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Sample records for cobas ampliprep-cobas taqman

  1. Comparison of the Hologic Aptima HIV-1 Quant Dx Assay to the Roche COBAS Ampliprep/COBAS TaqMan HIV-1 Test v2.0 for the quantification of HIV-1 RNA in plasma samples

    DEFF Research Database (Denmark)

    Schønning, Kristian; Johansen, Kim; Landt, Bodil

    2017-01-01

    of the Aptima HIV-1 Quant Dx Assay (Aptima) and the COBAS Ampliprep/COBAS TaqMan HIV-1 Test v2.0 (CAPCTMv2) for the quantification of HIV-1 RNA in plasma samples. STUDY DESIGN: The performance of the two tests was compared on 216 clinical plasma samples, on dilutions series in seven replicates of five clinical...

  2. Evaluation of the performance of Abbott m2000 and Roche COBAS Ampliprep/COBAS Taqman assays for HIV-1 viral load determination using dried blood spots and dried plasma spots in Kenya.

    Science.gov (United States)

    Zeh, Clement; Ndiege, Kenneth; Inzaule, Seth; Achieng, Rebecca; Williamson, John; Chih-Wei Chang, Joy; Ellenberger, Dennis; Nkengasong, John

    2017-01-01

    Routine HIV viral load testing is not widely accessible in most resource-limited settings, including Kenya. To increase access to viral load testing, alternative sample types like dried blood spots (DBS), which overcome the logistic barriers associated with plasma separation and cold chain shipment need to be considered and evaluated. The current study evaluated matched dried blood spots (DBS) and dried plasma spots (DPS) against plasma using the Abbott M 2000 (Abbott) and Roche Cobas Ampliprep/Cobas TaqMan (CAP/CTM) quantitative viral load assays in western Kenya. Matched plasma DBS and DPS were obtained from 200 HIV-1 infected antiretroviral treatment (ART)-experienced patients attending patient support centers in Western Kenya. Standard quantitative assay performance parameters with accompanying 95% confidence intervals (CI) were assessed at the assays lower detection limit (400cps/ml for CAP/CTM and 550cps/ml for Abbott) using SAS version 9.2. Receiver operating curves (ROC) were further used to assess viral-load thresholds with best assay performance (reference assay CAP/CTM plasma). Using the Abbott test, the sensitivity and specificity, respectively, for DPS were (97.3%, [95%CI: 93.2-99.2] and 98.1% [95%CI: 89.7-100]) and those for DBS (93.9% [95%CI: 88.8-97.2] and 88.0% [95%CI: 82.2-92.4]). The correlation and agreement using paired plasma and DPS/DBS were strong, with r2 = 90.5 and rc = 68.1. The Bland-Altman relative percent change was 95.3 for DPS, (95%CI: 90.4-97.7) and 73.6 (95%CI: 51.6-86.5) for DBS. Using the CAP/CTM assay, the sensitivity for DBS was significantly higher compared to DPS (100.0% [95% CI: 97.6-100.0] vs. 94.7% [95%CI: 89.8-97.7]), while the specificity for DBS was lower: 4%, [95% CI: 0.4-13.7] compared to DPS: 94.0%, [95% CI: 83.5-98.7]. When compared under different clinical relevant thresholds, the accuracy for the Abbott assay was 95% at the 1000cps/ml cut-off with a sensitivity and specificity of 96.6% [95% CI 91.8-98.7] and 90

  3. Field evaluation of Abbott Real Time HIV-1 Qualitative test for early infant diagnosis using dried blood spots samples in comparison to Roche COBAS Ampliprep/COBAS TaqMan HIV-1 Qual test in Kenya.

    Science.gov (United States)

    Chang, Joy; Omuomo, Kenneth; Anyango, Emily; Kingwara, Leonard; Basiye, Frank; Morwabe, Alex; Shanmugam, Vedapuri; Nguyen, Shon; Sabatier, Jennifer; Zeh, Clement; Ellenberger, Dennis

    2014-08-01

    Timely diagnosis and treatment of infants infected with HIV are critical for reducing infant mortality. High-throughput automated diagnostic tests like Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Qual Test (Roche CAPCTM Qual) and the Abbott Real Time HIV-1 Qualitative (Abbott Qualitative) can be used to rapidly expand early infant diagnosis testing services. In this study, the performance characteristics of the Abbott Qualitative were evaluated using two hundred dried blood spots (DBS) samples (100 HIV-1 positive and 100 HIV-1 negative) collected from infants attending the antenatal facilities in Kisumu, Kenya. The Abbott Qualitative results were compared to the diagnostic testing completed using the Roche CAPCTM Qual in Kenya. The sensitivity and specificity of the Abbott Qualitative were 99.0% (95% CI: 95.0-100.0) and 100.0% (95% CI: 96.0-100.0), respectively, and the overall reproducibility was 98.0% (95% CI: 86.0-100.0). The limits of detection for the Abbott Qualitative and Roche CAPCTM Qual were 56.5 and 6.9copies/mL at 95% CIs (p=0.005), respectively. The study findings demonstrate that the Abbott Qualitative test is a practical option for timely diagnosis of HIV in infants.

  4. Evaluation of an Upgraded Version of the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 Test for HIV-1 Load Quantification▿

    OpenAIRE

    Damond, F.; Avettand-Fenoel, V.; Collin, G.; Roquebert, B.; Plantier, J. C.; Ganon, A.; Sizmann, D.; Babiel, R. (Rainer); Glaubitz, J.; Chaix, M. L.; Brun-Vezinet, F.; Descamps, D; Rouzioux, C

    2010-01-01

    We evaluated the performance of the prototype Cobas AmpliPrep/Cobas TaqMan HIV-1 test, version 2.0, using prospective and archived clinical samples initially underquantitated by the Cobas AmpliPrep/Cobas TaqMan HIV-1 test. The performance of the new test was significantly improved, and the majority of the underquantitation observed with the first-version test was eliminated.

  5. Evaluation of an Upgraded Version of the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 Test for HIV-1 Load Quantification▿

    Science.gov (United States)

    Damond, F.; Avettand-Fenoel, V.; Collin, G.; Roquebert, B.; Plantier, J. C.; Ganon, A.; Sizmann, D.; Babiel, R.; Glaubitz, J.; Chaix, M. L.; Brun-Vezinet, F.; Descamps, D.; Rouzioux, C.

    2010-01-01

    We evaluated the performance of the prototype Cobas AmpliPrep/Cobas TaqMan HIV-1 test, version 2.0, using prospective and archived clinical samples initially underquantitated by the Cobas AmpliPrep/Cobas TaqMan HIV-1 test. The performance of the new test was significantly improved, and the majority of the underquantitation observed with the first-version test was eliminated. PMID:20129964

  6. Evaluation of an upgraded version of the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test for HIV-1 load quantification.

    Science.gov (United States)

    Damond, F; Avettand-Fenoel, V; Collin, G; Roquebert, B; Plantier, J C; Ganon, A; Sizmann, D; Babiel, R; Glaubitz, J; Chaix, M L; Brun-Vezinet, F; Descamps, D; Rouzioux, C

    2010-04-01

    We evaluated the performance of the prototype Cobas AmpliPrep/Cobas TaqMan HIV-1 test, version 2.0, using prospective and archived clinical samples initially underquantitated by the Cobas AmpliPrep/Cobas TaqMan HIV-1 test. The performance of the new test was significantly improved, and the majority of the underquantitation observed with the first-version test was eliminated.

  7. Evaluation of Cobas TaqMan MTB PCR for detection of Mycobacterium tuberculosis.

    Science.gov (United States)

    Kim, Jeong Hyun; Kim, Young Jae; Ki, Chang-Seok; Kim, Ji-Youn; Lee, Nam Yong

    2011-01-01

    Nucleic acid-based amplification tests allow the rapid detection of Mycobacterium tuberculosis. Recently, a real-time PCR assay for M. tuberculosis complex, the Cobas TaqMan MTB test (Roche Diagnostics, Basel, Switzerland), was introduced. We performed a prospective study to evaluate the diagnostic performance of the Cobas TaqMan MTB test system. A total of 406 specimens collected from 247 patients were simultaneously tested by conventional culture, Cobas Amplicor MTB PCR, and TaqMan MTB PCR. The cross-reactivity with other Mycobacterium species and the detection limit were also evaluated. Among 406 specimens, a total of 24 specimens (5.9%) were culture positive: 14 specimens were positive by both TaqMan and Amplicor MTB PCRs, while 5 specimens were positive by only TaqMan PCR. The remaining five specimens were negative by both PCR methods. Seven specimens with negative culture results were positive by TaqMan PCR, but five of these were negative by Amplicor MTB PCR. The sensitivity, specificity, and positive (PPV) and negative (NPV) predictive values were 79.1%, 98.2%, 73.1%, and 98.7% for TaqMan and 58.3%, 99.5%, 87.5%, and 97.4% for the Amplicor MTB PCR test, respectively. There was no cross-reactivity with M. tuberculosis and nontuberculous mycobacterial species. The detection limit for the Cobas TaqMan MTB PCR test was 4.0 copies/μl. The Cobas TaqMan MTB PCR test showed higher sensitivity for detection of the M. tuberculosis complex without disturbing the specificity and NPV than the Amplicor MTB PCR test.

  8. The Cobas AmpliPrep/Cobas TaqMan HCV Test, Version 2.0, Real-Time PCR Assay Accurately Quantifies Hepatitis C Virus Genotype 4 RNA

    OpenAIRE

    Chevaliez, Stéphane; Bouvier-Alias, Magali; Rodriguez, Christophe; Soulier, Alexandre; Poveda, Jean-Dominique; Pawlotsky, Jean-Michel

    2013-01-01

    Accurate hepatitis C virus (HCV) RNA quantification is mandatory for the management of chronic hepatitis C therapy. The first-generation Cobas AmpliPrep/Cobas TaqMan HCV test (CAP/CTM HCV) underestimated HCV RNA levels by >1-log10 international units/ml in a number of patients infected with HCV genotype 4 and occasionally failed to detect it. The aim of this study was to evaluate the ability of the Cobas AmpliPrep/Cobas TaqMan HCV test, version 2.0 (CAP/CTM HCV v2.0), to accurately quantify H...

  9. Performance evaluation of the new Roche cobas AmpliPrep/cobas TaqMan HCV test, version 2.0, for detection and quantification of hepatitis C virus RNA

    NARCIS (Netherlands)

    S.D. Pas (Suzan); R. Molenkamp (Richard); J. Schinkel (Janke); S. Rebers; C. Copra (Cederick); S. Seven-Deniz; D. Thamke (Diana); R.J. de Knegt (Robert); B.L. Haagmans (Bart); M. Schutten (Martin)

    2013-01-01

    textabstractTo evaluate the analytical performance and explore the clinical applicability of the new Roche cobas AmpliPrep/cobas TaqMan HCV test, v2.0 (CAP/CTM v2.0), a platform comparison was performed on panels and diagnostic samples with the Roche cobas AmpliPrep/cobas TaqMan HCV test (CAP/CTM v1

  10. Comparison of the COBAS/Ampliprep Taqman and Amplicor HIV-1 monitor tests in Lagos, Nigeria

    Directory of Open Access Journals (Sweden)

    Oluemi S. Amoo

    2013-03-01

    Full Text Available Background: The use of real-time Polymerase chain reaction (PCR technology options is increasing in resource-limited settings because they are faster, improve assay sensitivity,have higher throughput, larger dynamic ranges and reduced rates of contamination. In 2010, UNAIDS ranked Nigeria as the second highest population of people living with HIV and AIDS (2.98 million people in the world.Objective: The objective of this study was to compare the analytical performances of the Amplicor HIV-1 Monitor (version 1.5 and the COBAS Ampliprep/Taqman (version 2.0 usedin monitoring HIV disease progression in HIV-infected individuals.Method: In a cross-sectional study, HIV-1 RNA values obtained with the Amplicor HIV-1 monitor version 1.5 were compared with those of the COBAS/Ampliprep TaqMan HIV-1version 2.0 in a routine clinical setting. Between May and November 2011, 176 plasma samples collected were analysed in parallel using both techniques. Data analysis was done using statgraphics Centurion XVI and Medcalc version 12.0.Result: The correlation coefficient for the two assays was 0.83 and the level of agreement using a Bland–Altman plot was 94.2%.Conclusion: These findings suggest that the results from the two methods were comparable, hence the COBAS/Ampliprep Taqman version 2.0 is recommended for high-volume laboratories.

