reproducibly stable on one platform, and (3 using a larger volume when constructing serial dilution series reduces sampling error and enables calibration across a wider dynamic range.

  19. RNase H-dependent PCR (rhPCR: improved specificity and single nucleotide polymorphism detection using blocked cleavable primers

    Directory of Open Access Journals (Sweden)

    Powers Kristy M

    2011-08-01

    Full Text Available Abstract Background The polymerase chain reaction (PCR is commonly used to detect the presence of nucleic acid sequences both in research and diagnostic settings. While high specificity is often achieved, biological requirements sometimes necessitate that primers are placed in suboptimal locations which lead to problems with the formation of primer dimers and/or misamplification of homologous sequences. Results Pyrococcus abyssi (P.a. RNase H2 was used to enable PCR to be performed using blocked primers containing a single ribonucleotide residue which are activated via cleavage by the enzyme (rhPCR. Cleavage occurs 5'-to the RNA base following primer hybridization to the target DNA. The requirement of the primer to first hybridize with the target sequence to gain activity eliminates the formation of primer-dimers and greatly reduces misamplification of closely related sequences. Mismatches near the scissile linkage decrease the efficiency of cleavage by RNase H2, further increasing the specificity of the assay. When applied to the detection of single nucleotide polymorphisms (SNPs, rhPCR was found to be far more sensitive than standard allele-specific PCR. In general, the best discrimination occurs when the mismatch is placed at the RNA:DNA base pair. Conclusion rhPCR eliminates the formation of primer dimers and markedly improves the specificity of PCR with respect to off-target amplification. These advantages of the assay should find utility in challenging qPCR applications such as genotyping, high level multiplex assays and rare allele detection.

  20. Is real-time polymerase chain reaction (PCR) more useful than a conventional PCR for the clinical management of leishmaniasis?

    Science.gov (United States)

    Antinori, Spinello; Calattini, Sara; Piolini, Roberta; Longhi, Erika; Bestetti, Giovanna; Cascio, Antonio; Parravicini, Carlo; Corbellino, Mario

    2009-07-01

    It is currently unknown if the use of a real-time polymerase chain reaction (PCR) adds value to the diagnosis and follow-up prognosis of patients affected by leishmaniasis. We performed a study using a real-time PCR directed against the alpha-polymerase gene and a semiquantitative PCR that target the SSU ribosomal RNA (rRNA) gene as control for the diagnosis and quantification of parasites in patients with visceral (VL) and cutaneous (CL) leishmaniasis. Our single copy real-time PCR missed one diagnosis of VL compared with the conventional PCR, whereas both PCR methods were able to detect Leishmania parasites in CL. Under anti-leishmania treatment the kinetics of parasitemia were comparable with the two methods. The real-time PCR directed against alpha-polymerase of Leishmania despite being able to make a more accurate quantification of parasites does not add to the decision-making management compared with a semiquantitative PCR, and it is comparatively expensive.

  1. Critical appraisal of quantitative PCR results in colorectal cancer research: Can we rely on published qPCR results?

    NARCIS (Netherlands)

    Dijkstra, J.R.; Kempen, L.C.L.T. van; Nagtegaal, I.D.; Bustin, S.A.

    2014-01-01

    The use of real-time quantitative polymerase chain reaction (qPCR) in cancer research has become ubiquitous. The relative simplicity of qPCR experiments, which deliver fast and cost-effective results, means that each year an increasing number of papers utilizing this technique are being published. B

  2. Comparison of PCR-DGGE and Nested-PCR-DGGE Approach for Ammonia Oxidizers Monitoring in Membrane Bioreactors’ Activated Sludge

    Directory of Open Access Journals (Sweden)

    Ziembińska-Buczyńska Aleksandra

    2014-12-01

    Full Text Available Nitritation, the first stage of ammonia removal process is known to be limiting for total process performance. Ammonia oxidizing bacteria (AOB which perform this process are obligatory activated sludge habitants, a mixture consisting of Bacteria, Protozoa and Metazoa used for biological wastewater treatment. Due to this fact they are an interesting bacterial group, from both the technological and ecological point of view. AOB changeability and biodiversity analyses both in wastewater treatment plants and lab-scale reactors are performed on the basis of 16S rRNA gene sequences using PCR-DGGE (Polymerase Chain Reaction – Denaturing Gradient Gel Electrophoresis as a molecular biology tool. AOB researches are usually led with nested PCR. Because the application of nested PCR is laborious and time consuming, we have attempted to check the possibility of using only first PCR round to obtain DGGE fingerprinting of microbial communities. In this work we are comparing the nested and non-nested PCR-DGGE monitoring of an AOB community and presenting advantages and disadvantages of both methods used. The experiment revealed that PCR technique is a very sensitive tool for the amplification of even a minute amount of DNA sample. But in the case of nested-PCR, the sensitivity is higher and the template amount could be even smaller. The nested PCR-DGGE seems to be a better tool for AOB community monitoring and complexity research in activated sludge, despite shorter fragments of DNA amplification which seems to be a disadvantage in the case of bacteria identification. It is recommended that the sort of analysis approach should be chosen according to the aim of the study: nested-PCR-DGGE for community complexity analysis, while PCR-DGGE for identification of the dominant bacteria.

  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. Shape based kinetic outlier detection in real-time PCR

    Directory of Open Access Journals (Sweden)

    D'Atri Mario

    2010-04-01

    Full Text Available Abstract Background Real-time PCR has recently become the technique of choice for absolute and relative nucleic acid quantification. The gold standard quantification method in real-time PCR assumes that the compared samples have similar PCR efficiency. However, many factors present in biological samples affect PCR kinetic, confounding quantification analysis. In this work we propose a new strategy to detect outlier samples, called SOD. Results Richards function was fitted on fluorescence readings to parameterize the amplification curves. There was not a significant correlation between calculated amplification parameters (plateau, slope and y-coordinate of the inflection point and the Log of input DNA demonstrating that this approach can be used to achieve a "fingerprint" for each amplification curve. To identify the outlier runs, the calculated parameters of each unknown sample were compared to those of the standard samples. When a significant underestimation of starting DNA molecules was found, due to the presence of biological inhibitors such as tannic acid, IgG or quercitin, SOD efficiently marked these amplification profiles as outliers. SOD was subsequently compared with KOD, the current approach based on PCR efficiency estimation. The data obtained showed that SOD was more sensitive than KOD, whereas SOD and KOD were equally specific. Conclusion Our results demonstrated, for the first time, that outlier detection can be based on amplification shape instead of PCR efficiency. SOD represents an improvement in real-time PCR analysis because it decreases the variance of data thus increasing the reliability of quantification.

  5. Prenatal toxoplasmosis diagnosis from amniotic fluid by PCR

    Directory of Open Access Journals (Sweden)

    Vidigal Paula Vieira Teixeira

    2002-01-01

    Full Text Available Toxoplasmosis is one of the most common infections all over the world. Most cases are asymptomatic, except in immunosuppressed individuals and fetuses, which can be seriously damaged. Prenatal diagnosis should be made as soon as possible since treatment of the mother can minimize fetal sequelae. Our aim in this study was to test the polymerase chain reaction technique (PCR in 86 samples of amniotic fluid from women who seroconverted during pregnancy. DNA was amplified using external primers and, in a second step, internal primers, in a nested PCR system. Samples were also inoculated into mice and the newborn were evaluated by T. gondii serology, skull x-ray, transfontanel ultrasound, fundoscopic examination, lumbar puncture and clinical examination. PCR was positive in seven cases and negative in 79. Among PCR-positive cases, two were negative by inoculation into mice and by clinical evaluation; among PCR-negative ones, three had clinical evidence of toxoplasmosis and one was positive after inoculation into mice. PCR showed values of sensitivity = 62.5% and specificity = 97.4%; the values of inoculation into mice where 42.9% and 100%, respectively. Although PCR should not be used alone for prenatal diagnosis of congenital toxoplasmosis, it is a promising method and deserves more studies to improve its efficacy.

  6. Interaction of quantitative PCR components with polymeric surfaces.

    Science.gov (United States)

    Gonzalez, Asensio; Grimes, Ronan; Walsh, Edmond J; Dalton, Tara; Davies, Mark

    2007-04-01

    This study investigated the effect of exposing a polymerase chain reaction (PCR) mixture to capillary tubing of different materials and lengths, at different contact times and flow rates and the adsorption of major reaction components into the tubing wall. Using 0.5 mm ID tubing, lengths of 40 cm and residence times up to 45 min, none of the tested polymeric materials was found to affect subsequent PCR amplification. However, after exposure of the mixture to tubing lengths of 3 m or reduction of sample volume, PCR inhibition occurred, increasing with the volume to length ratio. Different flow velocities did not affect PCR yield. When the adsorption of individual PCR components was studied, significant DNA adsorption and even more significant adsorption of the fluorescent dye Sybr Green I was found. The results indicate that PCR inhibition in polymeric tubing results from adsorption of reaction components to wall surfaces, increasing substantially with tubing length or sample volume reduction, but not with contact time or flow velocities typical in dynamic PCR amplification. The data also highlight that chemical compatibility of polymeric capillaries with DNA dyes should be carefully considered for the design of quantitative microfluidic devices.

  7. Rapid and simple method of qPCR primer design.

    Science.gov (United States)

    Thornton, Brenda; Basu, Chhandak

    2015-01-01

    Quantitative real-time polymerase chain reaction (qPCR) is a powerful tool for analysis and quantification of gene expression. It is advantageous compared to traditional gel-based method of PCR, as gene expression can be visualized "real-time" using a computer. In qPCR, a reporter dye system is used which intercalates with DNA's region of interest and detects DNA amplification. Some of the popular reporter systems used in qPCR are the following: Molecular Beacon(®), SYBR Green(®), and Taqman(®). However, success of qPCR depends on the optimal primers used. Some of the considerations for primer design are the following: GC content, primer self-dimer, or secondary structure formation. Freely available software could be used for ideal qPCR primer design. Here we have shown how to use some freely available web-based software programs (such as Primerquest(®), Unafold(®), and Beacon designer(®)) to design qPCR primers.

  8. Development of a PCR test to diagnose Haemophilus parasuis infections.

    Science.gov (United States)

    Oliveira, S; Galina, L; Pijoan, C

    2001-11-01

    A polymerase chain reaction (PCR) test was developed in order to improve the accuracy and speed of diagnosis of Haemophilus parasuis, an economically important respiratory pathogen that affects swine. The gene sequence of the 16S small subunit ribosomal RNA of H. parasuis (GenBank M75065) was compared with 56 16S sequences of related bacteria, including those frequently isolated from pig tissues. Two species-specific primers were designed: HPS forward and HPS reverse. The predicted size of the amplified PCR product was 821 bp. The PCR test could detect a minimum of 102 bacteria and 0.69 pg of DNA. Thirty-one H. parasuis isolates, including 12 different serovars and 19 field isolates, were positive using the PCR test. No amplification was observed when the test was run using DNA from 15 other bacterial species commonly isolated from swine tissues. A weak band was observed when the PCR test was performed using Actinobacillus indolicus DNA as template. Clinical samples tested by PCR included tissues and swabs from 5 animals naturally infected with H. parasuis and 1 experimentally infected animal. The PCR was positive in 26 of 30 clinical samples. Four samples showed weak bands, and these results were not considered positive. Haemophilus parasuis was isolated from 18 of 30 of these samples. Tissues from specific pathogen-free (SPF) pigs and from unrelated species were negative for H. parasuis isolation and PCR. The developed PCR was successfully used in the diagnosis of H. parasuis infection, especially when compared with traditional microbiology techniques.

  9. A multiplex PCR for detection of six viruses in ducks.

    Science.gov (United States)

    Wang, Yongjuan; Zhu, Shanyuan; Hong, Weiming; Wang, Anping; Zuo, Weiyong

    2017-10-01

    In this study, six pairs of specific primers that can amplify DNA fragments of different sizes were designed and synthesized according to viral protein gene sequences published in GenBank. Then, a multiplex PCR method was established for rapid detection of duck hepatitis virus 1, duck plague virus, duck Tembusu virus, muscovy duck parvovirus, muscovy duck reovirus, and duck H9N2 avian influenza virus, and achieve simple and rapid detection of viral diseases in ducks. Single PCR was used to confirm primer specificity, and PCR conditions were optimized to construct a multiplex PCR system. Specificity and sensitivity assays were also developed. The multiplex PCR was used to detect duck embryos infected with mixed viruses and those with clinically suspected diseases to verify the feasibility of the multiplex PCR. Results show that the primers can specifically amplify target fragments, without any cross-amplification with other viruses. The multiplex PCR system can amplify six DNA fragments from the pooled viral genomes and specifically detect nucleic acids of the six duck susceptible viruses when the template amount is 10(2) copies/μl. In addition, the system can be used to detect viral nucleic acids in duck embryos infected with the six common viruses. The detection results for clinical samples are consistent with those detected by single PCR. Therefore, the established multiplex PCR method can perform specific, sensitive, and high-throughput detection of six duck-infecting viruses and can be applied to clinical identification and diagnosis of viral infection in ducks. Copyright © 2017. Published by Elsevier B.V.

  10. Buoyancy-Driven Polymerase Chain Reaction (PCR) Devices

    Energy Technology Data Exchange (ETDEWEB)

    Ness, K D; Wheeler, E K; Benett, W; Stratton, P; Christian, A; Chen, A; Ortega, J; Weisgraber, T H; Goodson, K E

    2004-09-28

    Polymerase chain reaction (PCR) facilitates DNA detection by significantly increasing the concentration of specific DNA segments. A new class of PCR instruments uses a buoyancy-driven re-circulating flow to thermally cycle the DNA sample and benefits from reduced cycle times, low sample volumes, a miniaturized format, and low power consumption. This paper analyzes a specific buoyancy PCR device in a micro-channel ''race-track'' geometry to determine key parameters about PCR cycle times and other figures of merit as functions of device dimensions. The 1-D model balances the buoyancy driving force with frictional losses. A hydrostatic pressure imbalance concept is used between the left and right sides of the fluid loop to calculate the buoyancy driving force. Velocity and temperature distributions within the channels are determined from two-dimensional analysis of the channel section, with developing region effects included empirically through scaled values of the local Nusselt number. Good agreement between four independent verification steps validate the 1-D simulation approach: (1) analytical expressions for the thermal entrance length are compared against, (2) comparison with a full 3-D finite element simulation, (3) comparison with an experimental flow field characterization, and (4) calculation of the minimum PCR runtime required to get a positive PCR signal from the buoyancy-driven PCR device. The 1-D approach closely models an actual buoyancy-driven PCR device and can further be used as a rapid design tool to simulate buoyancy PCR flows and perform detailed design optimizations studies.

  11. Development and Validation of a HPV-32 Specific PCR Assay

    Directory of Open Access Journals (Sweden)

    Leigh Janet

    2009-06-01

    Full Text Available Abstract Background Human Papillomavirus-32 (HPV-32 has traditionally been associated with focal-epithelial-hyperplasia (FEH. It is also present in 58% of oral warts of HIV-positive individuals whose prevalence is increasing. Current methods for the detection of HPV-32 are labor-intensive and insensitive so the goal of this work was to develop a highly sensitive and easy to use specific polymerase chain reaction (PCR assay. Materials and methods An HPV-32 L1 specific PCR assay was developed and optimized. The sensitivity and specificity was compared to previous assays utilized for detection (PGMY and MY09/11 PCR with dot blot hybridization using cloned HPV-32 L1, the closely related HPV-42 L1 as well as clinical samples (oral swabs and fluids from 89 HIV-positive subjects. Results The HPV-32 specific PCR assay showed improved sensitivity to 5 copies of HPV-32 as compared to the PGMY PCR, MY09/11 PCR and dot blot which had a limit of detection of approximately 3,000 copies. Using the HPV-32 dot blot hybridization assay as the gold standard, the HPV-32 specific PCR assay has a sensitivity of 95.8% and 88.9% by sample and subject, respectively, and specificity was 87.8% and 58.8% by sample and subject, respectively. The low sensitivity is due to the HPV-32 specific PCR assays ability to detect more HPV-32 positive samples and may be the new gold standard. Conclusion Due to the ease, sensitivity, and specificity the HPV-32 specific PCR assay is superior to previous assays and is ideal for detection of HPV-32 in large cohorts. This assay provides an excellent tool to study the natural history of HPV-32 infection and the development of oral warts.