  11. Validation of Cobas AmpliPrep/Cobas TaqMan HIV-1 Test on dried blood spots

    Directory of Open Access Journals (Sweden)

    N Ruiz

    2012-11-01

    Full Text Available The plasma specimen is the gold standard for viral load monitoring, the key method to assess the effect of antiviral chemotherapy and to monitor progression of the disease toward AIDS. Nevertheless, several works endorse the use of dried blood spots (DBS on filter paper for the reliable quantification of the levels needed to take therapeutic decisions, detect of treatment failure and monitor the occurrence of drug resistance. The purpose of this study was to validate the use of Cobas AmpliPrep/Cobas TaqMan HIV-1 test version 2.0, with DBS. To evaluate the performance of the above mentioned kit, three stages were involved: 1- Standardization of DBS working conditions, 2- Stability studies at three temperature conditions and 3- Performance evaluation of the kit using this alternative specimen. Additionally, the viral load was quantified in parallel (plasma and DBS to 43 genetically characterized samples, with different levels of viral load. The Pearson correlation coefficient was calculated and the prediction of the value of RNA in plasma starting from the obtained value in DBS was made. Linear regression analysis was performed and coefficients of variation in precision assays were calculated. The best conditions pickups to the work with DBS were: 100 µL of blood (2 spots/50 µl, dried time between 16 and 18 hours at room temperature and, elution of the blood, 2 hours, between 2 and 8°C; in TRIS-EDTA buffer. The samples on DBS proved to be stable during the study periods. A strong correlation was attained between the measurements of viral load in plasma and DBS samples (r=0.96. The detection rate was 90.7 and the coefficient of variation between the values obtained in plasma-DBS sample pairs averaged 3.42%. The CAP/CTM HIV-1 test provided a linear response in DBS, from 330 copies/mL to 420 000 copies/mL. Overall, coefficients of variation in precision tests were below 10%. Cobas AmpliPrep/Cobas TaqMan HIV-1 test version 2.0 had a good

  12. [Implementation of the COBAS Taqman HIV-1 Test, v1.0 for vertical transmission diagnosis].

    Science.gov (United States)

    Castro, Gonzalo M; Sosa, María P; Gallego, Sandra V; Sicilia, Paola; Marin, Ángeles L; Altamirano, Natalia; Kademian, Silvia; Barbás, María G; Cudolá, Analía

    2015-01-01

    Vertical transmission is the main route of HIV infection in childhood. Because of the persistence of maternal HIV antibodies, virologic assays that directly detect HIV are required to diagnose HIV infection in infants younger than 18 months of age. The sensitivity of HIV RNA/DNA assays increases as the child becomes older. These tests have specificity values greater than 95%. The aim of this study was to evaluate the performance of the COBAS Taqman HIV-1 Test, v1.0 assay (Roche) and its concordance with a Multiplex Nested-PCR. Of 341 samples processed, 15 were positive and 326 negative by both methods. Sensitivity and specificity overall values for the viral load assay were 88.2% and 100%, respectively. Our results indicate that the COBAS Taqman assay evaluated could be used as an alternative method to diagnose HIV congenital infection. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  13. Evaluation of the Cobas TaqMan MTB real-time PCR assay for direct detection of Mycobacterium tuberculosis in respiratory specimens.

    Science.gov (United States)

    Lee, Meng-Rui; Chung, Kuei-Pin; Wang, Hao-Chien; Lin, Chih-Bin; Yu, Chong-Jen; Lee, Jen-Jyh; Hsueh, Po-Ren

    2013-08-01

    The Cobas TaqMan MTB assay is a real-time PCR (qPCR) kit for rapid detection of Mycobacterium tuberculosis from clinical specimens. There are, however, limited studies validating its performance. We performed a prospective study in two hospitals in Taiwan on 586 respiratory specimens. By using culture as the reference method, the sensitivity and specificity of the Cobas TaqMan MTB assay were found to be 82.7 and 96.5 %, respectively. The sensitivity of the Cobas TaqMan MTB assay in acid-fast stain-negative respiratory specimens was only 34.9 %. Five specimens from five patients were positive for M. tuberculosis by the Cobas TaqMan MTB assay but were negative for M. tuberculosis by conventional culture methods. A diagnosis of pulmonary tuberculosis (TB) was made based on clinical and radiological findings as well as the response to anti-TB treatment in these five patients. Addition of data from these five specimens with discrepant results (PCR vs culture) from patients with symptoms clinically compatible with TB increased the sensitivity of the Cobas TaqMan MTB assay to 83.1 %. The Cobas TaqMan MTB assay is a rapid identification tool with a high degree of specificity for the direct detection of M. tuberculosis in respiratory specimens. The sensitivity for detecting acid-fast smear-negative respiratory specimens, however, is low.

  14. Comparison of AdvanSure TB/NTM PCR and COBAS TaqMan MTB PCR for Detection of Mycobacterium tuberculosis Complex in Routine Clinical Practice.

    Science.gov (United States)

    Cho, Won-Hyung; Won, Eun-Jeong; Choi, Hyun-Jung; Kee, Seung-Jung; Shin, Jong-Hee; Ryang, Dong-Wook; Suh, Soon-Pal

    2015-05-01

    The AdvanSure tuberculosis/non-tuberculous mycobacterium (TB/NTM) PCR (LG Life Science, Korea) and COBAS TaqMan Mycobacterium tuberculosis (MTB) PCR (Roche Diagnostics, USA) are commonly used in clinical microbiology laboratories. We aimed to evaluate these two commercial real-time PCR assays for detection of MTB in a large set of clinical samples over a two-year period. AdvanSure TB/NTM PCR and COBAS TaqMan MTB PCR were performed on 9,119 (75.2%) and 3,010 (24.8%) of 12,129 (9,728 respiratory and 2,401 non-respiratory) MTB specimens, with 361 (4.0%) and 102 (3.4%) acid-fast bacilli (AFB)-positive results, respectively. In MTB culture, 788 (6.5%) MTB and 514 (4.2%) NTM were identified. The total sensitivity and specificity of the AdvanSure assay were 67.8% (95% confidence interval [CI], 63.9-71.6) and 98.3% (95% CI, 98.0-98.6), while those of the COBAS TaqMan assay were 67.2% (95% CI, 60.0-73.8) and 98.4% (95% CI, 97.9-98.9), respectively. The sensitivities and specificities of the AdvanSure and COBAS TaqMan assays for AFB-positive and AFB-negative samples were comparable. Furthermore, the AdvanSure assay showed fewer invalid results compared with the COBAS TaqMan assay (5.0 vs. 20.4 invalid results/1,000 tests, P<0.001). AdvanSure assay represents a comparable yet more reliable method than COBAS TaqMan for the identification of mycobacteria in routine clinical microbiology.

  15. An in-house real-time polymerase chain reaction: standardisation and comparison with the Cobas Amplicor HBV monitor and Cobas AmpliPrep/Cobas TaqMan HBV tests for the quantification of hepatitis B virus DNA

    Science.gov (United States)

    Santos, Ana Paula de Torres; Levi, José Eduardo; Lemos, Marcilio Figueiredo; Calux, Samira Julien; Oba, Isabel Takano; Moreira, Regina Célia

    2016-01-01

    This study aimed to standardise an in-house real-time polymerase chain reaction (rtPCR) to allow quantification of hepatitis B virus (HBV) DNA in serum or plasma samples, and to compare this method with two commercial assays, the Cobas Amplicor HBV monitor and the Cobas AmpliPrep/Cobas TaqMan HBV test. Samples from 397 patients from the state of São Paulo were analysed by all three methods. Fifty-two samples were from patients who were human immunodeficiency virus and hepatitis C virus positive, but HBV negative. Genotypes were characterised, and the viral load was measure in each sample. The in-house rtPCR showed an excellent success rate compared with commercial tests; inter-assay and intra-assay coefficients correlated with commercial tests (r = 0.96 and r = 0.913, p < 0.001) and the in-house test showed no genotype-dependent differences in detection and quantification rates. The in-house assay tested in this study could be used for screening and quantifying HBV DNA in order to monitor patients during therapy. PMID:26872342

  16. Clinical performance of the new Roche COBAS (R) TaqMan HCV test and high pure system for extraction, detection and quantitation of HCV RNA in plasma and serum

    NARCIS (Netherlands)

    H.C. Gelderblom; S. Menting; M.G. Beld

    2006-01-01

    We evaluated the Roche COBAS (R) TaqMan HCV Test For Use With The High Pure System (TaqMan HPS; Roche Diagnostics), for the extraction, detection and quantitation of hepatitis C virus (HCV) RNA in serum or plasma of HCV-infected individuals. The TaqMan HPS is a real-time PCR assay with a reported li

  17. Evaluation of performances of VERSANT HCV RNA 1.0 assay (kPCR) and Roche COBAS AmpliPrep/COBAS TaqMan HCV test v2.0 at low level viremia.

    Science.gov (United States)

    Mazzuti, Laura; Lozzi, Maria Antonietta; Riva, Elisabetta; Maida, Paola; Falasca, Francesca; Antonelli, Guido; Turriziani, Ombretta

    2016-07-01

    We assess the concordance between low level HCV values obtained using the VERSANT HCV RNA 1.0 Assay (kPCR) and COBAS AmpliPrep/COBAS TaqMan HCV Quantitative Test v2.0. The correlation between the values obtained by the two RT-PCR assays for samples with quantifiable HCV RNA levels revealed that viral load measured by kPCR significantly correlated with that of the CAP/CTM (R=0.644, PHCV triple therapy or interferon- free regimens. It is therefore recommended to monitor individual patients with the same test throughout treatment.

  18. Sensitivity and specificity of Cobas TaqMan MTB real-time polymerase chain reaction for culture-proven Mycobacterium tuberculosis: meta-analysis of 26999 specimens from 17 Studies.

    Science.gov (United States)

    Horita, Nobuyuki; Yamamoto, Masaki; Sato, Takashi; Tsukahara, Toshinori; Nagakura, Hideyuki; Tashiro, Ken; Shibata, Yuji; Watanabe, Hiroki; Nagai, Kenjiro; Nakashima, Kentaro; Ushio, Ryota; Ikeda, Misako; Sakamaki, Kentaro; Yoshiyama, Takashi; Kaneko, Takeshi

    2015-12-09

    Since 2010, studies on the diagnostic accuracy of COBAS TaqMan MTB (CTM) have been frequently reported with an unignorable discrepancy. The key inclusion criterion for this systematic review was original studies that could provide sufficient data for calculating the sensitivity and the specificity of CTM for M tuberculosis (TB) or M tuberculosis complex. The reference test was Mycobacterium culture. We used bivariate model for meta-analyses. Of the 201 candidate articles, we finally identified 17 eligible articles.Concerning the respiratory specimens, 1900 culture positive specimens and 20983 culture negative specimens from 15 studies were assessed. This provided the summary estimate sensitivity of 0.808 (95% CI 0.758-0.850) and the summary estimate specificity of 0.990 (95% CI 0.981-0.994). The area under curve was 0.956. The diagnostic odds ratio was 459 (95% CI 261-805, I(2) 26%). For the smear positive respiratory specimens, the sensitivity was 0.952 (95% CI 0.926-0.969) and the specificity was 0.916 (95% CI 0.797-0.968). For the smear negative respiratory specimens, the sensitivity and the specificity were 0.600 (95% CI 0.459-0.726) and 0.989 (95% CI 0.981-0.993), respectively. The diagnostic accuracy was poorer for the non-respiratory specimens, than for the respiratory specimens, but was acceptable. We believe that the information obtained from this study will aid physicians' decision making.

  19. Implementación del ensayo de carga viral COBAS Taqman HIV-1 Test, v1.0, para el diagnóstico de la infección congénita por HIV-1

    Directory of Open Access Journals (Sweden)

    Gonzalo M Castro

    2015-03-01

    Full Text Available La transmisión vertical es la principal vía de contagio del HIV en la edad pediátrica. El diagnóstico de la infección congénita antes de los 18 meses se realiza mediante ensayos virológicos: detección de genoma viral como ARN plasmático y ADN proviral. La sensibilidad de estos ensayos varía según la edad del niño, con valores de especificidad mayores al 95 %. El objetivo de este trabajo fue evaluar el desempeño del ensayo de carga viral (CV COBAS Taqman HIV-1 Test, v1.0 (Roche, y su concordancia con una PCR múltiple anidada in-house para la detección del ADN proviral. De 341 muestras procesadas, 15 resultaron positivas y 326 negativas por ambas metodologías. Para la metodología de CV, la sensibilidad general fue del 88,2 % y la especificidad del 100 %. Nuestros resultados indican que la metodología de CV evaluada puede utilizarse como técnica alternativa para el diagnóstico de infección congénita por HIV.

  20. Correlación en la medición de la carga viral para VIH 1 entre las técnicas de PCR amplicor y PCR tiempo real

    OpenAIRE

    2009-01-01

    El objetivo es determinar el nivel de correlación de la cuantificación de carga viral para VIH-1 mediante las técnicas Amplicor HIV-1 Monitor Versión Estándar y Sistema COBAS Ampliprep/COBAS Taqman 48 HIV-1; los materiales y métodos due que se realizó un estudio correlacional, se incluyó 152 muestras de plasma, de personas viviendo con VIH/SIDA que se encuentran en el Sistema de Monitoreo de TARGA del INS. Las muestras incluidas tenían carga viral en rango de 400 a 750,000 copias/mL, y se a...

  1. The cobas p 630 instrument: a dedicated pre-analytic solution to optimize COBAS® AmpliPrep/COBAS® TaqMan® system workflow and turn-around-time.

    Science.gov (United States)

    Vallefuoco, L; Sorrentino, R; Spalletti Cernia, D; Colucci, G; Portella, G

    2012-12-01

    The cobas p 630, a fully automated pre-analytical instrument for primary tube handling recently introduced to complete the Cobas(®) TaqMan systems portfolio, was evaluated in conjunction with: the COBAS(®) AmpliPrep/COBAS(®) TaqMan HBV Test, v2.0, COBAS(®) AmpliPrep/COBAS(®) TaqMan HCV Test, v1.0 and COBAS(®) AmpliPrep/COBAS(®) TaqMan HIV Test, v2.0. The instrument performance in transferring samples from primary to secondary tubes, its impact in improving COBAS(®) AmpliPrep/COBAS(®) TaqMan workflow and hands-on reduction and the risk of possible cross-contamination were assessed. Samples from 42 HBsAg positive, 42 HCV and 42 HIV antibody (Ab) positive patients as well as 21 healthy blood donors were processed with or without automated primary tubes. HIV, HCV and HBsAg positive samples showed a correlation index of 0.999, 0.987 and of 0.994, respectively. To assess for cross-contamination, high titer HBV DNA positive samples, HCV RNA and HIV RNA positive samples were distributed in the cobas p 630 in alternate tube positions, adjacent to negative control samples within the same rack. None of the healthy donor samples showed any reactivity. Based on these results, the cobas p 630 can improve workflow and sample tracing in laboratories performing molecular tests, and reduce turnaround time, errors, and risks.

  2. Discordant rapid HIV tests: lessons from a low-resource community.

    Science.gov (United States)

    Adetunji, A A; Kuti, M A; Audu, R A; Muyibi, S A; Imhansoloeva, M; Mosuro, O A; Solanke, E A; Akpa, O M; Irabor, A E; Ladipo, Mma; Berzins, B; Robertson, K; Ogunniyi, A; Adewole, I F; Taiwo, B O

    2017-07-31

    HIV rapid antibody tests are widely used in Africa, but dual testing sometimes produces discordant results. It is not clear if discordant rapid HIV tests should always heighten suspicion by frontline health workers that early HIV infection is present. Some studies have reported that discordant rapid tests have value for identifying early HIV infection in high HIV prevalence populations. It is not known if rapid test performance influenced this conclusion, or if this observation will hold true for low HIV prevalence populations. We therefore explored the occurrence of discordant rapid HIV tests in a low-resource community. A cross-sectional sample of HIV status-unaware adults with recent exposure to unsafe sex was assessed using a validated risk-based tool (University of North Carolina (UNC)-Malawi Risk Screening Score) for acute HIV infection. Participants received rapid testing with Determine™ HIV 1/2 and Uni-Gold™ HIV assays, plus plasma HIV-1 antigen testing with the COBAS(®) Ampliprep/COBAS(®) Taqman(®) HIV-1 assay, followed by western blot in those with detected HIV-1 antigen. Of 408 participants, 1.0% were confirmed to have established HIV infection. The discordance between rapid tests at initial screening was 2.45 and 2.94% when the two assays were used sequentially and simultaneously, respectively. Discordant rapid tests were strongly associated with risk scores > 2 [odds ratio (OR) 10.88; 95% confidence interval (CI) 2.35-50.43], and with detected HIV-1 RNA (OR 26.06; 95% CI 3.91-173.60). When the sample occurrence of discordance between the first and second tests is below 5%, discordant rapid tests in an adult with sexual risk behaviour should trigger strong suspicion of early HIV infection in low HIV prevalence populations. © 2017 British HIV Association.

  3. Evaluation of the Whole-Blood Alere Q NAT Point-of-Care RNA Assay for HIV-1 Viral Load Monitoring in a Primary Health Care Setting in Mozambique.

    Science.gov (United States)

    Jani, Ilesh V; Meggi, Bindiya; Vubil, Adolfo; Sitoe, Nádia E; Bhatt, Nilesh; Tobaiwa, Ocean; Quevedo, Jorge I; Loquiha, Osvaldo; Lehe, Jonathan D; Vojnov, Lara; Peter, Trevor F

    2016-08-01

    Viral load testing is the WHO-recommended monitoring assay for patients on HIV antiretroviral therapy (ART). Point-of-care (POC) assays may help improve access to viral load testing in resource-limited settings. We compared the performance of the Alere Q NAT POC viral load technology (Alere Technologies, Jena, Germany), measuring total HIV RNA using finger prick capillary whole-blood samples collected in a periurban health center, with that of a laboratory-based plasma RNA test (Roche Cobas Ampliprep/Cobas TaqMan v2) conducted on matched venous blood samples. The whole-blood Alere Q NAT POC assay produced results with a bias of 0.8593 log copy/ml compared to the laboratory-based plasma assay. However, at above 10,000 copies/ml, the bias was 0.07 log copy/ml. Using the WHO-recommended threshold to determine ART failure of 1,000 copies/ml, the sensitivity and specificity of the whole-blood Alere Q NAT POC assay were 96.83% and 47.80%, respectively. A cutoff of 10,000 copies/ml of whole blood with the Alere Q NAT POC assay appears to be a better predictor of ART failure threshold (1,000 copies/ml of plasma), with a sensitivity of 84.0% and specificity of 90.3%. The precision of the whole-blood Alere Q NAT POC assay was comparable to that observed with the laboratory technology (5.4% versus 7.5%) between detectable paired samples. HIV POC viral load testing is feasible at the primary health care level. Further research on the value of whole-blood viral load to monitor antiretroviral therapy is warranted.

  4. Evaluation of the Whole-Blood Alere Q NAT Point-of-Care RNA Assay for HIV-1 Viral Load Monitoring in a Primary Health Care Setting in Mozambique

    Science.gov (United States)

    Meggi, Bindiya; Vubil, Adolfo; Sitoe, Nádia E.; Bhatt, Nilesh; Tobaiwa, Ocean; Quevedo, Jorge I.; Loquiha, Osvaldo; Lehe, Jonathan D.; Vojnov, Lara; Peter, Trevor F.

    2016-01-01

    Viral load testing is the WHO-recommended monitoring assay for patients on HIV antiretroviral therapy (ART). Point-of-care (POC) assays may help improve access to viral load testing in resource-limited settings. We compared the performance of the Alere Q NAT POC viral load technology (Alere Technologies, Jena, Germany), measuring total HIV RNA using finger prick capillary whole-blood samples collected in a periurban health center, with that of a laboratory-based plasma RNA test (Roche Cobas Ampliprep/Cobas TaqMan v2) conducted on matched venous blood samples. The whole-blood Alere Q NAT POC assay produced results with a bias of 0.8593 log copy/ml compared to the laboratory-based plasma assay. However, at above 10,000 copies/ml, the bias was 0.07 log copy/ml. Using the WHO-recommended threshold to determine ART failure of 1,000 copies/ml, the sensitivity and specificity of the whole-blood Alere Q NAT POC assay were 96.83% and 47.80%, respectively. A cutoff of 10,000 copies/ml of whole blood with the Alere Q NAT POC assay appears to be a better predictor of ART failure threshold (1,000 copies/ml of plasma), with a sensitivity of 84.0% and specificity of 90.3%. The precision of the whole-blood Alere Q NAT POC assay was comparable to that observed with the laboratory technology (5.4% versus 7.5%) between detectable paired samples. HIV POC viral load testing is feasible at the primary health care level. Further research on the value of whole-blood viral load to monitor antiretroviral therapy is warranted. PMID:27252459

  5. COBAS S201核酸检测系统在献血者血液筛查中的应用%Use of COBAS S201 nucleic acid detection system in screening blood donors

    Institute of Scientific and Technical Information of China (English)

    车嘉琳; 黄志森; 王德文; 梁兵; 师玲玲; 许惠芯; 朱毅瑜

    2010-01-01

    目的 采用罗氏COBAS S201核酸检测系统,调查东莞市现行血液筛查系统的残余风险,以评估开展核酸检测(nucleic acid amplification technique,NAT)的必要性和可行性.方法 对2008年7月31日至2009年3月31日期间经ELISA检测阴性的40 018份献血者血液样本,采用罗氏COBAS S201检测系统进行HBV DNA,HCV RNA,HIV RNA检测.COBAS S201检测为阳性的献血者样本,分别采用COBAS Ampliprep/Taqman平台做核酸定量检测和罗氏ECL电化学发光检测系统作乙肝"两对半"实验,以帮助分析判定样本的感染状态.结果 发现31例核酸反应性样本,阳性率为0.77‰,其中有17例为HBV核酸反应性,残余风险为1/2354~1/1291,COBAS S201核酸检测系统的临床特异性为99.97%.结论 现行的血液筛查策略为两遍ELISA检测,但仍然存在输血传播疾病的风险.COBAS S201系统操作安全简便,包含罗氏专利技术的防污染技术,可确保检测结果准确可靠,适合于对献血者血液常规筛查.%Objective To investigate the residual risk in the current blood screening system in Dongguan City by Roche COBAS S201 nucleic acid detection system, in order to assess the necessity and feasibility of nucleic acid amplification technique (NAT). Methods 40 018 ELISA-negative samples were detected for HBV DNA, HCV RNA as well as HIV RNA by Roche COBAS S201 detection system from July 31,2008 to March 31, 2009. Positive samples were under quantitative detection of nucleic acid by COBAS Ampliprep/Taqman platform as well as "two pairs of semi "-experiments of hepatitis B by Roche ECL electrochemiluminescence detection system, aiming at helping to analyze the infection status of samples.Results 31 NAT-reactive samples were found, and the positive rate was 0.77‰. 17 of 31 samples were HBV DNA-reactive, and the residual risk was 1/2354-1/1291. The clinical specificity of COBAS S201 nucleic acid detection system was 99.97%. Conclusions The current blood screening strategy

  6. Clinical Performance of Roche Cobas 4800 HPV Test

    Science.gov (United States)

    Cui, Miao; Chan, Nicholas; Liu, Momo; Thai, Khanh; Malaczynska, Joanna; Singh, Ila; Zhang, David

    2014-01-01

    Evaluation of the Cobas 4800 test demonstrated that Cobas had a low rate of cross-reactivity with low-risk human papillomavirus (lrHPV), a 3.74% disconcordance rate between prealiquots and postaliquots, and failure rates of 4.57% and 1.16%, respectively, after vortexing and swirling. This study demonstrated that the Cobas test has good sensitivity, accuracy, and reproducibility for detecting 14 high-risk HPV (hrHPV) genotypes. PMID:24719443

  7. Use of the cobas 4800 system for the rapid detection of toxigenic Clostridium difficile and methicillin-resistant Staphylococcus aureus.