  12. Identification and quantification of ice nucleation active microorganisms by digital droplet PCR (ddPCR)

    Science.gov (United States)

    Linden, Martin; Pöschl, Ulrich; Fröhlich-Nowoisky, Janine

    2015-04-01

    Several bioaerosol types, including bacteria, fungi, pollen and lichen, have been identified as sources of biological ice nucleators (IN) which induce ice formation already at temperatures as high as -10 °C or above. Accordingly, they potentially contribute widely to environmental ice nucleation in the atmosphere and are of great interest in the study of natural heterogenous ice nucleation processes. Ice nucleation active microorganisms have been found and studied among bacteria (Proteobacteria) and fungi (phyla Basidiomycota and Ascomycota). The mechanisms enabling the microorganisms to ice nucleation are subject to ongoing research. While it has been demonstrated that whole cells can act as ice nucleators in the case of bacteria due to the presence of specific membrane proteins, cell-free ice nucleation active particles seem to be responsible for this phenomenon in fungi and lichen. The identification and quantification of these ice nucleation active microorganisms and their IN in atmospheric samples is crucial to understand their contribution to the pool of atmospheric IN. This is not a trivial task since the respective microorganisms are often prevalent in lowest concentrations and a variety of states, be it viable cells, spores or cell debris from dead cells. Molecular biology provides tools to identify and quantify ice nucleation active microorganisms independent of their state by detecting genetic markers specific for the organism of interest. Those methods are not without their drawbacks in terms of sample material concentration required or reliable standardization. Digital Droplet Polymerase Chain Reaction (ddPCR) was chosen for our demands as a more elegant, quick and specific method in the investigation of ice nucleation active microorganisms in atmospheric samples. The advantages of ddPCR lie in the simultaneous detection and quantification of genetic markers and their original copy numbers in a sample. This is facilitated by the fractionation of the

  13. 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.

  14. PCR IN TRAUMATOLOGY AND ORTHOPAEDICS: METHOD DESCRIPTION AND APPLICABILITY

    Directory of Open Access Journals (Sweden)

    E. M. Polyakova

    2014-01-01

    Full Text Available Review brief presents description of polymerase chain reaction method (PCR and its most common variants. Three PCR-based lines of research, carried out in the traumatology and orthopaedics, include identifying a causative agents of the implant-associated infection after orthopaedic surgery; detection of antibiotic resistance genes and biofilm forming genes. It was shown that PCR can be used as additional method for detection of genetic disorders, significant for traumatology and orthopaedics, and for investigation of cartilage and bone regeneration.

  15. Forensic study of sex determination using PCR on teeth samples.

    OpenAIRE

    Murakami H; Yamamoto Y; Yoshitome K; Ono T; Okamoto O; Shigeta Y; Doi Y; Miyaishi S; Ishizu H

    2000-01-01

    In this study, sex determination using polymerase chain reaction (PCR) on tooth material was evaluated from the viewpoint of forensic medicine. The sensitivity of PCR for detection of the Y chromosome-specific alphoid repeat sequence and the X chromosome-specific alphoid repeat sequence was 0.5 pg of genomic DNA. Sex could be determined by PCR of DNA extracted from the pulp of 16 freshly extracted permanent teeth and dentine including the surface of the pulp cavity of 6 freshly extracted milk...

  16. 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.

  17. Surface effects on PCR reactions in multichip microfluidic platforms.

    Science.gov (United States)

    Panaro, Nicholas J; Lou, Xing Jian; Fortina, Paolo; Kricka, Larry J; Wilding, Peter

    2004-03-01

    We evaluated the compatibility of several common plastics, commercially available plastic tubing and disposable syringes which might be useful in the construction of microfluidic platforms with respect to the polymerase chain reaction (PCR). A simple and inexpensive plastic test module was constructed in order to evaluate some of the construction plastics. We also investigated the effect of addition of PEG 8000 to PCR reaction mixtures on the compatibility of materials. These studies identified several common plastics, plastic tubing, and disposable syringes which were compatible with the PCR reaction.

  18. Droplet digital polymerase chain reaction (PCR) outperforms real-time PCR in the detection of environmental DNA from an invasive fish species.

    Science.gov (United States)

    Doi, Hideyuki; Takahara, Teruhiko; Minamoto, Toshifumi; Matsuhashi, Saeko; Uchii, Kimiko; Yamanaka, Hiroki

    2015-05-01

    Environmental DNA (eDNA) has been used to investigate species distributions in aquatic ecosystems. Most of these studies use real-time polymerase chain reaction (PCR) to detect eDNA in water; however, PCR amplification is often inhibited by the presence of organic and inorganic matter. In droplet digital PCR (ddPCR), the sample is partitioned into thousands of nanoliter droplets, and PCR inhibition may be reduced by the detection of the end-point of PCR amplification in each droplet, independent of the amplification efficiency. In addition, real-time PCR reagents can affect PCR amplification and consequently alter detection rates. We compared the effectiveness of ddPCR and real-time PCR using two different PCR reagents for the detection of the eDNA from invasive bluegill sunfish, Lepomis macrochirus, in ponds. We found that ddPCR had higher detection rates of bluegill eDNA in pond water than real-time PCR with either of the PCR reagents, especially at low DNA concentrations. Limits of DNA detection, which were tested by spiking the bluegill DNA to DNA extracts from the ponds containing natural inhibitors, found that ddPCR had higher detection rate than real-time PCR. Our results suggest that ddPCR is more resistant to the presence of PCR inhibitors in field samples than real-time PCR. Thus, ddPCR outperforms real-time PCR methods for detecting eDNA to document species distributions in natural habitats, especially in habitats with high concentrations of PCR inhibitors.

  19. Sensitive simultaneous detection of seven sexually transmitted agents in semen by multiplex-PCR and of HPV by single PCR.

    Science.gov (United States)

    Gimenes, Fabrícia; Medina, Fabiana Soares; Abreu, André Luelsdorf Pimenta de; Irie, Mary Mayumi Taguti; Esquiçati, Isis Baroni; Malagutti, Natália; Vasconcellos, Vinícius Rodrigo Bulla; Discacciati, Michele Garcia; Bonini, Marcelo Gialluisi; Maria-Engler, Silvya Stuchi; Consolaro, Marcia Edilaine Lopes

    2014-01-01

    Sexually transmitted diseases (STDs) may impair sperm parameters and functions thereby promoting male infertility. To date limited molecular studies were conducted to evaluate the frequency and type of such infections in semen Thus, we aimed at conceiving and validating a multiplex PCR (M-PCR) assay for the simultaneous detection of the following STD pathogens in semen: Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis, Herpes virus simplex (HSV) -1 and -2, and Treponema pallidum; We also investigated the potential usefulness of this M-PCR assay in screening programs for semen pathogens. In addition, we aimed: to detect human Papillomavirus (HPV) and genotypes by single PCR (sPCR) in the same semen samples; to determine the prevalence of the seven STDs, HPV and co-infections; to assess the possibility that these infections affect semen parameters and thus fertility. The overall validation parameters of M-PCR were extremely high including agreement (99.2%), sensitivity (100.00%), specificity (99.70%), positive (96.40%) and negative predictive values (100.00%) and accuracy (99.80%). The prevalence of STDs was very high (55.3%). Furthermore, associations were observed between STDs and changes in semen parameters, highlighting the importance of STD detection in semen. Thus, this M-PCR assay has great potential for application in semen screening programs for pathogens in infertility and STD clinics and in sperm banks.

  20. Conventional PCR usage for the detection of Mycobacterium Tuberculosis complex in Cerebrospinal Fluid by MPB64-Target PCR

    Directory of Open Access Journals (Sweden)

    Sharma Narotam

    2011-12-01

    Full Text Available Tuberculous meningitis is a severe and potentially fatal form of Tuberculosis. Diagnosis of Tuberculous meningitis (TBM is challenging. Thus rapid, accurate and confirmatory diagnosis is necessary to initiate required therapy. The diagnostic workup involves detection of acidfast bacilli (AFB in the cerebrospinal fluid (CSF by microscopy or culture, however, the difficulty in detecting the organism poses a challenge to diagnosis. The use of the polymerase chain reaction (PCR in the diagnostic approach to Mycobacterium tuberculosis (MTB meningitis has been reported as a fast and accurate method using in- house protocols and several commercial kits available. We analysed the performance of PCR for the diagnosis of MTB meningitis in 64 consecutive patients, using MTB culture as gold standard.We used PCR as a tool for detecting mycobacterial mpb64 gene in CSF samples from suspected TB patient. 64 clinical specimens were taken from the patients attending SMI Hospital, Dehradun. 45 were PCR positive, 32 were culture positive and 2 were Zn smear positive.The sensitivity of PCR, culture and microscopy for CSF samples was, thus, 70%, 50% and 3% respectively. Therefore, compared to the conventional culture assay, the PCR can be considered a more useful and advanced technique for the rapid and accurate diagnosis of TBM. Thus PCR is better tool for the diagnosis of Tuberculous meningitis combined with the other concerned tests.

  1. Whole blood Nested PCR and Real-time PCR amplification of Talaromyces marneffei specific DNA for diagnosis.

    Science.gov (United States)

    Lu, Sha; Li, Xiqing; Calderone, Richard; Zhang, Jing; Ma, Jianchi; Cai, Wenying; Xi, Liyan

    2016-02-01

    Talaromyces marneffei is a dimorphic pathogenic fungus, which is a life-threatening invasive mycosis in the immunocompromised host. Prompt diagnosis of T. marneffei infection remains difficult although there has been progress in attempts to expedite the diagnosis of this infection. We previously demonstrated the value of nested polymerase chain reaction (PCR) to detect T. marneffei in paraffin embedded tissue samples with high sensitivity and specificity. In this study, this assay was used to detect the DNA of T. marneffei in whole blood samples. Real-time PCR assay was also evaluated to identify T. marneffei in the same samples. Twenty out of 30 whole blood samples (67%) collected from 23 patients were found positive by using the nested PCR assay, while 23/30 (77%) samples were found positive by using the real-time PCR assay. In order to express accurately the fungal loads, we used a normalized linearized plasmid as an internal control for real-time PCR. The assay results were correlated as the initial quantity (copies/μl) with fungal burden. These data indicate that combination of nested PCR and real-time PCR assay provides an attractive alternative for identification of T. marneffei DNA in whole blood samples of HIV-infected patients.

  2. Sensitive simultaneous detection of seven sexually transmitted agents in semen by multiplex-PCR and of HPV by single PCR.

    Directory of Open Access Journals (Sweden)

    Fabrícia Gimenes

    Full Text Available Sexually transmitted diseases (STDs may impair sperm parameters and functions thereby promoting male infertility. To date limited molecular studies were conducted to evaluate the frequency and type of such infections in semen Thus, we aimed at conceiving and validating a multiplex PCR (M-PCR assay for the simultaneous detection of the following STD pathogens in semen: Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis, Herpes virus simplex (HSV -1 and -2, and Treponema pallidum; We also investigated the potential usefulness of this M-PCR assay in screening programs for semen pathogens. In addition, we aimed: to detect human Papillomavirus (HPV and genotypes by single PCR (sPCR in the same semen samples; to determine the prevalence of the seven STDs, HPV and co-infections; to assess the possibility that these infections affect semen parameters and thus fertility. The overall validation parameters of M-PCR were extremely high including agreement (99.2%, sensitivity (100.00%, specificity (99.70%, positive (96.40% and negative predictive values (100.00% and accuracy (99.80%. The prevalence of STDs was very high (55.3%. Furthermore, associations were observed between STDs and changes in semen parameters, highlighting the importance of STD detection in semen. Thus, this M-PCR assay has great potential for application in semen screening programs for pathogens in infertility and STD clinics and in sperm banks.

  3. Clinical Performance of Aspergillus PCR for Testing Serum and Plasma: a Study by the European Aspergillus PCR Initiative.

    Science.gov (United States)

    White, P Lewis; Barnes, Rosemary A; Springer, Jan; Klingspor, Lena; Cuenca-Estrella, Manuel; Morton, C Oliver; Lagrou, Katrien; Bretagne, Stéphane; Melchers, Willem J G; Mengoli, Carlo; Donnelly, J Peter; Heinz, Werner J; Loeffler, Juergen

    2015-09-01

    Aspergillus PCR testing of serum provides technical simplicity but with potentially reduced sensitivity compared to whole-blood testing. With diseases for which screening to exclude disease represents an optimal strategy, sensitivity is paramount. The associated analytical study confirmed that DNA concentrations were greater in plasma than those in serum. The aim of the current investigation was to confirm analytical findings by comparing the performance of Aspergillus PCR testing of plasma and serum in the clinical setting. Standardized Aspergillus PCR was performed on plasma and serum samples concurrently obtained from hematology patients in a multicenter retrospective anonymous case-control study, with cases diagnosed according to European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) consensus definitions (19 proven/probable cases and 42 controls). Clinical performance and clinical utility (time to positivity) were calculated for both kinds of samples. The sensitivity and specificity for Aspergillus PCR when testing serum were 68.4% and 76.2%, respectively, and for plasma, they were 94.7% and 83.3%, respectively. Eighty-five percent of serum and plasma PCR results were concordant. On average, plasma PCR was positive 16.8 days before diagnosis and was the earliest indicator of infection in 13 cases, combined with other biomarkers in five cases. On average, serum PCR was positive 10.8 days before diagnosis and was the earliest indicator of infection in six cases, combined with other biomarkers in three cases. These results confirm the analytical finding that the sensitivity of Aspergillus PCR using plasma is superior to that using serum. PCR positivity occurs earlier when testing plasma and provides sufficient sensitivity for the screening of invasive aspergillosis while maintaining methodological simplicity.

  4. (PCR) for the identification of Mycoplasma capricolum subsp ...

    African Journals Online (AJOL)

    ajl yemi

    2011-10-07

    Oct 7, 2011 ... Ministry of Agriculture, Lanzhou Veterinary Research Institute, ... samples tested by PCR included lung and liquor pleurae from 14 goats artificially infected with Mccp. ... infectious disease of goats caused by Mycoplasma.

  5. Nanodroplet real-time PCR system with laser assisted heating.

    Science.gov (United States)

    Kim, Hanyoup; Dixit, Sanhita; Green, Christopher J; Faris, Gregory W

    2009-01-01

    We report the successful application of low-power (approximately 30 mW) laser radiation as an optical heating source for high-speed real-time polymerase chain reaction (PCR) amplification of DNA in nanoliter droplets dispersed in an oil phase. Light provides the heating, temperature measurement, and Taqman real-time readout in nanoliter droplets on a disposable plastic substrate. A selective heating scheme using an infrared laser appears ideal for driving PCR because it heats only the droplet, not the oil or plastic substrate, providing fast heating and completing the 40 cycles of PCR in 370 seconds. No microheaters or microfluidic circuitry were deposited on the substrate, and PCR was performed in one droplet without affecting neighboring droplets. The assay performance was quantitative and its amplification efficiency was comparable to that of a commercial instrument.

  6. Rapid PCR amplification of DNA utilizing Coriolis effects.

    Science.gov (United States)

    Mårtensson, Gustaf; Skote, Martin; Malmqvist, Mats; Falk, Mats; Asp, Allan; Svanvik, Nicke; Johansson, Arne

    2006-08-01

    A novel polymerase chain reaction (PCR) method is presented that utilizes Coriolis and centrifugal effects, produced by rotation of the sample disc, in order to increase internal circulatory rates, and with them temperature homogenization and mixing speeds. A proof of concept has been presented by testing a rapid 45-cycle PCR DNA amplification protocol. During the repeated heating and cooling that constitutes a PCR process, the 100 microL samples were rotated at a speed equivalent to an effective acceleration of gravity of 7,000 g. A cycle time of 20.5 s gave a total process time of 15 min to complete the 45 cycles. A theoretical and numerical analysis of the resulting flow, which describes the increased mixing and temperature homogenization, is presented. The device gives excellent reaction speed efficiency, which is beneficial for rapid PCR.

  7. Comparison of conventional culture and real-time quantitative PCR ...

    African Journals Online (AJOL)

    2009-10-28

    Oct 28, 2009 ... Results of real-time PCR were compared to con- ventional analysis .... water bath (Selecta 40W power, 40 kHz ultrasound fre- quency). ..... mental distributions of Legionella strains in France are different. J. clin. Microbiol.

  8. Bioinformatic tools and guideline for PCR primer design | Abd ...

    African Journals Online (AJOL)

    Bioinformatic tools and guideline for PCR primer design. ... AFRICAN JOURNALS ONLINE (AJOL) · Journals · Advanced Search · USING AJOL · RESOURCES ... Bioinformatics has become an essential tool not only for basic research but also ...

  9. Single-molecule emulsion PCR in microfluidic droplets.

    Science.gov (United States)

    Zhu, Zhi; Jenkins, Gareth; Zhang, Wenhua; Zhang, Mingxia; Guan, Zhichao; Yang, Chaoyong James

    2012-06-01

    The application of microfluidic droplet PCR for single-molecule amplification and analysis has recently been extensively studied. Microfluidic droplet technology has the advantages of compartmentalizing reactions into discrete volumes, performing highly parallel reactions in monodisperse droplets, reducing cross-contamination between droplets, eliminating PCR bias and nonspecific amplification, as well as enabling fast amplification with rapid thermocycling. Here, we have reviewed the important technical breakthroughs of microfluidic droplet PCR in the past five years and their applications to single-molecule amplification and analysis, such as high-throughput screening, next generation DNA sequencing, and quantitative detection of rare mutations. Although the utilization of microfluidic droplet single-molecule PCR is still in the early stages, its great potential has already been demonstrated and will provide novel solutions to today's biomedical engineering challenges in single-molecule amplification and analysis.

  10. Single base pair mutation analysis by PNA directed PCR clamping

    DEFF Research Database (Denmark)

    Ørum, H.; Nielsen, P.E.; Egholm, M.

    1993-01-01

    A novel method that allows direct analysis of single base mutation by the polymerase chain reaction (PCR) is described. The method utilizes the finding that PNAs (peptide nucleic acids) recognize and bind to their complementary nucleic acid sequences with higher thermal stability and specificity...... than the corresponding deoxyribooligonucleotides and that they cannot function as primers for DNA polymerases. We show that a PNA/DNA complex can effectively block the formation of a PCR product when the PNA is targeted against one of the PCR primer sites. Furthermore, we demonstrate that this blockage...... allows selective amplification/suppression of target sequences that differ by only one base pair. Finally we show that PNAs can be designed in such a way that blockage can be accomplished when the PNA target sequence is located between the PCR primers....

  11. Fluorescence Quantitative PCR Detected Infection of Condyloma Acuminatum

    Institute of Scientific and Technical Information of China (English)

    刘伟民; 杨华风; 高丽琴; 刁存英

    2002-01-01

    Objective: Infection of human papillomavirus in condylomaacuminatum (CA) was detected by real time fluorescencequantitative PCR (FQ-PCR) technique. Methods: Specimens of CA-DNA quantification from 94cases were examined by real time FQ-PCR technique and 32cases were compared with the same method after 10-daystreatment. Results: CA-DNA was found in all patients, with an averageof 4.0×106 copies/ul. After 10 days of treatment, the averagewas 2.1×105 copies/ul. There was a significant difference inthe average amount of CA-DNA before and after thetreatment. Conclusion: Real time FQ-PCR is a good method forexamining CA-DNA amount and it can direct the treatment of CA.

  12. Conjunctival swab PCR to detect Leishmania spp. in cats

    Directory of Open Access Journals (Sweden)

    Trícia Maria Ferreira de Sousa Oliveira

    Full Text Available The relevance of the dog as a source of visceral leishmaniasis infection is known, but the role of cats as reservoir hosts for leishmaniasis is not yet fully clear. This study assessed the efficacy of conjunctival swab PCR (CS-PCR in the detection of cats infected by Leishmania spp. The results were seven (13.5% cats positive for Leishmania spp. in the PCR, in 52 cats tested from Pirassunuga-SP and Ilha Solteira-SP. From the city of Pirassununga – SP 28.6% (2/7 were positive and from the city of Ilha Solteira – SP 11.1% (5/45 were positive. The results showed that CS-PCR was capable of detecting cats infected by this protozoan. Conjunctival swab samples proved easier to perform in cats, which might facilitate studies on the frequency and distribution of feline leishmaniasis.

  13. Conjunctival swab PCR to detect Leishmania spp. in cats.

    Science.gov (United States)

    Oliveira, Trícia Maria Ferreira de Sousa; Pereira, Vanessa Figueredo; Benvenga, Graziella Ulbricht; Martin, Maria Fernanda Alves; Benassi, Julia Cristina; da Silva, Diogo Tiago; Starke-Buzetti, Wilma Aparecida

    2015-01-01

    The relevance of the dog as a source of visceral leishmaniasis infection is known, but the role of cats as reservoir hosts for leishmaniasis is not yet fully clear. This study assessed the efficacy of conjunctival swab PCR (CS-PCR) in the detection of cats infected by Leishmania spp. The results were seven (13.5%) cats positive for Leishmania spp. in the PCR, in 52 cats tested from Pirassunuga-SP and Ilha Solteira-SP. From the city of Pirassununga - SP 28.6% (2/7) were positive and from the city of Ilha Solteira - SP 11.1% (5/45) were positive. The results showed that CS-PCR was capable of detecting cats infected by this protozoan. Conjunctival swab samples proved easier to perform in cats, which might facilitate studies on the frequency and distribution of feline leishmaniasis.

  14. Development of RT-PCR and Nested PCR for Detecting Four Quarantine Plant Viruses Belonging to Nepovirus

    Directory of Open Access Journals (Sweden)

    Siwon Lee

    2013-09-01

    Full Text Available For quarantine purpose, we developed the RT- and nested PCR module of Tomato black ring virus (TBRV, Arabis mosaic virus (ArMV, Cherry leafroll virus (CLRV and Grapevine fanleaf virus (GFLV. The PCR modules, developed in this study make diagnosis more convenient and speedy because of same PCR condition. And also, the methods are more accurate because it can check whether the result is contamination or not using the mutation-positive control. We discard or return the 27 cases of Nepovirus infection seed by employing the module past 3 years. This study provides a rapid and useful method for detection of four quarantine plant viruses.

  15. Comparison of PCR-ELISA and LightCycler real-time PCR assays for detecting Salmonella spp. in milk and meat samples

    DEFF Research Database (Denmark)

    Perelle, Sylvie; Dilasser, Françoise; Malorny, Burkhard

    2004-01-01

    , minced beef and raw milk, and 92 naturally-contaminated milk and meat samples. When using either PCR-ELISA or LC-PCR assays, only Salmonella strains were detected. PCR-ELISA and LC-PCR assays gave with pure Salmonella cultures the same detection limit level of 10(3) CFU/ml, which corresponds respectively...

  16. Identification of Phytophthora fragariae var. rubi by PCR.

    Science.gov (United States)

    Schlenzig, Alexandra

    2009-01-01

    The following chapter describes a PCR method for the identification of the raspberry root rot pathogen Phytophthora fragariae var. rubi. Furthermore, a nested PCR suitable for the detection of the pathogen in infected raspberry roots and validated against the "Duncan bait test" (EPPO Bull 35:87-91, 2005) is explained. Protocols for different DNA extraction methods are given which can be transferred to other fungal pathogens.

  17. A novel method of DNA shuffling without PCR process

    Institute of Scientific and Technical Information of China (English)

    WANG Qiang; LIU Qiuyun; LI Gang; LI Baojian

    2004-01-01

    Most DNA shuffling methods currently used require PCR process. A novel method of DNA shuffling without PCR process is described, taking advantage of the feature of some restriction enzymes whose recognition sites differ from their cleavage sites, thus giving rise to different cohesive ends.These cohesive ends can be rejoined at their native sites from different parental sequences, generating new sequences with various combinations of mutations.

  18. DNA Analysis of PCR-Inhibitory Forensic Samples

    OpenAIRE

    Hedman, Johannes

    2011-01-01

    DNA evidence, linking perpetrators to crime scenes, is central to many legal proceedings. However, crime scene samples often contain extraneous substances that may interfere with the PCR-based forensic analysis, resulting in partial or negative DNA profiles. Extensive DNA purification may remove inhibitors, but involves the risk of DNA loss. In this work, pre-PCR processing was applied to improve the success rate of forensic DNA analysis of “dirty” samples without interfering with the comp...

  19. Instrumentation and Fluorescent Chemistries Used in qPCR

    DEFF Research Database (Denmark)

    Josefsen, Mathilde Hartmann; Löfström, Charlotta; Hansen, Trine

    2012-01-01

    The polymerase chain reaction has revolutionized the world of scientific research and its broad application has caused a tremendous development of versatile PCR instruments and chemistries to fit its purpose. This chapter provides the reader with a general introduction to the basics of real-time ...... applied chemistries. The fluorophores and quenchers used for primer and probe labeling and their compatibility will be presented, and finally the future challenges and trends within the field of qPCR instrumentation will be discussed....

  20. Segmenting fluid effect on PCR reactions in microfluidic platforms.

    Science.gov (United States)

    Walsh, E J; King, C; Grimes, R; Gonzalez, A

    2005-12-01

    This paper evaluates the compatibility of segmenting fluids for two phase flow applications in biomedical microdevices. The evaluated fluids are chosen due to the variations in fluid properties and cost, while also reflecting their use in the recent literature. These segmenting fluids are examined to determine their compatibility with the Polymerase Chain Reaction (PCR), through controlled experiments. The results are the first to provide a quantitative measure of segmenting fluid compatibility with PCR.

  1. Template preparation for rapid PCR in Colletotrichum lindemuthianum

    Directory of Open Access Journals (Sweden)

    Roca M. Gabriela

    2003-01-01

    Full Text Available Isolation of DNA for PCR is time-consuming and involves many reagents. The aim of this work was to optimise a rapid and easy PCR methodology without previous DNA isolation. Different strains of the phytopathogenic fungus Colletotrichum lindemuthianum were used. Protoplasts were generated using lytic enzymes under high incubation temperatures using different methodologies to obtain the template. A rapid (10 minute methodology was successful for smaller amplicons (<750 bp.

  2. MPprimer: a program for reliable multiplex PCR primer design.

    Science.gov (United States)

    Shen, Zhiyong; Qu, Wubin; Wang, Wen; Lu, Yiming; Wu, Yonghong; Li, Zhifeng; Hang, Xingyi; Wang, Xiaolei; Zhao, Dongsheng; Zhang, Chenggang

    2010-03-18

    Multiplex PCR, defined as the simultaneous amplification of multiple regions of a DNA template or multiple DNA templates using more than one primer set (comprising a forward primer and a reverse primer) in one tube, has been widely used in diagnostic applications of clinical and environmental microbiology studies. However, primer design for multiplex PCR is still a challenging problem and several factors need to be considered. These problems include mis-priming due to nonspecific binding to non-target DNA templates, primer dimerization, and the inability to separate and purify DNA amplicons with similar electrophoretic mobility. A program named MPprimer was developed to help users for reliable multiplex PCR primer design. It employs the widely used primer design program Primer3 and the primer specificity evaluation program MFEprimer to design and evaluate the candidate primers based on genomic or transcript DNA database, followed by careful examination to avoid primer dimerization. The graph-expanding algorithm derived from the greedy algorithm was used to determine the optimal primer set combinations (PSCs) for multiplex PCR assay. In addition, MPprimer provides a virtual electrophotogram to help users choose the best PSC. The experimental validation from 2x to 5x plex PCR demonstrates the reliability of MPprimer. As another example, MPprimer is able to design the multiplex PCR primers for DMD (dystrophin gene which caused Duchenne Muscular Dystrophy), which has 79 exons, for 20x, 20x, 20x, 14x, and 5x plex PCR reactions in five tubes to detect underlying exon deletions. MPprimer is a valuable tool for designing specific, non-dimerizing primer set combinations with constrained amplicons size for multiplex PCR assays.

  3. MPprimer: a program for reliable multiplex PCR primer design

    Directory of Open Access Journals (Sweden)

    Wang Xiaolei

    2010-03-01

    Full Text Available Abstract Background Multiplex PCR, defined as the simultaneous amplification of multiple regions of a DNA template or multiple DNA templates using more than one primer set (comprising a forward primer and a reverse primer in one tube, has been widely used in diagnostic applications of clinical and environmental microbiology studies. However, primer design for multiplex PCR is still a challenging problem and several factors need to be considered. These problems include mis-priming due to nonspecific binding to non-target DNA templates, primer dimerization, and the inability to separate and purify DNA amplicons with similar electrophoretic mobility. Results A program named MPprimer was developed to help users for reliable multiplex PCR primer design. It employs the widely used primer design program Primer3 and the primer specificity evaluation program MFEprimer to design and evaluate the candidate primers based on genomic or transcript DNA database, followed by careful examination to avoid primer dimerization. The graph-expanding algorithm derived from the greedy algorithm was used to determine the optimal primer set combinations (PSCs for multiplex PCR assay. In addition, MPprimer provides a virtual electrophotogram to help users choose the best PSC. The experimental validation from 2× to 5× plex PCR demonstrates the reliability of MPprimer. As another example, MPprimer is able to design the multiplex PCR primers for DMD (dystrophin gene which caused Duchenne Muscular Dystrophy, which has 79 exons, for 20×, 20×, 20×, 14×, and 5× plex PCR reactions in five tubes to detect underlying exon deletions. Conclusions MPprimer is a valuable tool for designing specific, non-dimerizing primer set combinations with constrained amplicons size for multiplex PCR assays.

  4. PCR performance of a thermostable heterodimeric archaeal DNA polymerase

    Directory of Open Access Journals (Sweden)

    Tom eKillelea

    2014-05-01

    Full Text Available DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR, cDNA cloning, genome sequencing and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3’ primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications.

  5. PCR performance of a thermostable heterodimeric archaeal DNA polymerase

    Science.gov (United States)

    Killelea, Tom; Ralec, Céline; Bossé, Audrey; Henneke, Ghislaine

    2014-01-01

    DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing, and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3' primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications. PMID:24847315

  6. Quantification of HEV RNA by Droplet Digital PCR

    Directory of Open Access Journals (Sweden)

    Florence Nicot

    2016-08-01

    Full Text Available The sensitivity of real-time PCR for hepatitis E virus (HEV RNA quantification differs greatly among techniques. Standardized tools that measure the real quantity of virus are needed. We assessed the performance of a reverse transcription droplet digital PCR (RT-ddPCR assay that gives absolute quantities of HEV RNA. Analytical and clinical validation was done on HEV genotypes 1, 3 and 4, and was based on open reading frame (ORF3 amplification. The within-run and between-run reproducibilities were very good, the analytical sensitivity was 80 HEV RNA international units (IU/mL and linearities of HEV genotype 1, 3 and 4 were very similar. Clinical validation based on 45 samples of genotype 1, 3 or 4 gave results that correlated well with a validated reverse transcription quantitative PCR (RT-qPCR assay (Spearman rs = 0.89, p < 0.0001. The RT-ddPCR assay is a sensitive method and could be a promising tool for standardizing HEV RNA quantification in various sample types.

  7. PCR performance of a thermostable heterodimeric archaeal DNA polymerase.

    Science.gov (United States)

    Killelea, Tom; Ralec, Céline; Bossé, Audrey; Henneke, Ghislaine

    2014-01-01

    DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing, and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3' primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications.