    Science.gov (United States)

    Moure, Raquel; Cañizares, Ángeles; Muíño, María; Lobato, Margarita; Fernández, Ana; Rodríguez, María; Gude, Maria José; Tomás, Maria; Bou, Germán

    2016-01-01

    The new cobas® Cdiff and cobas® MRSA/SA tests were compared with conventional methods for the rapid detection of toxigenic Clostridium difficile and methicillin-resistant Staphylococcus aureus. The final concordance between cobas Cdiff Test and GDH/toxin gene screening was 97.62% and between cobas MRSA/SA Test and chromogenic culture, 91.30%, respectively.

  8. 乙型肝炎病毒核酸检测试剂临床应用的分析%Evaluation of multiplex nucleic acid testing assays for screening of hepatitis B virus DNA in blood donation process

    Institute of Scientific and Technical Information of China (English)

    周诚; 吴星; 黄维金; 蓝海云; 辜文洁; 祁自柏; 梁争论; 李河民

    2008-01-01

    Objective To evaluate the multiplex nucleic acid testing (NAT) assays for HBV,HCV and HIV in detecting HBV DNA in plasma samples. Methods 534 plasma samples collected form several areas were detected with Abbott Architect i2000 HBsAg, ani-HBs, HBeAg, anti-HBe, anti-HBc and anti-HBc IgM diagnostic kits. HBV DNA levels of those samples were detected with Roche COBAS AmpliPrep/ COBAS TaqMan HBV Test. Two kinds of multiplex NAT assays for HBV, HCV and HIV were used to test HBV DNA of those 534 samples. Results of serology-markers and quantitative HBV DNA levels with results of NAT were compared. Results HBV DNA was positive in all 81 HBsAg, HBeAg and anti-HBc positive samples,detected by both of NAT assays. HBV DNA was positive in 11 and 19 of 200 HBsAg negative samples when detected with the two kinds of NAT assays separately. Compared with the quantitative results detected by Roche COBAS AmpliPrep/COBAS TaqMan HBV Test, the HBV DNA positive rates were 96.9% and 94.3% in 193 samples of HBV DNA levels over 500 IU/ml while 40.2% and 45.3% in 117 samples of HBV DNA levels below 500 IU/ml while 99.3% and 96.0% in 151 samples of DNA negative HBV. Conclusion There are some occult low level HBV DNA carriers with HBsAg negative results in China. NAT assays for HBV, HCV and HIV may be useful to improve the transfusion safety.%目的 了解HBV/HCV/HIV联合核酸检测的临床应用.方法 使用Abbott Architecti2000化学发光检测盒对534份血浆样品进行血清学检测,Roche COBAS AmpliPrep/COBAS TaqManHBV Test试剂定量检测分别与2种联合核酸检测结果 进行比较分析.结果 81份HBsAg、HBeAg、抗-HBc 3项均阳性样品联合核酸检测均为HBV阳性,200份HBsAg阴性的样品中联合核酸检测试剂分别有11、19份检测为HBV阳性.HBV DNA定量检测>500 IU/ml的193份样品联合核酸检测试剂阳性符合率分别为96.9%、94.3%,117份样品<500 IU/ml阳性符合率分别为40.2%、45.3%,151份HBV DNA阴性样品联

  9. Real-time PCR per HBV DNA: valutazione del nuovo sistema automatizzato COBAS AMPLIPREP™/COBAS TAQMAN™ HBV

    Directory of Open Access Journals (Sweden)

    Tiziano Allice

    2007-12-01

    Full Text Available Success of antiviral therapy for chronic hepatitis B is supported by highly sensitive PCR-based assays for Hepatitis B virus (HBV DNA. Nucleic acid extraction from biologic specimens is technically demanding and reliable PCR results depend it. Performances of the fully automatic system COBAS AmpliPrep™/COBAS TaqMan™ 48 (CAP/CTM (Roche, Branchburg, NJ for HBV DNA extraction and real -time PCR quantification were assessed and compared with the end-point PCR COBAS AMPLICOR HBV Monitor (CAHBM, Roche. Analytical evaluation with a proficiency panel showed that CAP/CTM quantitated HBV DNA levels in one single run over a wide dynamic range (7 logs with a close correlation between expected and observed values (r=0.976, interassay variability below 5%. Clinical evaluation as tested with samples from 92 HBsAg-positive patients, demonstrated excellent correlation with CAHBM (r=0.966, mean difference in quantitation: 0.36 log10 IU/ml. CAP/CTM detected 10% more viremic patients and longer period of residual viremia in those on therapy. In lamivudine (LAM-resistant patients, reduction of HBV DNA after 12 months of Adefovir (ADF was higher in the combination (LAM+ADF schedule than in ADF monotherapy (5.1 vs. 3.5 logs suggesting a benefit in continuing LAM. In conclusion,CAP/CTM can improve the management of HBV infection, the assessment of antiviral therapy and drug resistance, supporting further insights in the emerging area of drug resistance.

  10. Evaluation of NGAL TestTM on Cobas 6000

    DEFF Research Database (Denmark)

    Hansen, Young B L; Damgaard, Anette; Poulsen, Jørgen H

    2014-01-01

    analyzed for method, anticoagulant, and freeze-thaw comparisons. Linearity was assessed using high NGAL samples diluted in urine, EDTA, and Li-Hep plasma. Commercial internal controls were used for the imprecision study. RESULTS: The Cobas 6000 measured identically with the Hitachi 917, however...... the Hitachi 917 in EDTA plasma. Though clinically insignificant, we found that the freeze-thaw process had a reduced effect. NGAL results were higher in Li-Hep tubes than in EDTA tubes. Thus, for blood samples we recommend use of EDTA tubes for NGAL measurements....

  11. Data of evolutionary structure change: 1COBA-1FUNF [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1COBA-1FUNF 1COB 1FUN A F ATKAVCVLKGDGPVQGTIHFEAKGD--TVVVTGSITGLT...VIGIAK ATKAVAVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVT... EEEEEEEE GGG EEEEE EEEEEEE EEEEE HHHH EEEEEEE EVID> 0 F 1FUNF EQKESNGPVKVW

  12. Cobas-6000 - Kliinisen kemian analysaattorin laitekoestus parathormonimäärityksen osalta

    OpenAIRE

    Lehtinen, Otto

    2011-01-01

    Laitekoestus on analysaattorin toimintakyvyn testaamista. Tutkimuksessa koestettavana analysaattorina oli Roche Cobas-6000. Koestus suoritettiin elektrokemiluminometrisen parathormonimäärityksen osalta. Tarkoituksena oli selvittää, kuinka toistettavia ja täsmääviä tuloksia Cobas-analysaattori antaa ja kuinka hyvin saadut tulokset täsmäävät aiemman palveluntarjoajan, Medixlaboratorion, tulostasoon. Tutkimus määriteltiin kvantitatiiviseksi. Saadut analyysitulokset on sen mukaisesti arvioitu...

  13. NT-proBNP on Cobas h 232 in point-of-care testing

    DEFF Research Database (Denmark)

    Gils, Charlotte; Ramanathan, R.; Breindahl, T.;

    2015-01-01

    Background. NT-proBNP may be useful for ruling out heart failure in primary health care. In this study we examined the analytical quality of NT-proBNP in primary health care on the Cobas h 232 point-of-care instrument compared with measurements performed in a hospital laboratory. Materials...... and methods. Blood samples requested for NT-proBNP were collected in primary health care (n = 95) and in a hospital laboratory (n = 107). NT-proBNP was measured on-site on Cobas h 232 instruments both in primary health care centres and at the hospital laboratory and all samples were also analyzed...... with a comparison method at the hospital. Precision, trueness, accuracy, and lot-variation were determined at different concentration levels and evaluated according to acceptance criteria. Furthermore user-friendliness was assessed by questionnaires. Results. For Cobas h 232 repeatability CV was 8...

  14. Prevalence of high-risk human papillomavirus by cobas 4800 HPV test in urban Peru

    Directory of Open Access Journals (Sweden)

    Ricardo Iwasaki

    2014-09-01

    Full Text Available Background: Molecular tests allow the detection of high-risk human papillomavirus in cervical samples, playing an important role in the prevention of cervical cancer. Objectives: We performed a study to determine the prevalence of HPV 16, HPV 18 and other high-risk human papillomavirus (pool 12 genotypes in Peruvian females from diverse urban areas using the cobas 4800 HPV test. Methods: Routine cervical samples collected in our laboratory were analyzed by cobas 4800 HPV test. Results: A total of 2247 samples from female patients aged 17–79 years were tested. high-risk human papillomavirus was positive in 775 (34.49% samples. Of these, 641 (82.71% were single infections and 134 (17.29% were multiple infections. The positivity rates for HPV 16, HPV 18, and other high-risk human papillomavirus were 10.77%, 2.0%, and 28.08%, respectively. In multiple high-risk human papillomavirus infections, the concomitance of HPV 16 and other high-risk human papillomavirus was more prevalent (13.42%. Conclusion: Our study showed high prevalence of high-risk human papillomavirus in urban Peru, mainly among young women. In both single and multiple infections other high-risk human papillomavirus were more prevalent than HPV 16 and HPV 18, which might influence vaccine impact in our country. Furthermore, the cobas 4800 HPV test may be considered a useful tool for HPV molecular diagnosis.

  15. Cross-reactivity profiles of hybrid capture II, cobas, and APTIMA human papillomavirus assays

    DEFF Research Database (Denmark)

    Preisler, Sarah Nørgaard; Rebolj, Matejka; Ejegod, Ditte Møller

    2016-01-01

    evaluated to what extent these can be explained by cross-reactivity, i.e. positive test results without evidence of high-risk HPV genotypes. The patterns of cross-reactivity have been thoroughly studied for hybrid capture II (HC2) but not yet for newer HPV assays although the manufacturers claimed...... no or limited frequency of cross-reactivity. In this independent study we evaluated the frequency of cross-reactivity for HC2, cobas, and APTIMA assays.METHODS:Consecutive routine cervical screening samples from 5022 Danish women, including 2859 from women attending primary screening, were tested with the three...... cytology and positive high-risk HPV test results were invited for repeated testing in 18 months.RESULTS:Cross-reactivity to low-risk genotypes was detected in 109 (2.2 %) out of 5022 samples on HC2, 62 (1.2 %) on cobas, and 35 (0.7 %) on APTIMA with only 10 of the samples cross-reacting on all 3 assays...

  16. Comparison of the COBAS AMPLICOR MTB and BDProbeTec ET assays for detection of Mycobacterium tuberculosis in respiratory specimens.

    NARCIS (Netherlands)

    W.H.F. Goessens (Wil); P. de Man (Peter); J.G. Koeleman; A. Luijendijk (Ad); R. te Witt (René); H.P. Endtz (Hubert); A.F. van Belkum (Alex)

    2005-01-01

    textabstractThe performances of the BDProbeTec ET (Becton Dickinson) and COBAS AMPLICOR MTB (Roche) were retrospectively evaluated for detecting Mycobacterium tuberculosis complex in various respiratory specimens. The BACTEC and MGIT liquid culture system (Becton Dickinson) was used as a reference m

  17. Hybrid Capture 2 and cobas human papillomavirus assays perform similarly on SurePath samples from women with abnormalities

    DEFF Research Database (Denmark)

    Fornari, D; Rebolj, M; Bjerregaard, B

    2016-01-01

    OBJECTIVE: In two laboratories (Departments of Pathology, Copenhagen University Hospitals of Herlev and Hvidovre), we compared cobas and Hybrid Capture 2 (HC2) human papillomavirus (HPV) assays using SurePath® samples from women with atypical squamous cells of undetermined significance (ASCUS) at...