  8. IDENTIFIKASI DAGING BABI MENGGUNAKAN METODE PCR-RFLP GEN Cytochrome b DAN PCR PRIMER SPESIFIK GEN AMELOGENIN (Pork Identification Using PCR-RFLP of Cytochrome b Gene and Species Specific PCR of Amelogenin Gene

    Directory of Open Access Journals (Sweden)

    Yuny Erwanto

    2013-03-01

    Full Text Available A polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP and species specific PCR methods had been applied for identifying pork in mixture of meat. Pork sample in various levels (1, 3, 5 and 10% was prepared in mixture with beef, chicken and mutton. The primary CYTb1 and CYTb2 were designed in the mitochondrial cytochrome b b (cytochrome b gene and PCR successfully amplified fragments of 359 bp. To distinguish pig species existence, the amplified PCR products of mitochondrial DNA were cut by BseDI restriction enzyme. The result showed that pig mitochondrial DNA was cut into 131 and 228 bp fragments. A polymerase chain reaction (PCR method based on the nucleotide sequence variation in the amelogenin gene has been chosen for the specific identification of pork DNAs in mixture meat. The primers designed generated specific fragments of 353 and 312 bp length for pork. The specificity of the primary designed was tested on 4 animal species including pig, cattle, chicken and goat species. Analysis of experimental mixture meat demonstrated that 1% of raw pork tissues could be detected using PCR-RFLP with BseDI restriction enzyme but detection using species-specific PCR showed the cross reactivity to beef, chicken and mutton. The cytochrome b PCR-RFLP species identification assay yielded excellent results for identification of pig species. PCR-RFLP is a potentially reliable technique for detection of the existence of pork in animal food product for Halal authentication. Keywords: Pork identification, cytochrome b, amelogenin, polymerase chain reaction   ABSTRAK   Penelitian ini dilakukan untuk mengaplikasikan metode deteksi daging babi dalam campuan daging dengan sapi, kambing dan ayam melalui PCR-RFLP dan PCR dengan primer spesifik untuk babi. Level kontaminasi daging babi dibuat sebesar 1, 3, 5 dan 10% dari total daging dalam campuran. Metode PCR-RFLP menggunakan sepasang primer yaitu gen cytochrome b dari mitokondria yang

  9. Absolute quantification of olive oil DNA by droplet digital-PCR (ddPCR): Comparison of isolation and amplification methodologies.

    Science.gov (United States)

    Scollo, Francesco; Egea, Leticia A; Gentile, Alessandra; La Malfa, Stefano; Dorado, Gabriel; Hernandez, Pilar

    2016-12-15

    Olive oil is considered a premium product for its nutritional value and health benefits, and the ability to define its origin and varietal composition is a key step towards ensuring the traceability of the product. However, isolating the DNA from such a matrix is a difficult task. In this study, the quality and quantity of olive oil DNA, isolated using four different DNA isolation protocols, was evaluated using the qRT-PCR and ddPCR techniques. The results indicate that CTAB-based extraction methods were the best for unfiltered oil, while Nucleo Spin-based extraction protocols showed greater overall reproducibility. The use of both qRT-PCR and ddPCR led to the absolute quantification of the DNA copy number. The results clearly demonstrate the importance of the choice of DNA-isolation protocol, which should take into consideration the qualitative aspects of DNA and the evaluation of the amplified DNA copy number.

  10. 基于随机引物的PCR-Walking技术%PCR-Walking Techniques Based on Random Primer

    Institute of Scientific and Technical Information of China (English)

    刘春香

    2010-01-01

    染色体步行技术越来越倾向于采用PCR技术,不同随机引物的应用产生了不同的方法.针对目前应用较广的热不对称交错PCR(TAIL-PCR)、单寡核苷酸巢式PCR(SON-PCR)、基于RAPD原理的单引物PCR、位点寻找PCR和自我形成接头式PCR进行了介绍,侧重说明了热不对称交错PCRTAIL-PCR的原理、优缺点及应用.

  11. Comparison of conventional PCR, multiplex PCR, and loop-mediated isothermal amplification assays for rapid detection of Arcobacter species.

    Science.gov (United States)

    Wang, Xiaoyu; Seo, Dong Joo; Lee, Min Hwa; Choi, Changsun

    2014-02-01

    This study aimed to develop a loop-mediated isothermal amplification (LAMP) method for the rapid detection of Arcobacter species. Specific primers targeting the 23S ribosomal RNA gene were used to detect Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii. The specificity of the LAMP primer set was assessed using DNA samples from a panel of Arcobacter and Campylobacter species, and the sensitivity was determined using serial dilutions of Arcobacter species cultures. LAMP showed a 10- to 1,000-fold-higher sensitivity than multiplex PCR, with a detection limit of 2 to 20 CFU per reaction in vitro. Whereas multiplex PCR showed cross-reactivity with Campylobacter species, the LAMP method developed in this study was more sensitive and reliable than conventional PCR or multiplex PCR for the detection of Arcobacter species.

  12. Comparative validation using quantitative real-time PCR (qPCR and conventional PCR of bovine semen centrifuged in continuous density gradient

    Directory of Open Access Journals (Sweden)

    M.V. Resende

    2011-06-01

    Full Text Available The objective of the present study was to determine the sperm enrichment with X-bearing spermatozoa, after one centrifugation in a Percoll or OptiPrep continuous density gradient, using quantitative real-time polymerase chain reaction (qPCR of sperm DNA and resultant in vitro-produced bovine embryos by PCR. Frozen/thawed sperm was layered on density gradients and the tubes were centrifuged. Supernatants were gently aspirated and the sperm recovered from the bottom of the tubes. Cleavage and blastocyst rates were determined through in vitro production of embryos and PCR was performed to identify the embryos' genetic sex. A difference in blastocyst rate was found in the Percoll treatment compared to OptiPrep (P<0.05. The percentage of female embryos in the Percoll and OptiPrep groups was 62.0% and 47.1%, respectively. These results were confirmed by qPCR of spermatozoa DNA and underestimation was seen only in the Percoll group. It was possible to sexing sperm using simple approach.

  13. Comparison of Droplet Digital PCR and qPCR for the Quantification of Shiga Toxin-Producing Escherichia coli in Bovine Feces.

    Science.gov (United States)

    Verhaegen, Bavo; De Reu, Koen; De Zutter, Lieven; Verstraete, Karen; Heyndrickx, Marc; Van Coillie, Els

    2016-05-18

    Cattle are considered to be the main reservoir for Shiga toxin-producing Escherichia coli (STEC) and are often the direct or indirect source of STEC outbreaks in humans. Accurate measurement of the concentration of shed STEC in cattle feces could be a key answer to questions concerning transmission of STEC, contamination sources and efficiency of treatments at farm level. Infected animals can be identified and the contamination level quantified by real-time quantitative PCR (qPCR), which has its specific limitations. Droplet digital PCR (ddPCR) has been proposed as a method to overcome many of the drawbacks of qPCR. This end-point amplification PCR is capable of absolute quantification independent from any reference material and is less prone to PCR inhibition than qPCR. In this study, the qPCR-based protocol described by Verstraete et al. (2014) for Shiga toxin genes stx1 and stx2 and the intimin gene eae quantification was optimized for ddPCR analysis. The properties of ddPCR and qPCR using two different mastermixes (EMM: TaqMan(®) Environmental Master Mix 2.0; UMM: TaqMan(®) Universal PCR Master Mix) were evaluated, using standard curves and both artificial and natural contaminated cattle fecal samples. In addition, the susceptibility of these assays to PCR-inhibitors was investigated. Evaluation of the standard curves and both artificial and natural contaminated cattle fecal samples suggested a very good agreement between qPCR using EMM and ddPCR. Furthermore, similar sensitivities and no PCR inhibition were recorded for both assays. On the other hand, qPCR using UMM was clearly prone to PCR inhibition. In conclusion, the ddPCR technique shows potential for the accurate absolute quantification of STEC on the farms, without relying on standardized reference material.

  14. Comparison of Droplet Digital PCR and qPCR for the Quantification of Shiga Toxin-Producing Escherichia coli in Bovine Feces

    Directory of Open Access Journals (Sweden)

    Bavo Verhaegen

    2016-05-01

    Full Text Available Cattle are considered to be the main reservoir for Shiga toxin-producing Escherichia coli (STEC and are often the direct or indirect source of STEC outbreaks in humans. Accurate measurement of the concentration of shed STEC in cattle feces could be a key answer to questions concerning transmission of STEC, contamination sources and efficiency of treatments at farm level. Infected animals can be identified and the contamination level quantified by real-time quantitative PCR (qPCR, which has its specific limitations. Droplet digital PCR (ddPCR has been proposed as a method to overcome many of the drawbacks of qPCR. This end-point amplification PCR is capable of absolute quantification independent from any reference material and is less prone to PCR inhibition than qPCR. In this study, the qPCR-based protocol described by Verstraete et al. (2014 for Shiga toxin genes stx1 and stx2 and the intimin gene eae quantification was optimized for ddPCR analysis. The properties of ddPCR and qPCR using two different mastermixes (EMM: TaqMan® Environmental Master Mix 2.0; UMM: TaqMan® Universal PCR Master Mix were evaluated, using standard curves and both artificial and natural contaminated cattle fecal samples. In addition, the susceptibility of these assays to PCR-inhibitors was investigated. Evaluation of the standard curves and both artificial and natural contaminated cattle fecal samples suggested a very good agreement between qPCR using EMM and ddPCR. Furthermore, similar sensitivities and no PCR inhibition were recorded for both assays. On the other hand, qPCR using UMM was clearly prone to PCR inhibition. In conclusion, the ddPCR technique shows potential for the accurate absolute quantification of STEC on the farms, without relying on standardized reference material.

  15. 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

  16. A survey of polymerase chain reaction (PCR) amplification studies of unicellular protists using single-cell PCR.

    Science.gov (United States)

    Lynn, Denis H; Pinheiro, Marcel

    2009-01-01

    We surveyed a variety of studies that have used single-cell polymerase chain reaction (SC-PCR) to examine the gene sequences of a diversity of unicellular protists. Representatives of all the Super-Groups of eukaryotes have been subjected to SC-PCR with ciliates and dinoflagellates being most commonly examined. The SC-PCR was carried out either by directly amplifying a single lysed cell or by first extracting DNA and following this with amplification of the DNA extract. Cell lysis methods included heating, freezing, mechanical rupture, and enzyme digestion. Cells fixed or preserved with ethanol, methanol, and Lugol's have also been used successfully. Heminested or seminested PCR might follow the initial PCR, whose products were then directly sequenced or cloned and then sequenced. The methods are not complicated. This should encourage protistologists to use SC-PCR in the description of new or revised taxa, especially rare and unculturable forms, and it should also enable the probing of gene expression in relation to life history stages.

  17. Sensitive detection of mRNA decay products by use of reverse-ligation-mediated PCR (RL-PCR).

    Science.gov (United States)

    Grange, Thierry

    2008-01-01

    Ligation-mediated PCR allows the detection and mapping of cleavage products of specific nucleic acid molecules out of complex nucleic acid mixtures. It can be applied to the detection of either degradation products of exogenously added nucleases in footprinting applications or natural decay products or reaction intermediates. We have developed various ligation-mediated PCR approaches to analyze mRNAs, all relying on RNA ligation, followed by reverse-transcription and PCR amplification. We have termed these approaches reverse-ligation-mediated PCR (RL-PCR). The ligation event involves either an RNA linker added to the 5'-end of cleaved RNA or RNA circularization, allowing, respectively, the mapping and quantification of the cleavage points or the simultaneous analysis of the presence or absence of the 5'-cap structure and the length of the poly(A) tail. These methods enabled us to develop a very efficient 5'-RACE procedure to map mRNA 5'-ends, to footprint in permeabilized cells the interaction of regulatory proteins with RNA, to detect the products of cellular ribozyme action and to analyze cellular decay pathways that involve deadenylation and/or decapping. I review herein the methodologic aspects and protocols of the various RL-PCR procedures we have developed.

  18. Inhibitory effect of common microfluidic materials on PCR outcome

    KAUST Repository

    Kodzius, Rimantas

    2012-02-20

    Microfluidic chips have a variety of applications in the biological sciences and medicine. In contrast with traditional experimental approaches, microfluidics entails lower sample and reagent consumption, allows faster reactions and enables efficient separation. Additionally microfluidics offers other advantages accruing from the fluids’ various distinct behaviors, such as energy dissipation, fluidic resistance, laminar flow, and surface tension. Biological molecules suspended in fluid and transported through microfluidics channels interact with the channel-wall material. This interaction is even stronger in high surface-area-to-volume ratio (SAVR) microfluidic channels. Adsorption and inhibition of biomolecules occur when these materials come in contact with biomolecular reaction components. Polymerase chain reaction (PCR) is a thermal cycling procedure for amplifying target DNA. The PCR compatibility of silicon, silicon dioxide (SiO2) and other surfaces have been studied; however the results are inconclusive. Usually for protein-surface interaction measurements, bulky and expensive equipment is used, such as Atomic Force Microscopy (AFM), Scanning or Transmission Electron Microscopy (SEM, TEM), spectrophotometric protein concentration measurement, Fourier transform infrared spectroscopy (FTIR) or X-Ray photoelectron spectroscopy (XPS). \\tThe PCR reaction components include the DNA template, primers, DNA polymerase (the main component), dNTPs, a buffer, divalent ions (MgCl2), and KCl. \\tWe designed a simple, relatively quick measurement that only requires a PCR cycler; thus it mimics actual conditions in PCR cycling. In our study, we evaluated the inhibitory affect of different materials on PCR, which is one of the most frequently used enzymatic reactions in microfluidics. PCR reaction optimization through choice of surface materials is of the upmost importance, as it enables and improves enzymatic reaction in microfluidics. Our assessment of the PCR

  19. 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

  20. Inverse PCR for subtyping of Acinetobacter baumannii carrying ISAba1.

    Science.gov (United States)

    Kim, Shukho; Park, Yun-Ju; Kim, Jungmin

    2016-05-01

    Acinetobacter baumannii has been prevalent in nosocomial infections, often causing outbreaks in intensive care units. ISAba1 is an insertion sequence that has been identified only in A. baumannii and its copy number varies among strains. It has been reported that ISAba1 provides a promoter for bla(OXA-51-like), bla(OXA-23-like), and bla(ampC), which are associated with the resistance of A. baumannii to carbapenems and cephalosporins. The main purpose of this study was to develop a novel inverse PCR method capable of typing A. baumannii strains. The method involves three major steps: cutting of genomic DNA with a restriction enzyme, ligation, and PCR. In the first step, bacterial genomic DNA was digested with DpnI. In the second step, the digested genomic DNAs were ligated to form intramolecular circular DNAs. In the last step, the ligated circular DNAs were amplified by PCR with primers specific for ISAba1 and the amplified PCR products were electrophoresed. Twenty-two clinical isolates of A. baumannii were used for the evaluation of the inverse PCR (iPCR) typing method. Dendrogram analysis revealed two major clusters, similar to pulsed-field gel electrophoresis (PFGE) results. Three ISAba1-associated genes--bla(ampC), bla(OXA-66-like), and csuD--were amplified and detected in the clinical isolates. This novel iPCR typing method is comparable to PFGE in its ability to discriminate A. baumannii strains, and is a promising molecular epidemiological tool for investigating A. baumannii carrying ISAba1.

  1. Droplet-based micro oscillating-flow PCR chip

    Science.gov (United States)

    Wang, Wei; Li, Zhi-Xin; Luo, Rong; Lü, Shu-Hai; Xu, Ai-Dong; Yang, Yong-Jun

    2005-08-01

    Polymerase chain reactions (PCR), thermally activated chemical reactions which are widely used for nucleic acid amplification, have recently received much attention in microelectromechanical systems and micro total analysis systems because a wide variety of DNA/RNA molecules can be enriched by PCR for further analyses. In the present work, a droplet-based micro oscillating-flow PCR chip was designed and fabricated by the silicon microfabrication technique. Three different temperature zones, which were stable at denaturation, extension and annealing temperatures and isolated from each other by a thin-wall linkage, were integrated with a single, simple and straight microchannel to form the chip's basic functional structure. The PCR mixture was injected into the chip as a single droplet and flowed through the three temperature zones in the main microchannel in an oscillating manner to achieve the temperature maintenance and transitions. The chip's thermal performance was theoretically analyzed and numerically simulated. The results indicated that the time needed for the temperature of the droplet to change to the target value is less than 1 s, and the root mean square error of temperature is less than 0.2 °C. A droplet of 1 µl PCR mixture with standard HPV (Human Papilloma Virus)-DNA sample inside was amplified by the present chip and the results were analyzed by slab gel electrophoresis with separation of DNA markers in parallel. The electrophoresis results demonstrated that the micro oscillating-flow PCR chip successfully amplified the HPV-DNA, with a processing time of about 15 min which is significantly reduced compared to that for the conventional PCR instrument.