  18. Flow-cytochemical differential leukocyte analysis with quantitation of neutrophil left shift. An evaluation of the Cobas-Helios analyzer.

    Science.gov (United States)

    Bentley, S A; Johnson, T S; Sohier, C H; Bishop, C A

    1994-08-01

    The Cobas-Helios (Roche Diagnostic Systems, Inc., Branchburg, NJ) is a new, fully automated hematology analyzer that performs a complete blood count and differential leukocyte count (DLC), classifying leukocytes by flow-cytochemical technology. The DLC component of the Cobas-Helios was evaluated according to the National Committee for Clinical Laboratory Standards H20-A protocol. Instrument performance was acceptable with respect to all parameters investigated, including imprecision, inaccuracy and clinical sensitivity for the identification of quantitative and qualitative leukocyte abnormalities. In a minority of samples with neutrophil left shift, neutrophils tended to overlap the monocyte domain, resulting in overestimation of monocytes and underestimation of neutrophils. This problem did not affect clinical sensitivity and was generally associated with a positive instrumental left-shift flag. Flags for the identification of specific qualitative abnormalities of the leukocyte population (atypical lymphoid cells, nucleated red cells, blast cells, immature granulocytes and neutrophil left shift) performed well. In addition to a conventional five-part DLC, the Cobas-Helios also identifies and quantitates atypical lymphoid cells and "large immature cells," the latter corresponding to bands and immature granulocytes. Counts of atypical lymphoid cells and large immature cells correlated well with the equivalent cell classes as enumerated by the reference method of the National Committee for Clinical Laboratory Standards. The Cobas-Helios offers the most reliable quantitative index of neutrophil left shift currently available in a commercial automated DLC analyzer.

  19. Prevalence of human papillomavirus in 5,072 consecutive cervical SurePath samples evaluated with the Roche cobas HPV real-time PCR assay.

    Directory of Open Access Journals (Sweden)

    Sarah Preisler

    Full Text Available New commercially available Human Papillomavirus (HPV assays need to be evaluated in a variety of cervical screening settings. Cobas HPV Test (cobas is a real-time PCR-based assay allowing for separate detection of HPV genotypes 16 and 18 and a bulk of 12 other high-risk genotypes. The aim of the present study, Horizon, was to assess the prevalence of high-risk HPV infections in an area with a high background risk of cervical cancer, where women aged 23-65 years are targeted for cervical screening. We collected 6,258 consecutive cervical samples from the largest cervical screening laboratory in Denmark serving the whole of Copenhagen. All samples were stored in SurePath media. In total, 5,072 samples were tested with cobas, Hybrid Capture 2 High Risk HPV DNA test (HC2 and liquid-based cytology. Of these, 27% tested positive on cobas. This proportion decreased by age, being 43% in women aged 23-29 years and 10% in women aged 60-65 years. HC2 assay was positive in 20% of samples, and cytology was abnormal (≥ atypical squamous cells of undetermined significance for 7% samples. When only samples without recent abnormalities were taken into account, 24% tested positive on cobas, 19% on HC2, and 5% had abnormal cytology. The proportion of positive cobas samples was higher than in the ATHENA trial. The age-standardized cobas positivity vs. cytology abnormality was 3.9 in our study and 1.7 in ATHENA. If in Copenhagen the presently used cytology would be replaced by cobas in women above age 30 years, an extra 11% of women would based on historical data be expected to have a positive cobas test without an underlying cervical intraepithelial lesion grade 3 or worse. Countries with a high prevalence of HPV infections should therefore proceed to primary HPV-based cervical screening with caution.

  20. Diagnostic value of Cobas Amplicor MTB and Rotorgene Real Time PCR for tuberculous meningitis: A six-year retrospective study

    Directory of Open Access Journals (Sweden)

    Gülnur Tarhan

    2015-12-01

    Full Text Available Objective: Tuberculous meningitis (TBM is the most severe and lethal form of tuberculosis (TB.Bacteriologic confirmation of TBM is difficult and slow. Therefore, most patients receive ntituberculosis treatment based only on clinical and cerebrospinal fluid (CSF characteristics. Rapid diagnosis of TBM is important to decrease morbidity and mortality. The aim of the study was to demonstrate that COBAS Amplicor MTB and Rotorgene Real Time (RT PCR is a rapid method of diagnosing TBM before and after initiating anti-tuberculosis treatment. Methods: A retrospective study was conducted between December 2002 and January 2009 in 468 patients with suspected TBM. Clinical specimens were collected from different hospitals in Ankara. All specimens were evaluated by smear microscopy and culture methods with Lowenstein-Jensen (LJ and MGIT culture system. Results: Using culture results as a gold standard, the sensitivity, specificity, positive predictive values (PPV, and negative predictive values (NPV were 71.0%, 98.8%, 97.8% and 75.0%, respectively, for COBAS Amplicor MTB and 80%, 98.9%, 99.0% and 80.0%, respectively, for Rotorgene RT PCR. Statistical analysis showed no significant differences between the COBAS Amplicor MTB and Rotorgene RT PCR (p≥0.05. All isolates were susceptible to isoniazid, rifampin, streptomycin, and ethambutol with proportion method in LJ medium. All isolates were defined as LAM7-TUR by spoligotyping. Conclusion: Retrospective analysis of COBAS Amplicor MTB and Rotorgene RT PCR found that both tests are effective in rapidly diagnosing MTB using CSF. It was concluded that Rotorgene RT PCR test is more sensitive (81.0% than COBAS Amplicor MTB (71.0%. J Microbiol Infect Dis 2015;5(4: 156-161

  1. TELAAH SOAL UJI COBA I BAHASA INDONESIA DI SMP KABUPATEN WONOSOBO TAHUN 2013

    Directory of Open Access Journals (Sweden)

    Mushoffa Mushoffa

    2015-10-01

    Full Text Available Penelitian ini bertujuan untuk mendeskripsikan ketepatan indikator dalam kisi-kisi soal, keterwakilan setiap kompetensi dasar dalam kisi-kisi soal, kesesuaian antara kisi-kisi soal dan soal, serta capaian daya serap setiap kompetensi dasar. Penelitian ini merupakan penelitian deskriptif kualitatif-kuantitatif. Subjek penelitian ini adalah kisi-kisi soal, soal, dan capaian daya serap dari soal uji coba I bahasa Indonesia di SMP kabupaten Wonosobo tahun 2013. Pengumpulan data dilakukan dengan teknik baca dan catat. Hasil penelitian ini adalah sebagai berikut: (1 ketepatan indikator dalam kisi-kisi soal termasuk kategori tepat (86,4% dengan rincian ketepatan indikator pada kategori sangat tepat sebesar 22%, tepat sebesar 68%, cukup tepat sebesar 8%, kurang tepat sebesar 2%, dan tidak tepat sebesar 0%; (2 keterwakilan setiap kompetensi dasar dalam kisi-kisi soal uji coba I bahasa Indonesia Kabupaten Wonosobo dalam kategori sangat terwakili (96,7%; (3 kesesuaian antara kisi-kisi soal dan soal uji coba I bahasa Indonesia Kabupaten Wonosobo tahun 2013 termasuk kategori sesuai (86%; dan capaian daya serap untuk seluruh kompetensi dasar dalam soal uji coba I bahasa Indonesia Kabupaten Wonosobo tahun 2013 termasuk kategori rendah yaitu sebesar 56,89% dengan rincian capaian pada kategori sangat tinggi sebesar 0%, tinggi sebesar 14%,  sedang sebesar 24%, rendah sebesar 30%, dan sangat rendah sebesar 32%. Kata Kunci: telaah, kisi-kisi soal, soal, daya serap   THE ANALYSIS OF THE FIRST TRY OUT OF INDONESIAN LANGUAGE TEST IN JUNIOR HIGH SCHOOLS OF WONOSOBO REGENCY IN 2013 Abstract This study aims to describe the accuracy of the indicators in the table of specifications, the representation of each basic competence in the table of specifications, the compatibility between table of specifications and test items, and the learning outcomes performance of each basic competence of the test items. This research is qualitative-quantitative descriptive. The subjects

  2. Reliability of Cobas Amplicor PCR test in detection of Mycobacterium tuberculosis in respiratory and nonorespiratory specimens

    Directory of Open Access Journals (Sweden)

    Lepšanović Zorica

    2009-01-01

    Full Text Available Background/Aim. Traditional methods for detection of mycobacteria, such as microscopic examination for the presence of acid-fast bacilli and isolation of the organism by culture, have either a low sensitivity and/or specificity, or take weeks before a definite result is available. Molecular methods, especially those based on nucleic acid amplification, are rapid diagnostic methods which combine high sensitivity and high specificity. The aim of this study was to determine the usefulness of the Cobas Amplicor Mycobacterium tuberculosis polymerase chain reaction (CAPCR assay in detecting the tuberculosis cause in respiratory and nonrespiratory specimens (compared to culture. Methods. Specimens were decontaminated by the N-acetyl-L-cystein- NaOH method. A 500 μL aliquot of the processed specimen were used for inoculation of Löwenstein-Jensen (L-J slants, a drop for acid-fast staining, and 100 μL for PCR. The Cobas Amplicor PCR was performed according to the manufacturer's instructions. Results. A total of 110 respiratory and 355 nonrespiratory specimens were investigated. After resolving discrepancies by reviewing medical history, overall sensitivity, specificity, and positive and negative predictive values for CA-PCR assay compared to culture, were 83%, 100%, 100%, and 96.8%, respectively. In comparison, they were 50%, 99.7%, 87.5%, and 98%, respectively, for the nonrespiratory specimens. The inhibition rate was 2.8% for respiratory, and 7.6% for nonrespiratory specimens. Conclusion. CA-PCR is a reliable assay that enables specialists to start treatment promptly on a positive test result. Lower value for specificity in a group of nonrespiratory specimens is a consequence of an extremely small number of mycobacteria in some of them.

  3. A Comparison of the Roche Cobas HPV Test With the Hybrid Capture 2 Test for the Detection of High-Risk Human Papillomavirus Genotypes

    National Research Council Canada - National Science Library

    Levi, Angelique W; Bernstein, Jane I; Hui, Pei; Duch, Kara; Schofield, Kevin; Chhieng, David C

    2016-01-01

    All Food and Drug Administration-approved methods in the United States for human papillomavirus testing including the Hybrid Capture 2 human papillomavirus assay and the Roche cobas human papilloma...

  4. Comparison of Architect i2000sr and Cobas e601 Systems for Determining Serum Human Chorionic Gonadotropin-Beta.

    Science.gov (United States)

    Guan, Xiaoyong; Sun, Yifan; Zhang, Hongyu; Liang, Ka; Long, Kang; Li, Jin; Tang, Shifu; Liu, Chunming

    2016-09-01

    Human chorionic gonadotropin-beta (β-hCG) is an important index used to monitor embryonic development following embryo transfer. Architect i2000sr and Cobas e601 are widely used automated immunoassay systems used to measure serum β-hCG concentrations; however, the correlations between serum β-hCG levels measured with these two immunoassays and the accuracy of the immunoassays have not been fully evaluated. Serum β-hCG levels were measured in 133 serum samples using the Architect i2000sr and Cobas e601 automated immunoassay systems. Passing-Bablok regression analysis was used to compare the correlation in serum β-hCG levels obtained using the two immunoassays. A Bland-Altman plot analysis was used to identify mean ratios and 95% CIs of the mean ratios of the β-hCG results between the two immunoassays. In this graphical method the mean ratios between the two techniques were plotted against the averages of the two techniques. The total coefficients of variations (CVs) of serum β-hCG ranged from 3.12 - 4.66% for Cobas e601 and 3.18 - 4.99% for Architect i2000sr. The measured value of serum β-hCG detected by the two immunoassays was statistically significant (p coefficient r was 0.9628. At a high concentration of serum β-hCG (> 10000 IU/L, n = 81), the correlation coefficient r was 0.8076. The Bland-Altman plot analysis showed that the measured value of serum β-hCG detected by Architect i2000sr was about 1.25 times higher than that of Cobas e601. The mean ratio was 1.12 at a low concentration of serum β-hCG, and it was 1.33 at a high concentration. Architect i2000sr and Cobas e601 have good concordance for determining serum β-hCG. However, the β-hCG values measured with Architect i2000sr were 25% higher than those obtained using Cobas e601.