  2. 侵染节瓜的3种病毒多重PCR检测体系的建立%Development of a Multiplex RT-PCR Protocol for the Detection of Three Viruses on Benincasa hispida. var. chieh-qua

    Institute of Scientific and Technical Information of China (English)

    赵芹; 李华平; 谢大森; 罗少波; 彭庆务

    2011-01-01

    According to the conserved nucleotide sequences of coat protein genes of Zucchini yellow mosaic virus(ZYMV),Papaya ring spot virus(PRSV)and Cucumber mosaic virus(CMV)infected chieh-qua specific primers were designed. Based on the establishment of the optimal RT-PCR for the single detection of ZYMV,PRSV and CMV,a multiplex RT-PCR protocol for the detection of three viruses on chieh-qua was developed,which provided a simple,rapid and economic method for simultaneous detection of three chieh-qua viruses to support the pathogen detection of chieh-qua technically and theoretically.%根据侵染节瓜3种病毒——小西葫芦黄花叶病毒(ZYMV)、番木瓜环斑病毒(PRSV)和黄瓜花叶病毒(CMV)的外壳蛋白基因保守区序列设计特异引物,在建立各种病毒单项PCR技术的基础上,通过优化多重PCR反应条件,初步建立了能同步、快速、灵敏地检测这3种病毒的多重PCR检测体系,为节瓜病毒病原的检测提供了理论与技术支持。

  3. PCR-based identification of Burkholderia pseudomallei Identificação de Burkholderia pseudomallei baseada em PCR

    Directory of Open Access Journals (Sweden)

    Adam Merritt

    2006-10-01

    Full Text Available DNA amplification techniques are being used increasingly in clinical laboratories to confirm the identity of medically important bacteria. A PCR-based identification method has been in use in our centre for 10 years for Burkholderia pseudomallei and was used to confirm the identity of bacteria isolated from cases of melioidosis in Ceará since 2003. This particular method has been used as a reference standard for less discriminatory methods. In this study we evaluated three PCR-based methods of B. pseudomallei identification and used DNA sequencing to resolve discrepancies between PCR-based results and phenotypic identification methods. The established semi-nested PCR protocol for B. pseudomallei 16-23s spacer region produced a consistent negative result for one of our 100 test isolates (BCC #99, but correctly identified all 71 other B. pseudomallei isolates tested. Anomalous sequence variation was detected at the inner, reverse primer binding site for this method. PCR methods were developed for detection of two other B. pseudomallei bacterial metabolic genes. The conventional lpxO PCR protocol had a sensitivity of 0.89 and a specificity of 1.00, while a real-time lpxO protocol performed even better with sensitivity and specificity of 1.00, and 1.00. This method identified all B. pseudomallei isolates including the PCR-negative discrepant isolate. The phaC PCR protocol detected the gene in all B. pseudomallei and all but three B. cepacia isolates, making this method unsuitable for PCR-based identification of B. pseudomallei. This experience with PCR-based B. pseudomallei identification methods indicates that single PCR targets should be used with caution for identification of these bacteria, and need to be interpreted alongside phenotypic and alternative molecular methods such as gene sequencing.As técnicas de amplificação de DNA estão sendo cada vez mais utilizadas em laboratórios clínicos para a confirmação da identificação de bact

  4. Efficacy of SSH PCR in isolating differentially expressed genes

    Directory of Open Access Journals (Sweden)

    Cai Li

    2002-05-01

    Full Text Available Abstract Background Suppression Subtractive Hybridization PCR (SSH PCR is a sophisticated cDNA subtraction method to enrich and isolate differentially expressed genes. Despite its popularity, the method has not been thoroughly studied for its practical efficacy and potential limitations. Results To determine the factors that influence the efficacy of SSH PCR, a theoretical model, under the assumption that cDNA hybridization follows the ideal second kinetic order, is proposed. The theoretical model suggests that the critical factor influencing the efficacy of SSH PCR is the concentration ratio (R of a target gene between two cDNA preparations. It preferentially enriches "all or nothing" differentially expressed genes, of which R is infinite, and strongly favors the genes with large R. The theoretical predictions were validated by our experiments. In addition, the experiments revealed some practical limitations that are not obvious from the theoretical model. For effective enrichment of differentially expressed genes, it requires fractional concentration of a target gene to be more than 0.01% and concentration ratio to be more than 5 folds between two cDNA preparations. Conclusion Our research demonstrated theoretical and practical limitations of SSH PCR, which could be useful for its experimental design and interpretation.

  5. Electrochemistry-based real-time PCR on a microchip.

    Science.gov (United States)

    Yeung, Stephen S W; Lee, Thomas M H; Hsing, I-Ming

    2008-01-15

    The development of handheld instruments for point-of-care DNA analysis can potentially contribute to the medical diagnostics and environmental monitoring for decentralized applications. In this work, we demonstrate the implementation of a recently developed electrochemical real-time polymerase chain reaction (ERT-PCR) technique on a silicon-glass microchip for simultaneous DNA amplification and detection. This on-chip ERT-PCR process requires the extension of an oligonucleotide in both solution and at solid phases and intermittent electrochemical signal measurement in the presence of all the PCR reagents. Several important parameters, related to the surface passivation and electrochemical scanning of working electrodes, were investigated. It was found that the ERT-PCR's onset thermal cycle ( approximately 3-5), where the analytical signal begins to be distinguishable from the background, is much lower than that of the fluorescence-based counterparts for high template DNA situations (3 x 10(6) copies/microL). By carefully controlling the concentrations of the immobilized probe and the enzyme polymerase, improvements have been made in obtaining a meaningful electrochemical signal using a lower initial template concentration. This ERT-PCR technique on a microchip platform holds significant promise for rapid DNA detection for point-of-care testing applications.

  6. Development of internal controls for PCR detection of Bacillus anthracis.

    Science.gov (United States)

    Brightwell, G; Pearce, M; Leslie, D

    1998-12-01

    This work describes the development and evaluation of a multiplex polymerase chain reaction (PCR) for the detection of Bacillus anthracis strains harbouring plasmid pX02. The multiplex also incorporated an internal control (IC) to avoid false negative reactions. Internal controls consisted of plasmids containing modified PCR target sequences, corresponding to the capC and BA813 genes of B. anthracis, which were then co-amplified with the original target sequences using the same set of amplimers. The initial IC construct comprised of an internally deleted form of the genomic target sequence cloned into pUC19. A series of nested DNA fragments corresponding to the 23S rRNA sequences of Bacillus cereus were then subcloned into the point of deletion, producing a number of IC constructs with similar sequences but increasing product size on PCR amplification. Neither the presence of IC DNA template or IC PCR product size affected the specificity or non-specific cross-reactivity of the original PCR assay. The concentration of IC was critical, too much IC DNA template would out compete the genomic DNA template, thus giving a false negative result. However, when the concentration of IC was optimal assay sensitivity was not compromised.

  7. Multiplex PCR: Optimization and Application in Diagnostic Virology

    Science.gov (United States)

    Elnifro, Elfath M.; Ashshi, Ahmed M.; Cooper, Robert J.; Klapper, Paul E.

    2000-01-01

    PCR has revolutionized the field of infectious disease diagnosis. To overcome the inherent disadvantage of cost and to improve the diagnostic capacity of the test, multiplex PCR, a variant of the test in which more than one target sequence is amplified using more than one pair of primers, has been developed. Multiplex PCRs to detect viral, bacterial, and/or other infectious agents in one reaction tube have been described. Early studies highlighted the obstacles that can jeopardize the production of sensitive and specific multiplex assays, but more recent studies have provided systematic protocols and technical improvements for simple test design. The most useful of these are the empirical choice of oligonucleotide primers and the use of hot start-based PCR methodology. These advances along with others to enhance sensitivity and specificity and to facilitate automation have resulted in the appearance of numerous publications regarding the application of multiplex PCR in the diagnosis of infectious agents, especially those which target viral nucleic acids. This article reviews the principles, optimization, and application of multiplex PCR for the detection of viruses of clinical and epidemiological importance. PMID:11023957

  8. Digital PCR using micropatterned superporous absorbent array chips.

    Science.gov (United States)

    Wang, Yazhen; Southard, Kristopher M; Zeng, Yong

    2016-06-21

    Digital PCR (dPCR) is an emerging technology for genetic analysis and clinical diagnostics. To facilitate the widespread application of dPCR, here we developed a new micropatterned superporous absorbent array chip (μSAAC) which consists of an array of microwells packed with highly porous agarose microbeads. The packed beads construct a hierarchically porous microgel which confers superior water adsorption capacity to enable spontaneous filling of PDMS microwells for fluid compartmentalization without the need of sophisticated microfluidic equipment and operation expertise. Using large λ-DNA as the model template, we validated the μSAAC for stochastic partitioning and quantitative digital detection of DNA molecules. Furthermore, as a proof-of-concept, we conducted dPCR detection and single-molecule sequencing of a mutation prevalent in blood cancer, the chromosomal translocation t(14;18), demonstrating the feasibility of the μSAAC for analysis of disease-associated mutations. These experiments were carried out using the standard molecular biology techniques and instruments. Because of its low cost, ease of fabrication, and equipment-free liquid partitioning, the μSAAC is readily adaptable to general lab settings, which could significantly facilitate the widespread application of dPCR technology in basic research and clinical practice.

  9. Simultaneous detection of HBV and HCV by multiplex PCR normalization

    Institute of Scientific and Technical Information of China (English)

    Ning Wang; Xue-Qin Gao; Jin-Xiang Han

    2004-01-01

    AIM: To design and establish a method of multiplex PCR normalization for simultaneously detecting HBV and HCV.METHODS: Two pairs of primers with a 20 bp joint sequence were used to amplify the target genes of HBV and HCV by two rounds of amplification. After the two rounds of amplification all the products had the joint sequence. Then the joint sequence was used as primers to finish the last amplification. Finally multiplex PCR was normalized to a single PCR system to eliminate multiplex factor interference. Four kinds of nucleic acid extraction methods were compared and screened. A multiplex PCR normalization method was established and optimized by orthogonal design of 6 key factors. Then twenty serum samples were detected to evaluate the validity and authenticity of this method.RESULTS: The sensitivity, specificity, diagnostic index and efficiency were 83.3%, 70%, 153.3% and 72.2%,respectively for both HBsAg and anti-HCV positive patients,and were 78.6%, 80%, 258.6% and 79.2%, respectively for HBsAg positive patients, and were 75%, 90%, 165%and 83.3%, respectively for anti-HCV positive patients.CONCLUSION: The multiplex PCR normalization method shows a broad prospect in simultaneous amplification of multiple genes of different sources. It is practical, correct and authentic, and can be used to prevent and control HBV and HCV.

  10. Design and optimization of reverse-transcription quantitative PCR experiments.

    Science.gov (United States)

    Tichopad, Ales; Kitchen, Rob; Riedmaier, Irmgard; Becker, Christiane; Ståhlberg, Anders; Kubista, Mikael

    2009-10-01

    Quantitative PCR (qPCR) is a valuable technique for accurately and reliably profiling and quantifying gene expression. Typically, samples obtained from the organism of study have to be processed via several preparative steps before qPCR. We estimated the errors of sample withdrawal and extraction, reverse transcription (RT), and qPCR that are introduced into measurements of mRNA concentrations. We performed hierarchically arranged experiments with 3 animals, 3 samples, 3 RT reactions, and 3 qPCRs and quantified the expression of several genes in solid tissue, blood, cell culture, and single cells. A nested ANOVA design was used to model the experiments, and relative and absolute errors were calculated with this model for each processing level in the hierarchical design. We found that intersubject differences became easily confounded by sample heterogeneity for single cells and solid tissue. In cell cultures and blood, the noise from the RT and qPCR steps contributed substantially to the overall error because the sampling noise was less pronounced. We recommend the use of sample replicates preferentially to any other replicates when working with solid tissue, cell cultures, and single cells, and we recommend the use of RT replicates when working with blood. We show how an optimal sampling plan can be calculated for a limited budget. .

  11. Sensitive Detection of Giardia Cysts by Polymerase Chain Reaction (PCR

    Directory of Open Access Journals (Sweden)

    M Nikaeen

    2003-07-01

    Full Text Available Giardia is one of the most common human parasites and causes a lengthly course of nonbacterial diarrhea. Disease outbreaks due to Giardia infection are often attributed to contaminated water supplies. A major problem associated with detection for this organism is the lack of sensitive and reliable methods. PCR has the potential to address many of the limitations.We have performed a PCR-based method for sensitive detection of Giardia cysts. Because the sensitivity of PCR is a function of the efficiency of DNA extraction from cysts, we have also compared some different methods for DNA extraction from the cysts. Giardia cysts were collected from infected human, partially purified and serially diluted samples were prepared. DNA was extracted by 3 different methods and we found that simple repeated freezing and thawing was the best method for extraction of DNA from the cysts. A 163 bp conserved fragment related to the giardial heat shock protein (HSP70 gene was used as the target for PCR amplification. We were able to detect as few as 5 cysts in the samples. The results suggest the potential utilities of PCR for sensitive detection of Giardia in water sources.

  12. Result Variation and Efficiency Kinetics in Real-Time PCR

    Directory of Open Access Journals (Sweden)

    Reza Shahsiah

    2010-10-01

    Full Text Available Fluorescent monitoring of DNA amplification is the basis of real-time PCR. Absolute quantification can be achieved using a standard curve method. The standard curve is constructed by amplifying known amounts of standards under identical conditions to that of the samples.The objective of the current study is to propose a mathematical model to assess the acceptability of PCR resulys.Four commercial standards for HCV-RNA (hepatitis C virus RNA along with 6 patient samples were measured by real-time PCR, using two different RT-PCR reagents. The standard deviation of regression (Sy,x was calculated for each group of standard and compared by F-Test. The efficiency kinetics was computed by logistic regression, c2 goodness of fit test was preformed to assess the appropriateness of the efficiency curves.Calculated efficiencies were not significantly different from the value predicted by logistic regression model. Reactions with more variation showed less stable efficiency curves, with wider range of amplification efficiencies.Amplification efficiency kinetics can be computed by fitting a logistic regression curve to the gathered fluorescent data of each reaction. This model can be employed to assess the acceptability of PCR results calculated by standard curve method.

  13. Application of PCR and real-time PCR for monitoring cyanobacteria, Microcystis spp. and Cylindrospermopsis raciborskii in Macau freshwater reservoir

    Science.gov (United States)

    Zhang, Weiying; Lou, Inchio; Ung, Wai Kin; Kong, Yijun; Mok, Kai Meng

    2014-06-01

    Freshwater algal blooms have become a growing concern world-wide. They are caused by a high level of cyanobacteria, predominantly Microcystis spp. and Cylindrospermopsis raciborskii, which can produce microcystin and cylindrospermopsin, respectively. Longtime exposure to these cyanotoxins may affect public health, thus reliable detection, quantification, and enumeration of these harmful algae species has become a priority in water quality management. Traditional manual enumeration of algal bloom cells primarily involves microscopic identification which limited by inaccuracy and time-consumption.With the development of molecular techniques and an increasing number of microbial sequences available in the Genbank database, the use of molecular methods can be used for more rapid, reliable, and accurate detection and quantification. In this study, multiplex polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR) techniques were developed and applied for monitoring cyanobacteria Microcystis spp. and C. raciborskii in the Macau Storage Reservoir (MSR). The results showed that the techniques were successful for identifying and quantifying the species in pure cultures and mixed cultures, and proved to be a potential application for water sampling in MSR. When the target species were above 1 million cells/L, similar cell numbers estimated by microscopic enumeration and qPCR were obtained. Further quantification in water samples indicated that the ratio of the estimated number of cell by microscopy and qPCR was 0.4-12.9 for cyanobacteria and 0.2-3.9 for C. raciborskii. However, Microcystis spp. was not observed by manual enumeration, while it was detected at low levels by qPCR, suggesting that qPCR is more sensitive and accurate. Thus the molecular approaches provide an additional reliable monitoring option to traditional microscopic enumeration for the ecosystems monitoring program.