  5. Neisseria gonorrhoeae False-Positive Result Obtained from a Pharyngeal Swab by Using the Roche cobas 4800 CT/NG Assay in New Zealand in 2012

    OpenAIRE

    Upton, Arlo; Bromhead, Collette; Whiley, David M

    2013-01-01

    The Roche cobas 4800 CT/NG assay is a commonly used commercial system for screening for Neisseria gonorrhoeae infection, and previous studies have shown the method to be highly sensitive and specific for urogenital samples. We present the first confirmed clinical N. gonorrhoeae false-positive result using the cobas 4800 NG assay, obtained from testing a pharyngeal swab sample and caused by cross-reaction with a commensal Neisseria strain.

  6. Prevalence of human papillomavirus in 5,072 consecutive cervical SurePath samples evaluated with the Roche cobas HPV real-time PCR assay

    DEFF Research Database (Denmark)

    Preisler, Sarah; Rebolj, Matejka; Untermann, Anette

    2013-01-01

    in Denmark serving the whole of Copenhagen. All samples were stored in SurePath media. In total, 5,072 samples were tested with cobas, Hybrid Capture 2 High Risk HPV DNA test (HC2) and liquid-based cytology. Of these, 27% tested positive on cobas. This proportion decreased by age, being 43% in women aged 23......-29 years and 10% in women aged 60-65 years. HC2 assay was positive in 20% of samples, and cytology was abnormal (≥ atypical squamous cells of undetermined significance) for 7% samples. When only samples without recent abnormalities were taken into account, 24% tested positive on cobas, 19% on HC2, and 5......% had abnormal cytology. The proportion of positive cobas samples was higher than in the ATHENA trial. The age-standardized cobas positivity vs. cytology abnormality was 3.9 in our study and 1.7 in ATHENA. If in Copenhagen the presently used cytology would be replaced by cobas in women above age 30...

  7. CLSI-Based Validation of Manufacturer-Derived Reference Intervals on the Cobas 8000 Platform.

    Science.gov (United States)

    Leitner-Ferenc, Veronika; Atamaniuk, Johanna; Jansen-Skoupy, Sonja; Stöckelmeier, Brigitta; Grohs, Katharina; Födinger, Manuela

    2017-05-01

    Reference intervals provided by diagnostic test manufacturers should be transferred to clinical laboratories after validation. Although protocols exist, laboratories rarely perform and report on results of validation studies. We validated reference intervals (RIs) of 87 analytes on a Cobas 8000 platform according to standards published by the Clinical and Laboratory Standards Institute (CLSI). For 8 analytes, decision limits were provided in the package inserts. Among the 79 RIs subjected to transference validation, 8 were found not valid for transference, including lactate dehydrogenase (LDH) among women, and the following among both sexes: potassium, homocysteine, immunoglobulin E (IgE), free lambda light chain (FLC λ), C3 complement (C3c), folate, and 25-hydroxy vitamin D (25[(OH]D). For LDH, potassium, homocysteine, C3c, folate, and 25(OH)D, RIs or thresholds suitable for transference were available in the literature; however, this was not the case for IgE and FLC λ. The present study demonstrates that validation of RIs provided in the manufacturer provided package inserts is indispensable.

  8. Optical kinetic method for calibration of spectrophotometer temperature: demonstration with the Cobas-Bio analyzer.

    Science.gov (United States)

    Armitage, E K; Miller, W G

    1987-10-01

    The pseudo-first-order rate constant for the Jaffé reaction with creatinine varies logarithmically with temperature and was calibrated in the range 25 to 37 degrees C to measure the temperature of the liquid in the lightpath of spectrophotometric instrumentation. The reagent concentrations can be adjusted to permit rate-constant measurements in time intervals from a few seconds to several minutes. The temperature increment that can be resolved is limited only by the analytical imprecision of the instrumentation used to measure the rate constant and the calibration temperature. In this investigation, a temperature SD of 0.03 degrees C could be measured. Two Cobas-Bio centrifugal analyzers, used to demonstrate the utility of this technique, were found to have temperature errors from -1.0 to -1.7 degrees C in the 30 to 37 degrees C range and overall temperature SD of 0.19 and 0.36 degree C, respectively, at 37 degrees C. Analysis of variance gave between-rotor SD of 0.14 and 0.34 degrees C and within-rotor SD of 0.13 and 0.11 degree C, respectively. We found temperature differences of 0.3 degree C between cuvets in a rotor, and gradients of 0.3 and 0.4 degree C, respectively, from the top to bottom of an individual cuvet in the two instruments.

  9. Interim Report on Multiple Sequence Alignments and TaqMan Signature Mapping to Phylogenetic Trees

    Energy Technology Data Exchange (ETDEWEB)

    Gardner, S; Jaing, C

    2012-03-27

    The goal of this project is to develop forensic genotyping assays for select agent viruses, addressing a significant capability gap for the viral bioforensics and law enforcement community. We used a multipronged approach combining bioinformatics analysis, PCR-enriched samples, microarrays and TaqMan assays to develop high resolution and cost effective genotyping methods for strain level forensic discrimination of viruses. We have leveraged substantial experience and efficiency gained through year 1 on software development, SNP discovery, TaqMan signature design and phylogenetic signature mapping to scale up the development of forensics signatures in year 2. In this report, we have summarized the Taqman signature development for South American hemorrhagic fever viruses, tick-borne encephalitis viruses and henipaviruses, Old World Arenaviruses, filoviruses, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus and Japanese encephalitis virus.

  10. Performance verification of Roche COBAS6000 automatic electrochemiluminescence immunoassay analyzer%罗氏COBAS6000全自动电化学发光免疫分析仪性能验证

    Institute of Scientific and Technical Information of China (English)

    谭晓辉; 王勇

    2011-01-01

    目的 按ISO15189要求对罗氏COBAS 6000全自动电化学发光免疫分析仪的性能进行验证. 方法 对甲胎蛋白(alpha-fetoprotein,AFP)的精密度、准确度、临床可报告范围(clinical reportable range,CRR)、分析测量范围(analytical measurement range,AMR)、参考区间进行验证实验. 结果 批内精密度变异系数(coefficient variation,CV)高低值分别为3.28%和3.46%;日间精密度CV高低值分别为4.39%和5.13%,均小于厂家提供的CV(10%).相对偏差为0.862%,小于5%.分析测量范围为0.80-1 200 ng/ml,参考区间为0-20.00 ng/ml,临床可报告范围为0-60 000 ng/ml. 结论 罗氏COBAS 6000全自动电化学发光免疫分析仪的性能与厂家提供的资料基本一致,故可用其进行临床标本的检验工作,所得结果具有可信性.%Objeaive To test and verify the system performance of Roche COBAS 6000 automatic electrochemiluminescence immunoassay analyzer according to the requirements of IS015189. Methods Verification experiments were taken to measure the precision.accuracy , clinical reportable range( CRR) , analytical measurement range ( AMR ) , reference interval of alpha-feloprotein ( AFP) . Re-sults The high and low values of coefficient variation( CV) of inter-assay were 3. 28% and 3. 46% , and those of between-day precision were 4. 39% and 5. 13% , which were all less than the CV provided by the manufacturer( 1O% ) . Relative bias was 0. 862% . Analytical measurement range was 0.80 - 1 200 ng/ml.the reference interval was O - 20. 00 ng/ml, and the clinical reportable range was O 60 000 ng/ml. Conclusion The basic performances of Roche COBAS6000 automatic electrochemiluminescence immunoassay analyzer are consistent wich the data provided by the manufacturer,so it can be used to inspect the clinical samples and the results are credible.

  11. Evaluation of Roche Cobas E601 for Determining CEA%罗氏Cobas E601检测癌胚抗原的方法学性能评价

    Institute of Scientific and Technical Information of China (English)

    苏维; 王淑仙; 陈占良; 冯惠清; 段琳; 李逸阳

    2013-01-01

    目的:对罗氏Cobas E601全自动电化学发光免疫分析仪检测癌胚抗原(CEA)的分析性能进行验证。方法对CEA的精密度、准确度、测量线性范围、参考区间和交叉污染率进行验证实验。结果批内精密度高低两种浓度的(CV)分别为4.96%和4.38%,日间精密度高低两种浓度的CV分别为4.99%和4.81%;5份室间质控品的检测结果与靶值的偏倚在1.49%~3.57%;测量线性范围与厂家提供的范围相近;CEA的测量数值有96.3%在提供的参考区间内;交叉污染率为0.12%。结论罗氏Cobas E601检测CEA的方法学性能良好,检验结果准确可靠,能够满足临床检测的要求。%Objective To evaluate the effects of Roche Cobas E601 automatic electrochemiluminescence immunoassay analyzer for determining CEA. Methods To analyze E601’s measurement precision, accuracy, measurement range, reference interval and carryover rate of CEA, verification experiments were taken to measure. Results The high and low values of CV of inter-assay were 4.96%and 4.38%, and between-day CV were 4.99% and 4.81%; 5 quality-control serum’s relative bias were between 1.49%-3.57%; there was good linear relationship between measured values and expected values;there was 96.3%of the measurement data in the recommended reference interval;carryover rate range was 0.12%. Conclusion Roche Cobas E601 analyzer is stable, precise and accurate.

  12. Validation of methods performance for routine biochemistry analytes at Cobas 6000 analyzer series module c501.

    Science.gov (United States)

    Supak Smolcic, Vesna; Bilic-Zulle, Lidija; Fisic, Elizabeta

    2011-01-01

    Cobas 6000 (Roche, Germany) is biochemistry analyzer for spectrophotometric, immunoturbidimetric and ion-selective determination of biochemical analytes. Hereby we present analytical validation with emphasis on method performance judgment for routine operation. Validation was made for 30 analytes (metabolites, enzymes, trace elements, specific proteins and electrolytes). Research included determination of within-run (N = 20) and between-run imprecision (N = 30), inaccuracy (N = 30) and method comparison with routine analyzer (Beckman Coulter AU640) (N = 50). For validation of complete analytical process we calculated total error (TE). Results were judged according to quality specification criteria given by European Working Group. Within-run imprecision CVs were all below 5% except for cholesterol, triglycerides, IgA and IgM. Between-run CVs for all analytes were below 10%. Analytes that did not meet the required specifications for imprecision were: total protein, albumin, calcium, sodium, chloride, immunoglobulins and HDL cholesterol. Analytes that did not fulfill requirements for inaccuracy were: total protein, calcium, sodium and chloride. Analytes that deviated from quality specifications for total error were: total protein, albumin, calcium, sodium, chloride and IgM. Passing-Bablok regression analysis provided linear equation and 95% confidence interval for intercept and slope. Complete accordance with routine analyzer Beckman Coulter AU640 showed small number of analytes. Other analytes showed small proportional and/or small constant difference and therefore need to be adjusted for routine operation. Regarding low CV values, tested analyzer has satisfactory accuracy and precision and is extremely stable. Except for analytes that are coherent on both analyzers, some analytes require adjustments of slope and intercept for complete accordance.