  14. Rapid detection and ruling out of neonatal sepsis by PCR coupled with Electrospray Ionization Mass Spectrometry (PCR/ESI-MS).

    Science.gov (United States)

    Delcò, Cristina; Karam, Oliver; Pfister, Riccardo; Gervaix, Alain; Renzi, Gesuele; Emonet, Stéphane; Schrenzel, Jacques; Posfay-Barbe, Klara M

    2017-05-01

    Sepsis is an important cause of morbidity and mortality in neonates and clinicians are typically required to administer empiric antibiotics while waiting for blood culture results. However, prolonged and inappropriate use of antibiotics is associated with various complications and adverse events. Better tools to rapidly rule out bacterial infections are therefore needed. We aimed to assess the negative predictive value of PCR coupled with Electrospray Ionization Mass Spectrometry (PCR/ESI-MS) compared to conventional blood cultures in neonatal sepsis. Prospective observational study. All consecutive neonates (PCR/ESI-MS analysis were collected at the same time as samples for the blood culture, before the initiation of antibiotics. Our primary objective was to evaluate the negative predictive value of PCR/ESI-MS for the detection of bacteria in the bloodstream of newborns with suspected sepsis. Our secondary objective was the evaluation of the sensitivity, specificity and positive predictive value of the PCR/ESI-MS in such a neonatal population. We analysed 114 samples over 14months. The median age and weight were 32weeks+3days and 1840g, respectively. Two patients had negative PCR/ESI-MS results, but positive blood cultures. Overall, the negative predictive value was 98% (95%CI: 92% to 100%). Based on these results, PCR/ESI-MS analysis of blood samples of neonates with suspected sepsis appears to have a very good negative predictive value when compared to blood cultures as gold standard. This novel test might allow for early reassessment of the need for antibiotics. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Evaluation of PCR, DNA hybridization and immunomagnetic separation - PCR for detection of Burkholderia mallei in artificially inoculated environmental samples.

    Science.gov (United States)

    Merwyn, S; Kumar, S; Agarwal, G S; Rai, G P

    2010-06-01

    Glanders is highly contagious disease of equines, caused by Burkholderia mallei. The disease though rare, can be transmitted to humans. Here, we report a strategy for rapid detection of B. mallei from environmental samples. Different bacteriological media were evaluated and brain heart infusion broth medium with selective supplements (BHIB-SS) of penicillin (200 U/ml) and crystal violet (1:10,00000) was found to support the maximum growth of B. mallei even in the presence of other bacteria like Escherichia coli and Staphylococcus aureus. A polymerase chain reaction (PCR) and a DNA hybridization method was standardized for 823 bp specific dNA sequence of B. mallei. To enable the quicker and direct enrichment of B. mallei bacteria from environmental samples, an immunomagnetic separation (IMS) method was also standardized. Water, husk, grass and gram samples were artificially contaminated by B. mallei bacteria and after enrichment of B. mallei in BHIB-SS, detection was carried out by PCR and DNA hybridization. PCR was found to be a better method of the two with a detection limit of 10(4)-10(6) CFU/ml (6 h enrichment in BHIB-SS) in water and other particulate matrices. Detection by PCR in the above samples without enrichment in BHIBSS was carried out following IMS where the detection limit was about 1-2 log higher than PCR following enrichment in BHIB-SS. We recommend PCR for 823 bp for detection of B. mallei from environmental samples either following enrichment in BHIB-SS or IMS. IMS-PCR method may be preferred in situations where numbers of B. mallei bacteria are expected to be high and results are required in short time.

  16. Identification of neotropical felid faeces using RCP-PCR.

    Science.gov (United States)

    Roques, S; Adrados, B; Chavez, C; Keller, C; Magnusson, W E; Palomares, F; Godoy, J A

    2011-01-01

    Faeces similarity among sympatric felid species has generally hampered their use in distributional, demographic and dietary studies. Here, we present a new and simple approach based on a set of species-specific primers, for the unambiguous identification of faeces from sympatric neotropical felids (i.e. puma, jaguar, jaguarundi and ocelot/ margay). This method, referred to as rapid classificatory protocol-PCR (RCP-PCR), consists of a single-tube multiplex PCR yielding species-specific banding patterns on agarose gel. The method was optimized with samples of known origin (14 blood and 15 fresh faeces) and validated in faecal samples of unknown origin (n = 138), for some of which (n = 40) we also obtained species identification based on mtDNA sequencing. This approach proved reliable and provides high identification success rates from faeces. Its simplicity and cost effectiveness should facilitate its application for routine surveys of presence and abundance of these species. © 2010 Blackwell Publishing Ltd.

  17. CUTANEOUS MYCOBACTERIUM MARINUM INFECTION DIAGNOSED BY PCR-RFLP ANALYSIS

    Institute of Scientific and Technical Information of China (English)

    LI Xiao-jie; WANG Hong-sheng; TAO Shi-qin; WU Qin-xue; LIU Wei-da

    2009-01-01

    Objective To identify Mycobacterium marinum (M. marinum) inducing misdiagnosis and treatment failure.Methods The lesional specimen of patient with cutaneous M. marinum were cultivated on Lwenstein-Jensen medium. The isolate was identified by biochemical tests and polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis of the hsp65 gene.Results Smooth and non-pigmented colonies were noted after incubation at 32 ℃ for 2 weeks. The isolate was acid-fast bacilli and confirmed as M. marinum by biochemical tests and PCR-RFLP.Conclusion For a correct diagnosis of cutaneous M. marinum infection, it is crucial for clinicians to have a high index of suspicion, obtain the history of exposure and trauma and understand growth characteristics of the organism. Compared with conventional biochemical techniques, PCR-RFLP analysis is a more rapid, accurate and reliable method for mycobacterial identification to species level.

  18. Noninvasive genotyping of common marmoset (Callithrix jacchus) by fingernail PCR.

    Science.gov (United States)

    Takabayashi, Shuji; Katoh, Hideki

    2015-07-01

    The common marmoset (Callithrix jacchus) is a New World primate that is a useful model for medical studies. In this study, we report a convenient, reliable, and noninvasive procedure to genotype a living common marmoset by using fingernails. This method was used to successfully genotype DNA by restriction fragment length polymorphism (RFLP) PCR without prior purification, by using the KOD FX PCR enzyme kit. Additionally, there is no sample contamination from hematopoietic chimera derived from fused placenta in utero. We compared chimeric levels between various tissues in females with male littermates using quantitative fluorescent (QF)-PCR to prepare a reliable DNA source for genetic analyses, such as genotyping, gene mapping, or genomic sequencing. The chimerism detected appeared to be restricted to lymphatic tissues, such as bone marrow, thymus, spleen, lymph nodes and blood cells. As a result, DNA from fingernails with the quick is the best DNA source for genetic research in living marmosets.

  19. DNA extraction protocol for rapid PCR detection of pathogenic bacteria.

    Science.gov (United States)

    Brewster, Jeffrey D; Paoli, George C

    2013-11-01

    Twelve reagents were evaluated to develop a direct DNA extraction method suitable for PCR detection of foodborne bacterial pathogens. Many reagents exhibited strong PCR inhibition, requiring significant dilution of the extract with a corresponding reduction in sensitivity. Most reagents also exhibited much lower recovery of DNA from the gram-positive test organism (Listeria monocytogenes) than from the gram-negative organism (Escherichia coli O157:H7), preventing unbiased detection and quantitation of both organisms. The 5× HotSHOT+Tween reagent exhibited minimal inhibition and high extraction efficiency for both test organisms, providing a 15-min single-tube DNA-extraction protocol suitable for highly sensitive quantitative PCR assays. Published by Elsevier Inc.

  20. [Application of rapid PCR to authenticate medicinal snakes].

    Science.gov (United States)

    Chen, Kang; Jiang, Chao; Yuan, Yuan; Huang, Lu-Qi; Li, Man

    2014-10-01

    To obtained an accurate, rapid and efficient method for authenticate medicinal snakes listed in Chinese Pharmacopoeia (Zaocysd humnades, Bungarus multicinctus, Agkistrodon acutus), a rapid PCR method for authenticate snakes and its adulterants was established based on the classic molecular authentication methods. DNA was extracted by alkaline lysis and the specific primers were amplified by two-steps PCR amplification method. The denatured and annealing temperature and cycle numbers were optimized. When 100 x SYBR Green I was added in the PCR product, strong green fluorescence was visualized under 365 nm UV whereas adulterants without. The whole process can complete in 30-45 minutes. The established method provides the technical support for authentication of the snakes on field.

  1. Multiplex PCR for identification of herpes virus infections in adolescents.

    Science.gov (United States)

    Durzyńska, Julia; Pacholska-Bogalska, Joanna; Kaczmarek, Maria; Hanć, Tomasz; Durda, Magdalena; Skrzypczak, Magdalena; Goździcka-Józefiak, Anna

    2011-02-01

    The aim of the study was to develop a multiplex PCR (mPCR) for a rapid and simultaneous detection of herpes simplex 1 (HSV-1), herpes simplex 2 (HSV-2), and human cytomegalovirus (HCMV) DNA in squamous oral cells obtained from adolescents. Accuracy of the method was tested in a group of 513 adolescents, almost 11% of subjects were positive for infection with herpes viruses. Correlations with gender, age, and place of residence were sought. A similar incidence of HSV-2 and HCMV was found (4.3% and 5.4%, respectively) and the incidence of HSV-1 was the lowest (1%) in the study group. Conversely to HSV-2, HCMV was detected mostly in the youngest individuals. The same occurrence of all viruses was observed in boys and girls. The mPCR method described is suggested as a useful tool for epidemiologic studies of active herpes infections.

  2. Automated 5 ' nuclease PCR assay for identification of Salmonella enterica

    DEFF Research Database (Denmark)

    Hoorfar, Jeffrey; Ahrens, Peter; Rådström, P.

    2000-01-01

    A simple and ready-to-go test based on a 5' nuclease (TaqMan) PCR technique was developed for identification of presumptive Salmonella enterica isolates. The results were compared with those of conventional methods. The TaqMan assay was evaluated for its ability to accurately detect 210 S. enterica...... isolates, including 100 problematic "rough" isolates. An internal positive control was designed to use the same Salmonella primers for amplification of a spiked nonrelevant template (116 bp) in the sample tube. The PCR test correctly identified all the Salmonella strains by resulting in positive end...... Salmonella strains tested resulted in positive FAM and TET signals. In addition, it was found that the complete PCR mixture, predispensed in microwell plates, could be stored for up to 3 months at -20 degrees C, Thus, the diagnostic TaqMan assay developed can be a useful and simple alternative method...

  3. Oligoribonucleotide (ORN) Interference-PCR (ORNi-PCR): A Simple Method for Suppressing PCR Amplification of Specific DNA Sequences Using ORNs

    OpenAIRE

    Naoki Tanigawa; Toshitsugu Fujita; Hodaka Fujii

    2014-01-01

    Polymerase chain reaction (PCR) amplification of multiple templates using common primers is used in a wide variety of molecular biological techniques. However, abundant templates sometimes obscure the amplification of minor species containing the same primer sequences. To overcome this challenge, we used oligoribonucleotides (ORNs) to inhibit amplification of undesired template sequences without affecting amplification of control sequences lacking complementarity to the ORNs. ORNs were effect...

  4. Quantitative PCR for detection of the OT-1 transgene

    Directory of Open Access Journals (Sweden)

    Crispe Nicholas I

    2005-08-01

    Full Text Available Abstract Background Transgenic TCR mice are often used experimentally as a source of T cells of a defined specificity. One of the most widely used transgenic TCR models is the OT-1 transgenic mouse in which the CD8+ T cells express a TCR specific for the SIINFEKL peptide of ovalbumin presented on kb. Although OT-1 CD8+ can be used in a variety of different experimental settings, we principally employ adoptive transfer and peptide-driven expansion of OT-1 cells in order to explore the distribution and fate of these antigen-specific OT-1 T cells. We set out to develop a quantitative PCR assay for OT-1 cells in order to assess the distribution of OT-1 CD8+ T cells in tissues that are either intrinsically difficult to dissociate for flow cytometric analysis or rendered incompatible with flow cytometric analysis through freezing or fixation. Results We show excellent correlation between flow cytometric assessment of OT-1 cells and OT-1 signal by qPCR assays in cell dilutions as well as in in vivo adoptive transfer experiments. We also demonstrate that qPCR can be performed from archival formalin-fixed paraffin-embedded tissue sections. In addition, the non-quantitative PCR using the OT-1-specific primers without the real-time probe is a valuable tool for OT-1 genotyping, obviating the need for peripheral blood collection and subsequent flow cytometric analysis. Conclusion An OT-1 specific qPCR assay has been developed to quantify adoptively transferred OT-1 cells. OT-1 qPCR to determine cell signal is a valuable adjunct to the standard flow cytometric analysis of OT-1 cell number, particularly in experimental settings where tissue disaggregation is not desirable or in tissues which are not readily disassociated

  5. Evaluation of blood PCR in the diagnosis of pulmonary tuberculosis

    Directory of Open Access Journals (Sweden)

    Aliakbar Heydari

    2012-05-01

    Full Text Available Background and Aim: Culture and specific staining (including Zeil-Nelson and fluorescent methods are standard measures for the diagnosis of tuberculosis (TB.Because these methods are time-consuming and, sometimes, due to their low accuracy faster and more accurate methods are necessitated. Methods, which can substitute invasive procedures, when obtaining smear samples and culture is not possible and in addition to being simple and fast, they have an acceptable diagnostic accuracy. The aim of the present study was to verify the diagnostic value of blood PCR in pulmonary TB. Materials and Methods: This case-control study included 64 proven pulmonary TB cases (according to The National TB Protocol and 28 subjects who were completely healthy. 4.5ml of blood was derived from each participant and then mixed with 0.5ml EDTA. Finally, DNA extraction and PCR testing using SI 6110 primers was performed for all blood samples. Results: Mean age of the cases and controls was 49.8±18.6 and 48.2±18.5, respectively. 49.2% of the cases and 25% of controls were male. Blood PCR in 23 patients with TB was positive, but none of the controls had a positive PCR (thus, sensitivity of 35.7% and specificity of 100%. Conclusion: With regard to specificity of 100% in PCR method (despite its low sensitivity, in conditions where there is no access to an appropriate specimen, a positive blood PCR can obviate invasive procedures and rapid and definitive diagnosis of the disorder and timely treatment of the patient, his life is saved.

  6. Employing Real Time PCR for the Diagnosis of Huntington Disease

    Directory of Open Access Journals (Sweden)

    Frouzandeh Mahjoubi

    2013-07-01

    Full Text Available Background: Huntington disease (HD is a dominantly inherited, neurodegenerative disease characterized by choreiform movement disturbances and dementia. The onset age of this disease is varied but usually is between the ages 40-50. Huntington's disease is caused by a triplet-repeat expansion in the IT15 gene (also known as huntingtin or HD which is located on chromosome 4p3.1. Since many clinical picture of HD are indistinguishable from other distinct genetic disorders molecular test such as PCR is the only way to confirm the disease. The aim of this study was to introduce a new and fast technique for the diagnosis of Huntington disease.Materials and Methods: Blood specimens were collected from individuals suspected for Huntington disease and also people with no symptoms and family history of this disease. DNAs were extracted according to standard protocol. Using conventional PCR, patient positive for Huntington disease were diagnosed. Then employing real time PCR on the basis of difference between melting temperature (Tm a new and fast diagnostic method was introduced.Results: Among 29 patients suspected to be HD only 8 HD patients were confirmed using PCR and real time PCR. The numbers of CAG repeat were between 42-50 and melting temperatures were between 89-92.Conclusion: The concept of using melting temperature in real time PCR protocol presented in here could be employed for the rapid diagnosis of the diseases caused by the increased in triple repeat sequences. It is fast, robust and has the potential use for the prenatal diagnosis.

  7. Monitoring of geosmin producing Anabaena circinalis using quantitative PCR.

    Science.gov (United States)

    Tsao, Hsiang-Wei; Michinaka, Atsuko; Yen, Hung-Kai; Giglio, Steven; Hobson, Peter; Monis, Paul; Lin, Tsair-Fuh

    2014-02-01

    Geosmin is one of the most commonly detected off-flavor chemicals present in reservoirs and drinking water systems. Quantitative real-time PCR (qPCR) is useful for quantifying geosmin-producers by focusing on the gene encoding geosmin synthase, which is responsible for geosmin synthesis. In this study, several primers and probes were designed and evaluated to detect the geosmin synthase gene in cyanobacteria. The specificity of primer and probe sets was tested using 21 strains of laboratory cultured cyanobacteria isolated from surface waters in Australia (18) and Taiwan (2), including 6 strains with geosmin producing ability. The results showed that the primers designed in this study could successfully detect all geosmin producing strains tested. The selected primers were used in a qPCR assay, and the calibration curves were linear from 5 × 10(1) to 5 × 10(5) copies mL(-1), with a high correlation coefficient (R(2) = 0.999). This method was then applied to analyze samples taken from Myponga Reservoir, South Australia, during a cyanobacterial bloom event. The results showed good correlations between qPCR techniques and traditional methods, including cell counts determined by microscopy and geosmin concentration measured using gas chromatography (GC) coupled with a mass selective detector (MSD). Results demonstrate that qPCR could be used for tracking geosmin-producing cyanobacteria in drinking water reservoirs. The qPCR assay may provide water utilities with the ability to properly characterize a taste and odor episode and choose appropriate management and treatment options.

  8. Specific PCR product primer design using memetic algorithm.

    Science.gov (United States)

    Yang, Cheng-Hong; Cheng, Yu-Huei; Chuang, Li-Yeh; Chang, Hsueh-Wei

    2009-01-01

    To provide feasible primer sets for performing a polymerase chain reaction (PCR) experiment, many primer design methods have been proposed. However, the majority of these methods require a relatively long time to obtain an optimal solution since large quantities of template DNA need to be analyzed. Furthermore, the designed primer sets usually do not provide a specific PCR product size. In recent years, evolutionary computation has been applied to PCR primer design and yielded promising results. In this article, a memetic algorithm (MA) is proposed to solve primer design problems associated with providing a specific product size for PCR experiments. The MA is compared with a genetic algorithm (GA) using an accuracy formula to estimate the quality of the primer design and test the running time. Overall, 50 accession nucleotide sequences were sampled for the comparison of the accuracy of the GA and MA for primer design. Five hundred runs of the GA and MA primer design were performed with PCR product lengths of 150-300 bps and 500-800 bps, and two different methods of calculating T(m) for each accession nucleotide sequence were tested. A comparison of the accuracy results for the GA and MA primer design showed that the MA primer design yielded better results than the GA primer design. The results further indicate that the proposed method finds optimal or near-optimal primer sets and effective PCR products in a dry dock experiment. Related materials are available online at http://bio.kuas.edu.tw/ma-pd/. 2009 American Institute of Chemical Engineers

  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. Detection of DNA double-strand breaks and chromosome translocations using ligation-mediated PCR and inverse PCR.

    Science.gov (United States)

    Singh, Sheetal; Shih, Shyh-Jen; Vaughan, Andrew T M

    2014-01-01

    Current techniques for examining the global creation and repair of DNA double-strand breaks are restricted in their sensitivity, and such techniques mask any site-dependent variations in breakage and repair rate or fidelity. We present here a system for analyzing the fate of documented DNA breaks, using the MLL gene as an example, through application of ligation-mediated PCR. Here, a simple asymmetric double-stranded DNA adapter molecule is ligated to experimentally induced DNA breaks and subjected to seminested PCR using adapter- and gene-specific primers. The rate of appearance and loss of specific PCR products allows detection of both the break and its repair. Using the additional technique of inverse PCR, the presence of misrepaired products (translocations) can be detected at the same site, providing information on the fidelity of the ligation reaction in intact cells. Such techniques may be adapted for the analysis of DNA breaks and rearrangements introduced into any identifiable genomic location. We have also applied parallel sequencing for the high-throughput analysis of inverse PCR products to facilitate the unbiased recording of all rearrangements located at a specific genomic location.

  11. Differentiation Between Bacillus thuringiensis and Bacillus cereus by 16S rDNA-PCR and ERIC-PCR

    Institute of Scientific and Technical Information of China (English)

    LI Haitao; LIU Dongming; GAO Jiguo

    2011-01-01

    16S rDNA and ERIC (Enterobacteia Repetitive Intergenic Consensus Sequences) based on PCR method were tested for the effectiveness of the differentiation of B. thuringiensis and B. cereus. 16S rDNA-PCR primers were designed based on the sequence difference in variable regions of B. cereus 16S rDNA and B. thuringiensis 16S rDNA, 16S rDNA-PCR showed no obvious difference between B. cereus and B. thuringiensis. The only difference was that one 1600-bp amplificon could be obtained from all the three B. Cereus strains, and none amplificon from any B. thuringiensis strains. ERIC was optimized based on previous reports. The genonlic DNA was used for the template of ER1C-PCR, and the following DNA fingerprints were analyzed by the agarose gel electrophoresis. The results showed that DNA fingerprint of three B. thuringiensis strains had a unique amplicon less than 100-bp, while DNA fingerprint of three B. cereus" strains had none. Moreover, DNA fingerprint of B. cereus showed a 700-bp amplicon, but didn't have any DNA fingerprints ofB. thuringiensis genome. Therefore, ERIC-PCR technique should be able to be used for the differentiation of B. thuringiensis and B. cereus.

  12. Development of duplex-PCR for identification of Aeromonas species

    Directory of Open Access Journals (Sweden)

    Carina Lucena Mendes-Marques

    2013-10-01

    Full Text Available Introduction The number of reports of intestinal infections caused by Aeromonas spp. has increased significantly in recent years. In most clinical laboratories, identification of these bacteria is carried out by general phenotypic tests that sometimes do not accurately differentiate Aeromonas and Vibrio. Methods A duplex-polymerase chain reaction (PCR was developed directed to 2 targets identifying Aeromonas spp. pathogenic to humans. Results The duplex-PCR results were reproducible and specific for Aeromonas spp. pathogenic to humans. Conclusions This method will allow differentiation between Vibrio and Aeromonas spp. in patients with in cholera-like symptoms and can also be used in water quality monitoring.

  13. [Basics of PCR and related techniques applied in veterinary parasitology].

    Science.gov (United States)

    Ben Abderrazak, S

    2004-01-01

    We attempte through the following overall review pertaining to the basics of PCR techniques (Polymerase Chain Reaction), to introduce the main applications used in veterinary parasitology. A major problem restricting the application possibilities of molecular biology techniques is of quantitative nature. Amplification techniques represent a real revolution, for it makes possible the production of tens, even hundreds of nanogrammes of sequences when starting from very small quantities. The PCR technique has dramatically transformed the strategies used so far in molecular biology and subsequently research and medical diagnosis.

  14. Wolbachia detection: an assessment of standard PCR protocols.

    Science.gov (United States)

    Simões, P M; Mialdea, G; Reiss, D; Sagot, M-F; Charlat, S

    2011-05-01

    Wolbachia is a large monophyletic genus of intracellular bacteria, traditionally detected using PCR assays. Its considerable phylogenetic diversity and impact on arthropods and nematodes make it urgent to assess the efficiency of these screening protocols. The sensitivity and range of commonly used PCR primers and of a new set of 16S primers were evaluated on a wide range of hosts and Wolbachia strains. We show that certain primer sets are significantly more efficient than others but that no single protocol can ensure the specific detection of all known Wolbachia infections.

  15. Typing of Y chromosome SNPs with multiplex PCR methods

    DEFF Research Database (Denmark)

    Sanchez Sanchez, Juan Jose; Børsting, Claus; Morling, Niels

    2005-01-01

    We describe a method for the simultaneous typing of Y-chromosome single nucleotide polymorphism (SNP) markers by means of multiplex polymerase chain reaction (PCR) strategies that allow the detection of 35 Y chromosome SNPs on 25 amplicons from 100 to 200 pg of chromosomal deoxyribonucleic acid...... factors for the creation of larger SNP typing PCR multiplexes include careful selection of primers for the primary amplification and the SBE reaction, use of DNA primers with homogenous composition, and balancing the primer concentrations for both the amplification and the SBE reactions....

  16. Novel PCR assay for determining the genetic sex of mice.

    Science.gov (United States)

    McFarlane, L; Truong, V; Palmer, J S; Wilhelm, D

    2013-01-01

    A number of studies require the determination of the genetic sex of mouse embryos before sexual differentiation and/or of mutant mice that display partial or complete sex reversal. The majority of current methods for sexing by PCR involve multiplexing of 2 primer pairs. We have developed a novel sexing PCR using a single primer pair that amplifies fragments from the X and the Y chromosome with a clear size difference between the respective amplicons. This assay provides a rapid and reliable method to identify the genetic sex of mice across different mouse strains.

  17. Sex Determination in Ostrich (Struthio camelus Using PCR Technique

    Directory of Open Access Journals (Sweden)

    M Alipanah

    2011-12-01

    Full Text Available The important of ostrich breeding have been increased in the Iran as other country around the world. Lack of sexual dimorphism in many species of birds including Ostrich (Struthio camelus, making it difficult to differentiate between males and females, especially in young birds. This can be problematic for breeding programs. Blood samples from randomly chicks in the first days were collected and then DNA was extracted from blood. For sex typing used PCR by OSFES primer that produced a 423 fragment. Our results shown that sex determination using PCR method for chicks in the first days is useful, easy and inexpensive.

  18. Development of duplex-PCR for identification of Aeromonas species

    Directory of Open Access Journals (Sweden)

    Carina Lucena Mendes-Marques

    2013-05-01

    Full Text Available Introduction The number of reports of intestinal infections caused by Aeromonas spp. has increased significantly in recent years. In most clinical laboratories, identification of these bacteria is carried out by general phenotypic tests that sometimes do not accurately differentiate Aeromonas and Vibrio. Methods A duplex-polymerase chain reaction (PCR was developed directed to 2 targets identifying Aeromonas spp. pathogenic to humans. Results The duplex-PCR results were reproducible and specific for Aeromonas spp. pathogenic to humans. Conclusions This method will allow differentiation between Vibrio and Aeromonas spp. in patients with in cholera-like symptoms and can also be used in water quality monitoring.

  19. [Allele-specific PCR and its application in forensic science].

    Science.gov (United States)

    Nie, Yan-chai; Wang, Bin; Zhao, Zi-qin; Zhou, Huai-gu

    2014-08-01

    Allele-specific polymerase chain reaction (AS-PCR) is a technique based on allele-specific primers, which can be used to analyze single nucleotide polymorphism (SNP) effectively including the transition, transversion and insertion/deletion polymorphism and has been exploited in the study of diseases research, molecular diagnosis, and forensic biological evidence. The article systematically reviews the principle, the detection methods, improvement of AS-PCR, and its research updates in the fields of autosome, Y chromosome and mitochondrial SNP, as well as its application in forensic science.

  20. PCR for detection of Shigella spp. in mayonnaise.

    Science.gov (United States)

    Villalobo, E; Torres, A

    1998-04-01

    The use of PCR to amplify a specific virA gene fragment serves as a highly specific and sensitive method to detect virulent bacteria of the genus Shigella and enteroinvasive Escherichia coli. Amplification of a 215-bp DNA band was obtained by using isolated genomic DNA of Shigella, individual cells of Shigella dysenteriae, and mayonnaise contaminated with S. dysenteriae. Moreover, a multiplex PCR with specific (virA) and bacterium-restricted (16S ribosomal DNA) primers generated an amplification product of approximately 755 bp for all bacteria tested and an additional 215-bp product for Shigella and enteroinvasive E. coli.

  1. PCR Amplicon Prediction from Multiplex Degenerate Primer and Probe Sets

    Energy Technology Data Exchange (ETDEWEB)

    2013-08-08

    Assessing primer specificity and predicting both desired and off-target amplification products is an essential step for robust PCR assay design. Code is described to predict potential polymerase chain reaction (PCR) amplicons in a large sequence database such as NCBI nt from either singleplex or a large multiplexed set of primers, allowing degenerate primer and probe bases, with target mismatch annotates amplicons with gene information automatically downloaded from NCBI, and optionally it can predict whether there are also TaqMan/Luminex probe matches within predicted amplicons.

  2. Real-time PCR in Food Science: Introduction.

    Science.gov (United States)

    Rodriguez-Lazaro, David; Hernandez, Marta

    2013-01-01

    Food safety and quality control programmes are increasingly applied throughout the production food chain in order to guarantee added value products as well as to minimize the risk of infection for the consumer. The development of real-time PCR has represented one of the most significant advances in food diagnostics as it provides rapid, reliable and quantitative results. These aspects become increasingly important for the agricultural and food industry. Different strategies for real-time PCR diagnostics have been developed including unspecific detection independent of the target sequence using fluorescent dyes such as SYBR Green, or by sequence-specific fluorescent oligonucleotide probes such as TaqMan probes or molecular beacons.

  3. Fecal specimens preparation methods for PCR diagnosis of human taeniosis

    Directory of Open Access Journals (Sweden)

    Nunes Cáris Maroni

    2006-01-01

    Full Text Available Sample preparation and DNA extraction protocols for DNA amplification by PCR, which can be applied in human fecal samples for taeniasis diagnosis, are described. DNA extracted from fecal specimens with phenol/chloroform/isoamilic alcohol and DNAzol® reagent had to be first purified to generate fragments of 170 pb and 600 pb by HDP2-PCR. This purification step was not necessary with the use of QIAmp DNA stool mini kit®. Best DNA extraction results were achieved after eggs disruption with glass beads, either with phenol/chloroform/isoamilic alcohol, DNAzol® reagent or QIAmp DNA stool mini kit®.

  4. Clinical nursing of HFOV plus CMV on premature newborns with hyaline membrane disease%高频振荡通气加常频通气治疗早产儿肺透明膜病的临床和护理研究

    Institute of Scientific and Technical Information of China (English)

    叶蕾; 杨小红; 孙小娟

    2012-01-01

    Objective To study the effect and nursing strategies of high frequency oscillatory ventilation (HFOV) plus conventional mandatory ventilation (CMV)on treating critical hyaline membrane disease(HMD). Methods A total of 18 infants with critical HMD in the NICU from January 2009 to December 2010 were enrolled and divided into treatment group(HFOV plus CMV) and control group(CMV). Clinical efficacy, ventilator parameters, blood gas analysis and complications (pneumothorax, intracranial hemorrhage) were compared between the two groups. Nursing strategies were summarized accordingly. Results After 24 h treatment, PO2, MAP, OI and outcome of the treatment group were better than those of the control group (P<0. 05). Compared with the control group, the incidence of pneumothorax in the treatment group was significantly low-er(P<0.05). But no statistical significance was found in incidence of intracranial hemorrhage between the two groups(P<0. 05). Conclusion HFOV plus CMV can improve lung oxygenation and clinical outcome, and reduce the incidence rate of pneumothorax without increasing the risk of intracranial hemorrhage. The effect is superior to conventional mandatory ventilation. During the treatment process, nursing cooperation related to high - quality mechanical ventilation is paramount to an successful treatment.%目的 研究高频振荡通气加常频通气治疗早产儿肺透明膜病的临床疗效和护理特点.方法 以2009年1月~2010年12月在本院新生儿重症监护室(NICU)住院采用机械通气治疗的18例早产儿肺透明膜病(HMD)患儿为研究对象,随机分为研究组10例和对照组8例,研究组通气模式为高频振荡通气加常频通气,对照组采用为常频通气模式,比较两组患儿上机后临床疗效、呼吸机参数、氧合指标以及气胸、颅内出血等并发症的发生率,同时总结高频振荡通气加常频通气的护理特点和体会.结果 研究组上机24 h后吸氧浓度(FiO2)、平均气道

  5. Low predictive value of polymerase chain reaction for diagnosis of cytomegalovirus disease in liver transplant recipients.