  13. An Evaluation of the Cobas4800 HPV Test on Cervico-Vaginal Specimens in Liquid versus Solid Transport Media.

    Directory of Open Access Journals (Sweden)

    Hongxue Luo

    Full Text Available Determine the ability of the Cobas 4800 assay to detect high-risk human papillomavirus (HrHPV and high-grade cervical lesions when using cervico-vaginal samples applied to liquid medium and solid media cards compared to a direct cervical sample.Two cervico-vaginal specimens (pseudo self-collected were obtained from 319 women. One was applied to an iFTA Card (FTA then the brush placed in liquid-based medium (LSELF; the other was applied to a new solid media: POI card (POI. The clinical performance of Cobas4800 assay using the three aforementioned specimens was compared to direct collected endocervical specimens in liquid media (LDOC.The overall agreements of HrHPV detection were 84.2% (LSELF vs. LDOC, 81.0% (FTA vs. LDOC, and 82.3% (POI vs. LDOC. LSELF, FTA and POI identified 98.0%, 79.6%, and 97.5% positive cases of LDOC. Sensitivity to identify CIN2+ were 98.4% (LSELF, 73.8% (FTA, 95.1% (POI, and 93.4% (LDOC respectively. FTA had 78.1% and 90.4% agreement with the LSELF samples for all HrHPV and HPV16/18 detection respectively, while POI had 91.6% for both.Cobas4800 HPV test combined with cervico-vaginal specimens applied to both liquid media and POI solid card are accurate to detect HrHPV infection and high-grade cervical lesions as compared with direct endocervical samples in liquid media.

  14. Comparison of Cobas 6500 and Iris IQ200 fully-automated urine analyzers to manual urine microscopy.

    Science.gov (United States)

    Bakan, Ebubekir; Ozturk, Nurinnisa; Baygutalp, Nurcan Kilic; Polat, Elif; Akpinar, Kadriye; Dorman, Emrullah; Polat, Harun; Bakan, Nuri

    2016-10-15

    Urine screening is achieved by either automated or manual microscopic analysis. The aim of the study was to compare Cobas 6500 and Iris IQ200 urine analyzers, and manual urine microscopic analysis. A total of 540 urine samples sent to the laboratory for chemical and sediment analysis were analyzed on Cobas 6500 and Iris IQ200 within 1 hour from sampling. One hundred and fifty three samples were found to have pathological sediment results and were subjected to manual microscopic analysis performed by laboratory staff blinded to the study. Spearman's and Gamma statistics were used for correlation analyses, and the McNemar test for the comparison of the two automated analyzers. The comparison of Cobas u701 to the manual method yielded the following regression equations: y = - 0.12 (95% CI: - 1.09 to 0.67) + 0.78 (95% CI: 0.65 to 0.95) x for WBC and y = 0.06 (95% CI: - 0.09 to 0.25) + 0.66 (95% CI: 0.57 to 0.73) x for RBC. The comparison of IQ200 Elite to manual method the following equations: y = 0.03 (95% CI: - 1.00 to 1.00) + 0.88 (95% CI: 0.66 to 1.00) x for WBC and y = - 0.22 (95% CI: - 0.80 to 0.20) + 0.40 (95% CI: 0.32 to 0.50) x for RBC. IQ200 Elite compared to Cobas u701 yielded the following equations: y = - 0.95 (95% CI: - 2.13 to 0.11) + 1.25 (95% CI: 1.08 to 1.44) x for WBC and y = - 1.20 (95% CI: - 1.80 to -0.30) + 0. 80 (95% CI: 0.55 to 1.00) x for RBC. The two analyzers showed similar performances and good compatibility to manual microscopy. However, they are still inadequate in the determination of WBC, RBC, and EC in highly-pathological samples. Thus, confirmation by manual microscopic analysis may be useful.

  15. Comparison of a highly automated 5-h susceptibility testing system, the Cobas-Bact, with two reference methods: Kirby-Bauer disk diffusion and broth microdilution.

    OpenAIRE

    Murray, P R; Niles, A C; Heeren, R L

    1987-01-01

    The results of susceptibility tests performed with the Cobas-Bact system were compared with those of the Kirby-Bauer disk diffusion and the broth microdilution methods. The evaluation included tests with 24 antibiotics against 250 isolates of the family Enterobacteriaceae and 13 antibiotics against 100 gram-positive cocci. Complete agreements between the Cobas-Bact and Kirby-Bauer methods were 82.8 and 84.5% for gram-positive cocci and gram-negative bacilli, respectively. Agreements between t...

  16. Development and evaluation of Taqman assays for the differentiation of Dickeya (sub)species

    NARCIS (Netherlands)

    Wolf, van der J.M.; Haas, de B.H.; Hoof, van R.A.; Haan, de E.G.; Bovenkamp, van den G.W.

    2014-01-01

    TaqMan assays were developed for the detection of seven Dickeya species, namely D. dianthicola, D. dadantii, D. paradisiaca, D. chrysanthemi, D. zeae, D. dieffenbachiae and D. solani. Sequences of the gene coding for dnaX were used for the design of primers and probes. In studies with axenic culture

  17. Effect of specimen type on free immunoglobulin light chains analysis on the Roche Diagnostics cobas 8000 analyzer.

    Science.gov (United States)

    Nelson, Louis S; Steussy, Bryan; Morris, Cory S; Krasowski, Matthew D

    2015-01-01

    The measurement of free immunoglobulin light chains is typically performed on serum; however, the use of alternative specimen types has potential benefits. Using the Freelite™ kappa and lambda free light chains assay on a Roche Diagnostics cobas 8000 c502 analyzer, we compared three specimen types (serum, EDTA-plasma and lithium heparin plasma separator gel-plasma) on 100 patients. Using Deming regression and eliminating outliers (limiting data to light chain concentrations below 400 mg/L), the three specimen types showed comparable results for kappa light chain concentration, lambda light chain concentration, and kappa/lambda ratio with slopes close to 1.0 and y-intercepts close to zero. EDTA-plasma showed slightly more positive bias relative to serum than lithium heparin. Analysis using EDTA-plasma and lithium heparin plasma showed comparable linearity, precision, and temperature stability. A single sample showing hook effect (not in the comparison set) gave comparable results using either plasma specimen type. For the Freelite™ kappa and lambda free light chains assay, both EDTA-plasma or lithium heparin-plasma can serve as acceptable substitutes for serum, at least for the Roche cobas 8000 analyzer.

  18. Detection of BRAF V600 mutations in melanoma: evaluation of concordance between the Cobas® 4800 BRAF V600 mutation test and the methods used in French National Cancer Institute (INCa) platforms in a real-life setting.

    Science.gov (United States)

    Mourah, Samia; Denis, Marc G; Narducci, Fabienne Escande; Solassol, Jérôme; Merlin, Jean-Louis; Sabourin, Jean-Christophe; Scoazec, Jean-Yves; Ouafik, L'Houcine; Emile, Jean-François; Heller, Remy; Souvignet, Claude; Bergougnoux, Loïc; Merlio, Jean-Philippe

    2015-01-01

    Vemurafenib is approved for the treatment of metastatic melanoma in patients with BRAF V600 mutation. In pivotal clinical trials, BRAF testing has always been done with the approved cobas 4800 BRAF test. In routine practice, several methods are available and are used according to the laboratories usual procedures. A national, multicenter, non-interventional study was conducted with prospective and consecutive collection of tumor samples. A parallel evaluation was performed in routine practice between the cobas 4800 BRAF V600 mutation test and home brew methods (HBMs) of 12 national laboratories, labelled and funded by the French National Cancer Institute (INCa). For 420 melanoma samples tested, the cobas method versus HBM showed a high concordance (93.3%; kappa = 0.86) in BRAF V600 genotyping with similar mutation rates (34.0% versus 35.7%, respectively). Overall, 97.4% and 98.6% of samples gave valid results using the cobas and HBM, respectively. Of the 185 samples strictly fulfilling the cobas guidelines, the concordance rate was even higher (95.7%; kappa = 0.91; 95%CI [0.85; 0.97]). Out of the 420 samples tested, 28 (6.7%) showed discordance between HBM and cobas. This prospective study shows a high concordance rate between the cobas 4800 BRAF V600 test and home brew methods in the routine detection of BRAF V600E mutations.

  19. Detection of BRAF V600 mutations in melanoma: evaluation of concordance between the Cobas® 4800 BRAF V600 mutation test and the methods used in French National Cancer Institute (INCa platforms in a real-life setting.

    Directory of Open Access Journals (Sweden)

    Samia Mourah

    Full Text Available Vemurafenib is approved for the treatment of metastatic melanoma in patients with BRAF V600 mutation. In pivotal clinical trials, BRAF testing has always been done with the approved cobas 4800 BRAF test. In routine practice, several methods are available and are used according to the laboratories usual procedures. A national, multicenter, non-interventional study was conducted with prospective and consecutive collection of tumor samples. A parallel evaluation was performed in routine practice between the cobas 4800 BRAF V600 mutation test and home brew methods (HBMs of 12 national laboratories, labelled and funded by the French National Cancer Institute (INCa. For 420 melanoma samples tested, the cobas method versus HBM showed a high concordance (93.3%; kappa = 0.86 in BRAF V600 genotyping with similar mutation rates (34.0% versus 35.7%, respectively. Overall, 97.4% and 98.6% of samples gave valid results using the cobas and HBM, respectively. Of the 185 samples strictly fulfilling the cobas guidelines, the concordance rate was even higher (95.7%; kappa = 0.91; 95%CI [0.85; 0.97]. Out of the 420 samples tested, 28 (6.7% showed discordance between HBM and cobas. This prospective study shows a high concordance rate between the cobas 4800 BRAF V600 test and home brew methods in the routine detection of BRAF V600E mutations.

  20. Comparison of COBAS AMPLICOR Neissefia gonorrhoeae PCR, including confirmation with N-gonorrhoeae-specific 16S rRNA PCR, with traditional culture

    NARCIS (Netherlands)

    Luijt, DS; Bos, PAJ; van Zwet, AA; Vader, PCV; Schirm, J

    A total of 3,023 clinical specimens were tested for Neisseria gonorrhoeae by using COBAS AMPLICOR (CA) PCR and confirmation of positives by N. gonorrhoeae-specific 16S rRNA PCR. The sensitivity of CA plus 16S rRNA PCR was 98.8%, compared to 68.2% for culture. Confirmation of CA positives increased

  1. Comparison of COBAS AMPLICOR Neisseria gonorrhoeae PCR, including confirmation with N. gonorrhoeae-specific 16S rRNA PCR, with traditional culture

    NARCIS (Netherlands)

    Luijt, D.S.; Bos, P.A.; van Zwet, A.A.; Voorst-Vader, P.C.; Schirm, J.

    2005-01-01

    : A total of 3,023 clinical specimens were tested for Neisseria gonorrhoeae by using COBAS AMPLICOR (CA) PCR and confirmation of positives by N. gonorrhoeae-specific 16S rRNA PCR. The sensitivity of CA plus 16S rRNA PCR was 98.8%, compared to 68.2% for culture. Confirmation of CA positives increased

  2. Comparison of COBAS AMPLICOR Neissefia gonorrhoeae PCR, including confirmation with N-gonorrhoeae-specific 16S rRNA PCR, with traditional culture

    NARCIS (Netherlands)

    Luijt, DS; Bos, PAJ; van Zwet, AA; Vader, PCV; Schirm, J

    2005-01-01

    A total of 3,023 clinical specimens were tested for Neisseria gonorrhoeae by using COBAS AMPLICOR (CA) PCR and confirmation of positives by N. gonorrhoeae-specific 16S rRNA PCR. The sensitivity of CA plus 16S rRNA PCR was 98.8%, compared to 68.2% for culture. Confirmation of CA positives increased t

  3. Comparison of COBAS AMPLICOR Neissefia gonorrhoeae PCR, including confirmation with N-gonorrhoeae-specific 16S rRNA PCR, with traditional culture

    NARCIS (Netherlands)

    Luijt, DS; Bos, PAJ; van Zwet, AA; Vader, PCV; Schirm, J

    2005-01-01

    A total of 3,023 clinical specimens were tested for Neisseria gonorrhoeae by using COBAS AMPLICOR (CA) PCR and confirmation of positives by N. gonorrhoeae-specific 16S rRNA PCR. The sensitivity of CA plus 16S rRNA PCR was 98.8%, compared to 68.2% for culture. Confirmation of CA positives increased t

  4. Comparison of COBAS AMPLICOR Neisseria gonorrhoeae PCR, including confirmation with N. gonorrhoeae-specific 16S rRNA PCR, with traditional culture

    NARCIS (Netherlands)

    Luijt, D.S.; Bos, P.A.; van Zwet, A.A.; Voorst-Vader, P.C.; Schirm, J.