    Science.gov (United States)

    Delgado, R; Lumbreras, C; Alba, C; Pedraza, M A; Otero, J R; Gómez, R; Moreno, E; Noriega, A R; Payá, C V

    1992-07-01

    The polymerase chain reaction (PCR) and viral culture techniques were prospectively compared for the detection of cytomegalovirus (CMV) in blood samples from 24 liver transplant recipients. Nine patients had one or more episodes of viremia, seven of which were clinically symptomatic infections. All samples in which CMV was isolated by culture were positive by the PCR. However, the PCR result was also positive for one or more samples from 11 patients who never developed CMV-related symptoms. Although the PCR is a very sensitive technique for CMV detection in blood samples from liver transplant recipients, it is not useful as a marker of symptomatic CMV disease.

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

    Science.gov (United States)

    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.

  7. 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.

  8. Construction of Recombinant Plasmid pTracer-CMV-HSP47 in vitro and its Expression in NIH/3T3%大鼠热休克蛋白47真核表达载体的体外构建

    Institute of Scientific and Technical Information of China (English)

    程杰; 王佐林

    2007-01-01

    目的:体外构建大鼠热休克蛋白47(47 kDa heat shock protein,HSP47)的荧光真核表达载体,并检测其在鼠胚胎成纤维细胞NIH/3T3中的表达.方法:应用RT-PCR方法从大鼠肝脏中分离、扩增目的片断,双酶切定向克隆到真核表达载体pTracer-CMV中,构成重组质粒pTracer-CMV-HSP47,测序验证.通过脂质体介导质粒瞬时转染NIH/3T3细胞,Western b1otting和免疫细胞化学检测HSP47及Ⅰ型胶原蛋白(collagen Ⅰ)的表达变化.结果:通过RT-PCR获得1.3kb的cDNA片断,测序发现除个别碱基差异外,其余cDNA序列与基因库(Genebank)中序列一致,且编码的氨基酸序列完全一致.转染后经Western blotting和免疫细胞化学检测证实HSP47和collagen Ⅰ表达明显增强.结论:本研究成功构建了大鼠HSP47荧光真核表达载体pTracer-CMV-HSP47,并能在NIH/3T3细胞中表达,同时证实HSP47能促进collagen Ⅰ的表达.

  9. Designing multiplex PCR system of Campylobacter jejuni for efficient typing by improving monoplex PCR binary typing method.

    Science.gov (United States)

    Yamada, Kazuhiro; Ibata, Ami; Suzuki, Masahiro; Matsumoto, Masakado; Yamashita, Teruo; Minagawa, Hiroko; Kurane, Ryuichiro

    2015-01-01

    Campylobacter jejuni is responsible for the majority of Campylobacter infections. As the molecular epidemiological study of outbreaks, pulsed-field gel electrophoresis (PFGE) is performed in general. But PFGE has several problems. PCR binary typing (P-BIT) method is a typing method for Campylobacter spp. that was recently developed, and was reported to have a similar discriminatory power and stability to those of PFGE. We modified the P-BIT method from 18 monoplex PCRs to two multiplex PCR systems (mP-BIT). The same results were obtained from monoplex PCRs using original primers and multiplex PCR in the representative isolates. The mP-BIT can analyze 48 strains at a time by using 96-well PCR systems and can identify C. jejuni because mP-BIT includes C. jejuni marker. The typing of the isolates by the mP-BIT and PFGE demonstrated generally concordant results and the mP-BIT method (D = 0.980) has a similar discriminatory power to that of PFGE with SmaI digest (D = 0.975) or KpnI digest (D = 0.987) as with original article. The mP-BIT method is quick, simple and easy, and comes to be able to perform it at low cost by having become a multiplex PCR system. Therefore, the mP-BIT method with two multiplex PCR systems has high potential for a rapid first-line surveillance typing assay of C. jejuni and can be used for routine surveillance and outbreak investigations of C. jejuni in the future. Copyright © 2014 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  10. Critical appraisal of quantitative PCR results in colorectal cancer research: can we rely on published qPCR results?

    Science.gov (United States)

    Dijkstra, J R; van Kempen, L C; Nagtegaal, I D; Bustin, S A

    2014-06-01

    The use of real-time quantitative polymerase chain reaction (qPCR) in cancer research has become ubiquitous. The relative simplicity of qPCR experiments, which deliver fast and cost-effective results, means that each year an increasing number of papers utilizing this technique are being published. But how reliable are the published results? Since the validity of gene expression data is greatly dependent on appropriate normalisation to compensate for sample-to-sample and run-to-run variation, we have evaluated the adequacy of normalisation procedures in qPCR-based experiments. Consequently, we assessed all colorectal cancer publications that made use of qPCR from 2006 until August 2013 for the number of reference genes used and whether they had been validated. Using even these minimal evaluation criteria, the validity of only three percent (6/179) of the publications can be adequately assessed. We describe common errors, and conclude that the current state of reporting on qPCR in colorectal cancer research is disquieting. Extrapolated to the study of cancer in general, it is clear that the majority of studies using qPCR cannot be reliably assessed and that at best, the results of these studies may or may not be valid and at worst, pervasive incorrect normalisation is resulting in the wholesale publication of incorrect conclusions. This survey demonstrates that the existence of guidelines, such as MIQE, is necessary but not sufficient to address this problem and suggests that the scientific community should examine its responsibility and be aware of the implications of these findings for current and future research.

  11. Detection and subtyping (H5 and H7) of avian type A influenza virus by reverse transcription-PCR and PCR-ELISA

    DEFF Research Database (Denmark)

    Munch, M.; Nielsen, L.P.; Handberg, Kurt

    2001-01-01

    Avian influenza virus infections are a major cause of morbidity and rapid identification of the virus has important clinical, economical and epidemiological implications. We have developed a one-tube Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) for the rapid diagnosis of avian influenza...... catching probe confirming the NP influenza A origin. The PCR-ELISA was about 100 times more sensitive than detection of PCR products by agarose gel electrophoresis. RT-PCR and detection by PCR-ELISA is comparable in sensitivity to virus propagation in eggs. We also designed primers for the detection...... A. A panel of reference influenza strains from various hosts including avian species, human, swine and horse were evaluated in a one tube RT-PCR using primers designed for the amplification of a 218 bp fragment of the NP gene. The PCR products were detected by PCR-ELISA by use of an internal...

  12. Identification of Helicobacter pylori DNA in gallstones using Multiplex PCR

    Directory of Open Access Journals (Sweden)

    Shohreh Farshad

    2006-02-01

    Full Text Available Background: Recent studies showed a possible relationship between infections caused by some of Helicobacter members and gallstones formation. The aim of this study was identification of Helicobacter members in gallstones from patients with biliary diseases. Methods: Gallstones and bile samples from 33 patients were subjected to rapid urease test, culture and Multiplex-PCR using primers based on 16s rRNA and isocitrate dehydrogenase genes to identify Helicobacter genus and H. pylori species genes, respectively. This PCR was also done on bile samples from 40 autopsied gallbladders with normal pathology (as a control group. Results: In 18.1% of stones and 12.1% of bile samples, H. pylori DNA was detected using PCR. Rapid urease and cultures tests were negative for all samples. The genome of H. pylori was not detected in control group using PCR. Conclusion: H. pylori DNA was detected in gallstone, however, we are not sure of H. pylori viability in these samples. To clarify the clinical role of Helicobacter in gallbladder diseases, more investigations are needed to ascertain whether this microorganism is innocent bystander or active participant in gallstone formation.

  13. High resolution positron tomography using PCR-I

    Energy Technology Data Exchange (ETDEWEB)

    Brownell, G.L.; Burnham, C.A.; Sandrew, B.; Elmaleh, D.R.; Livni, E.; Kizuka, H.

    1984-01-01

    PCR-I is a high resolution positron tomograph developed by the Physics Research Laboratory of the Massachusetts General Hospital to explore resolution limits of positron tomographs. PCR-I currently obtains images with 4.8 mm FWHM resolution at the center. Plane thickness may be varied between 5 and 10 mm. The instrument uses analog coding to obtain high resolution images without mechanical motion. This permits rapid dynamic imaging and gated cardiac imaging as well as conventional high resolution imaging. A series of studies has been carried out to demonstrate the ability of PCR-I to image structures in small animals. F-18 in the rat skeleton is clearly defined and various structures such as the spinal processes can be clearly resolved. A sequence of images at different spacing provides a three-dimensional reconstruction of the rat skeleton. Blood volume and palmitic acid have been imaged in the dog heart. Again, the sequence of images provides a clear delineation of the three dimensional nature of the blood pools and of the surrounding musculature. Blood flow, blood volume and glucose metabolism have been studied in the monkey brain. Structures within the brain of the Resus monkey can be clearly resolved. Increased activity resulting from induced seizures in the squirrel monkey have been observed and delineated. All of these studies indicate areas of future animal and clinical research using the high resolution tomograph, PCR-I.

  14. Animal Species Identification by PCR – RFLP of Cytochrome b

    Directory of Open Access Journals (Sweden)

    Tomáš Minarovič

    2010-05-01

    Full Text Available An alternative DNA detection system is based on the polymerase chain reaction (PCR amplification of a segment of the mitochondrial cytochrome b gene. Subsequent cleavage by a restriction enzymes gives rise to a specie-specific pattern on an agarose gel. We used five animal species (Mustela vison, Mustela putorius furo, Sus scrofa domesticus, Oryctolagus cuninculus, Anser anser. Length of PCR product was 359 bp and we used universal primers. Restriction fragment length polymorphism was analyzed by using the restriction endonuclease AluI. Results of cleavage were visualized by using electrophoresis and UV transiluminator. Every animal specie has a unique combination of restriction fragments i.e. Mustela vison 81 bp, 109 bp and 169 bp, Mustela putorius furo 169 bp and 190 bp, Sus scrofa domesticus 115 bp and 244 bp, Oryctolagus cunninculus is not cleaved by AluI so it has whole 359 bp fragment on agarose gel, Anser anser 130 bp and 229 bp. The results suggest that the method of PCR - RFLP is rapid and simple method for identification of species. PCR – RFLP can reliably identify chosen species. Application of genetic methods is very useful for breeding of livestock and protection of biodiversity.

  15. PCR-Internal Transcribed Spacer (ITS) genes sequencing and ...

    African Journals Online (AJOL)

    2. Department of Pure and Applied Zoology, Federal University of Agriculture, Abeokuta, Nigeria. 3. ... Keywords: Internal transcribed spacer genes, phylogenetic, genetic ... ization of fungi by polymerase chain reaction (PCR) am- .... Basic Local Alignment search Tool (BLAST) to establish ..... Population structure and.

  16. Evaluation of nested PCR in diagnosis of fungal rhinosinusitis

    Directory of Open Access Journals (Sweden)

    Parisa Badiee

    2015-10-01

    Full Text Available Background and Objective: Given the importance of rapid diagnosis for fungal rhinosinusitis, this study aimed to evaluate the use of nested PCR to identify Aspergillus and Mucor species in clinical samples from patients with suspected fungal rhinosinusitis.Methods: Functional endoscopic sinus surgery specimens were collected from 98 patients with rhinosinusitis from 2012 to 2013. All samples were ground and cultured on sabouraud dextrose agar. The isolated fungi were identified based on their macroscopic and microscopic features. Fungal DNA was extracted from the tissue samples and nested PCR was performedwith two sets of primers for Mucor and Aspergillus.Results: Direct microscopic showed that 5.1% contained fungal components and 9.2% exhibited growth of fungi in culture. The most common agents isolated were Aspergillus fumigatus (n= 3 , Aspergillus flavus (n=2, Penicillium sp (n=3 and Alternaria sp. (n=1. Mucor sp. was identified in the pathology smear from 1 patient. Positive results for fungal rhinosinusitis were obtained for a total of 10.2% by culture or pathology smear. Positive PCR results were obtained in 72 samples for Aspergillus and 31 samples for Mucor.Conclusion: Our results suggest that endoscopic sinus surgery specimens are not suitable for nested PCR, probably because of the accumulation of fungi that contaminate the environmental air. This drawback is a limiting factor for diagnosis with nasal cavity specimens. Therefore, molecular methods and conventional culture techniques are helpful complementarydiagnostic methods to detect fungal rhinosinusitis and determine appropriate management for these patients.

  17. Detection of mitochondrial DNA deletion by a modified PCR method

    Institute of Scientific and Technical Information of China (English)

    汪振诚; 王学敏; 缪明永; 章卫平; 焦炳华; 倪庆桂

    2003-01-01

    Objective: To develop a simple and efficient method for detecting small populations of mitochondrial DNA deletion. Methods: Peripheral blood cell DNA was obtained from a victim who was accidently exposed to a 60Co radiation source 11 years ago. Using the DNA as template, PCR was performed to generate multiple products including true deletions and artifacts. The full length product was recovered and used as template of secondary PCR. The suspicious deletion product of mtDNA could be confirmed if it was only yielded by first PCR. Using either original primers or their nested primers, the suspicious deletion product was amplified and authenticated as true deletion product. The template was recovered and determined to be a deletion by sequencing directly. Results: A new mtDNA deletion, spanning 889 bp from nt11688 to nt12576, was detected in the peripheral blood cells of the victim. Conclusion: The new PCR-based method is more efficient in detecting small populations of mtDNA deletion than other routine methods. MtDNA deletion is found in the victim, suggesting there is relationship between the deletion and phenotypes of the disease.

  18. Qualitative PCR method for Roundup Ready soybean: interlaboratory study.

    Science.gov (United States)

    Kodama, Takashi; Kasahara, Masaki; Minegishi, Yasutaka; Futo, Satoshi; Sawada, Chihiro; Watai, Masatoshi; Akiyama, Hiroshi; Teshima, Reiko; Kurosawa, Yasunori; Furui, Satoshi; Hino, Akihiro; Kitta, Kazumi

    2011-01-01

    Quantitative and qualitative methods based on PCR have been developed for genetically modified organisms (GMO). Interlaboratory studies were previously conducted for GMO quantitative methods; in this study, an interlaboratory study was conducted for a qualitative method for a GM soybean, Roundup Ready soy (RR soy), with primer pairs designed for the quantitative method of RR soy studied previously. Fourteen laboratories in Japan participated. Each participant extracted DNA from 1.0 g each of the soy samples containing 0, 0.05, and 0.10% of RR soy, and performed PCR with primer pairs for an internal control gene (Le1) and RR soy followed by agarose gel electrophoresis. The PCR product amplified in this PCR system for Le1 was detected from all samples. The sensitivity, specificity, and false-negative and false-positive rates of the method were obtained from the results of RR soy detection. False-negative rates at the level of 0.05 and 0.10% of the RR soy samples were 6.0 and 2.3%, respectively, revealing that the LOD of the method was somewhat below 0.10%. The current study demonstrated that the qualitative method would be practical for monitoring the labeling system of GM soy in kernel lots.

  19. Introducing Undergraduate Students to Real-Time PCR

    Science.gov (United States)

    Hancock, Dale; Funnell, Alister; Jack, Briony; Johnston, Jill

    2010-01-01

    An experiment is conducted, which in four 3 h laboratory sessions, introduces third year undergraduate Biochemistry students to the technique of real-time PCR in a biological context. The model used is a murine erythroleukemia cell line (MEL cells). These continuously cycling, immature red blood cells, arrested at an early stage in erythropoiesis,…

  20. Real-Time PCR Quantification of Methanobrevibacter oralis in Periodontitis

    OpenAIRE

    Bringuier, Amélie; Khelaifia, Saber; Richet, Hervé; Aboudharam, Gérard; Drancourt, Michel

    2013-01-01

    A real-time PCR assay developed to quantify Methanobrevibacter oralis indicated that its inoculum significantly correlated with periodontitis severity (P = 0.003), despite a nonsignificant difference in prevalence between controls (3/10) and patients (12/22) (P = 0.2, Fisher test). The M. oralis load can be used as a biomarker for periodontitis.