    2005-01-01

    : A total of 3,023 clinical specimens were tested for Neisseria gonorrhoeae by using COBAS AMPLICOR (CA) PCR and confirmation of positives by N. gonorrhoeae-specific 16S rRNA PCR. The sensitivity of CA plus 16S rRNA PCR was 98.8%, compared to 68.2% for culture. Confirmation of CA positives increased

  5. Detection of hepatitis B virus infection: A systematic review

    Institute of Scientific and Technical Information of China (English)

    Mallika; Ghosh; Srijita; Nandi; Shrinwanti; Dutta; Malay; Kumar; Saha

    2015-01-01

    AIM: To review published methods for detection of hepatitis B virus(HBV) infection.METHODS: A thorough search on Medline database was conducted to find original articles describing different methods or techniques of detection of HBV, which are published in English in last 10 years. Articles outlining methods of detection of mutants or drug resistance were excluded. Full texts and abstracts(if full text not available) were reviewed thoroughly. Manual search of references of retrieved articles were also done. We extracted data on different samples and techniques of detection of HBV, their sensitivity(Sn), specificity(Sp) and applicability.RESULTS: A total of 72 studies were reviewed. HBV was detected from dried blood/plasma spots, hepatocytes, ovarian tissue, cerumen, saliva, parotid tissue, renal tissue, oocytes and embryos, cholangiocarcinoma tissue, etc. Sensitivity of dried blood spot for detecting HBV was > 90% in all the studies. In case of seronegative patients, HBV DNA or serological markers have been detected from hepatocytes or renal tissue in many instances. Enzyme linked immunosorbent assay and Chemiluminescent immunoassay(CLIA) are most commonly used serological tests for detection. CLIA systems are also used for quantitation. Molecular techniques are used qualitatively as well as for quantitative detection. Among the molecular techniques version 2.0 of the Cobas Ampliprep/Cobas Taq Man assay and Abbott’s real time polymerase chain reaction kit were found to be most sensitive with a lower detection limit of only 6.25 IU/m L and 1.48 IU/m L respectively. CONCLUSION: Serological and molecular assays are predominant and reliable methods for HBV detection. Automated systems are highly sensitive and quantify HBV DNA and serological markers for monitoring.

  6. Clinical performances of two real-time PCR assays and bDNA/TMA to early monitor treatment outcome in patients with chronic hepatitis C.

    Science.gov (United States)

    Martinot-Peignoux, Michelle; Khiri, Hacène; Leclere, Laurence; Maylin, Sarah; Marcellin, Patrick; Halfon, Philippe

    2009-11-01

    Early viral monitoring is essential for the management of treatment outcome in patients with chronic hepatitis C. A variety of commercially available assays are now available to quantify HCV-RNA in routine clinical practice. Compare the clinical results of 3 commercially available assays to evaluate the positive predictive value (PPV) and the negative predictive value (NPV) of rapid virological response (RVR) at week 4 and early virological response (EVR) at week 12. 287 patients treated with standard care regimen combination therapy were studied. HCV-RNA values measured at baseline, week 4, week 12 with VERSANT HCV 3.0 Assay (bDNA), and VERSANT HCV-RNA Qualitative Assay (TMA) (bDNA/TMA); COBAS Ampliprep/COBAS/TaqMan (CAP/CTM) and Abbott m2000sp extraction/m2000rt amplification system (ART). RVR was defined as undetectable serum HCV-RNA and EVR as a > OR =2 log decline in baseline viral load (BLV). Median (range) BVLs were: 5.585(2.585-6.816), 5.189(2.792-7.747) and 4.804(2.380-6.580) log(10)IU/ml, with bDNA/TMA, CAP/CTM and ART, respectively (pTMA, CAP/CTM and ART, respectively (p=0.317). EVR was observed in 76%, 73% and 67% of the patients and NPVs were 93%, 83% and 79% with bDNA/TMA, CAP/CTM and ART, respectively (p=0.09). Treatment monitoring should include both detection of serum HCV-RNA at week 4 to predict SVR and at week 12 to predict non-SVR. The value of all 3 assays was similar for evaluating RVR or EVR. Because of viral load discrepancies the same assay should be used throughout patient treatment follow-up.

  7. Comparison of GMT presto assay and Roche cobas® 4800 CT/NG assay for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in dry swabs.

    Science.gov (United States)

    de Waaij, Dewi J; Dubbink, Jan Henk; Peters, Remco P H; Ouburg, Sander; Morré, Servaas A

    2015-11-01

    Urogenital Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are the most prevalent bacterial STIs worldwide. Molecular tests are the standard for the detection of CT and NG, as these are difficult to culture. The recently introduced CE-IVD marked GMT Presto assay promises to be a valuable addition in CT and NG diagnostics. The advantage of the Presto assay is that it works on many PCR systems and the DNA can be isolated by any system.We compared the Presto assay to the widely used Roche cobas® 4800 CT/NG test for the detection of CT and NG in 612 vaginal and rectal dry collected swabs. Discrepant samples were tested by the TIB MOLBIOL Lightmix Kit 480 HT CT/NG assay. The alloyed gold standard was defined as two concurring Presto and cobas® 4800 results, or, with discrepant Presto and cobas® results, two concurring results of either test together with the Lightmix Kit 480 HT CT/NG assay. For the Presto assay,we observed 77 CT positive (13%) and 22 NG positive (3,6%) vaginal samples, and 41 CT positive (6,7%) and 11 NG positive (1,8%) rectal samples. For the cobas® 4800 assay,we observed 77 CT positive (13%) and 21NG positive (3,4%) vaginal samples, and 39 CT positive (6,4%) and 11 NG positive (1,8%) rectal samples. Ten CT samples were discrepant between Presto and cobas® 4800 CT/NG assays, while two NG samples were discrepant. CT sensitivity in both assays was 100% compared to the alloyed gold standard. The sensitivity was 100% for both vaginal and rectal dry swabs, underlining the suitability of these sample types for detection of CT and NG. The Presto assay is therefore valuable for molecular detection of CT and NG in dry vaginal and rectal swabs.

  8. Detection of EGFR Mutations by TaqMan Mutation Detection Assays Powered by Competitive Allele-Specific TaqMan PCR Technology

    Directory of Open Access Journals (Sweden)

    Cristin Roma

    2013-01-01

    Full Text Available Epidermal growth factor receptor (EGFR mutations in non-small-cell lung cancer (NSCLC are predictive of response to treatment with tyrosine kinase inhibitors. Competitive Allele-Specific TaqMan PCR (castPCR is a highly sensitive and specific technology. EGFR mutations were assessed by TaqMan Mutation Detection Assays (TMDA based on castPCR technology in 64 tumor samples: a training set of 30 NSCLC and 6 colorectal carcinoma (CRC samples and a validation set of 28 NSCLC cases. The sensitivity and specificity of this method were compared with routine diagnostic techniques including direct sequencing and the EGFR Therascreen RGQ kit. Analysis of the training set allowed the identification of the threshold value for data analysis (0.2; the maximum cycle threshold (Ct=37; and the cut-off ΔCt value (7 for the EGFR TMDA. By using these parameters, castPCR technology identified both training and validation set EGFR mutations with similar frequency as compared with the Therascreen kit. Sequencing detected rare mutations that are not identified by either castPCR or Therascreen, but in samples with low tumor cell content it failed to detect common mutations that were revealed by real-time PCR based methods. In conclusion, our data suggest that castPCR is highly sensitive and specific to detect EGFR mutations in NSCLC clinical samples.

  9. Performance of the NG OligoGen kit for the diagnosis of Neisseria gonorrhoeae: comparison with cobas 4800 assay.

    Science.gov (United States)

    Parra-Sánchez, M; García-Rey, S; Marcuello, A; Zakariya-Yousef, I; Bernal, S; Pueyo, I; Martín-Mazuelos, E; Palomares, J C

    2016-01-01

    PCR assays are nowadays between the most sensitive and reliable methods for screening and diagnosing sexually transmitted infections (STIs). The aim of this study was to analyze the reliability, accuracy, and usefulness of the new NG OligoGen kit in comparison with the cobas 4800 assay for the detection of Neisseria gonorrhoeae in clinical samples. A prospective study was designed for detection of N. gonorrhoeae including urine samples (n=152), rectal (n=80), endocervical (n=67), pharyngeal (n=41), and urethral swabs (n=5) that were sent from a regional STI clinic in Seville, Spain. Samples were collected from 255 (73.9%) men and 90 women. Sensitivity, specificity, positive and negative predicative values, and kappa value for N. gonorrhoeae detection using the NG OligoGen kit were 99.6%, 100%, 100%, 99.1%, and 0.99, respectively. Statistical data obtained in this study confirm the usefulness and reliable results of this new assay.

  10. Comparison of real-time polymerase chain reaction with the COBAS Amplicor test for quantitation of hepatitis B virus DNA in serum samples

    Institute of Scientific and Technical Information of China (English)

    Ming Shi; Yong Zhang; Ying-Hua Zhu; Jing Zhang; Wei-Jia Xu

    2008-01-01

    AIM: To compare the clinical performance of a real-time PCR assay with the COBAS Amplicor Hepatitis B Virus (HBV) Monitor test for quantitation of HBV DNA in serum samples. METHODS: The reference sera of the Chinese National Institute for the Control of Pharmaceutical and Biological Products and the National Center for Clinical Laboratories of China, and 158 clinical serum samples were used in this study. The linearity, accuracy, reproducibility, assay time, and costs of the real-time PCR were evaluated and compared with those of the Cobas Amplicor test. RESULTS: The intra-assay and inter-assay variations of the real-time PCR ranged from 0.3% to 3.8% and 1.4% to 8.1%, respectively. The HBV DNA levels measured by the real-time PCR correlated very well with those obtained with the COBAS Amplicor test (r = 0.948). The real-time PCR HBV DNA kit was much cheaper and had a wider dynamic range. CONCLUSION: The real-time PCR assay is an excellent tool for monitoring of HBV DNA levels in patients with chronic hepatitis B.

  11. Verification of analytical measurement range of serum creatinine detected by Roche Cobas 501 Biochemistry Analyzer%Roche Cobas 501生化分析仪血清肌酐分析测量范围的验证

    Institute of Scientific and Technical Information of China (English)

    陈永传; 崔亚利; 李艳; 任飒爽

    2016-01-01

    目的:通过对血清肌酐分析测量范围(AMR)的验证,探讨临床实验室如何按照国际标准要求进行生化分析仪定量检测项目分析测量范围的验证,保证检验结果准确、可靠。方法采用酶法在Roche Cobas 501生化分析仪上检测7个浓度水平美国病理学家协会(CAP)线性范围能力测试样品,这7个样品靶值涵盖厂家说明书标示肌酐分析测量范围低、中、高值,每个样品检测两次取其均值,计算其与靶值的偏倚。另外参照美国临床和实验室标准协会(CLSI )指南文件 EP6‐P的要求,收集含高值肌酐的新鲜患者血清,按一定比例混合、离心,计算混合物的浓度并将之作为高值样品(H ),与经同样处理获得的低值样品(L )分别按5L、4L+1H、3L+2H、2L+3H、1L+4H、5H的关系配制,形成系列样品,在Roche Cobas 501生化分析仪上对各样品的肌酐进行检测,每个样品检测4次,数据进行回归分析。结果7个水平的CAP样品与靶值的偏倚均小于北京善方医院检验科设定的允许误差±7.5%[(1/2×TE)%]。新鲜患者混合血清样品回归方程为Y=0.9886X+16.614,b=0.9886,介于0.97~1.03,截距a与0经 t检验,ta< t0.05,P>0.05,说明截距与0无明显差异,回归直线事实上通过0点。结论厂家说明书标示的血清肌酐分析测量范围验证通过,实验室可以采用。%Objective To investigate how the clinical laboratory conducting the verification of analytical measurement range (AM R) of quantitative items detected by the biochemical analyzer according to the requirements of the international standards by verifying the serum creatinine AMR for ensuring the accuracy and reliability of detection results .Methods The enzyme method was adopted to detect the 7‐concentration levels test specimens of CAP linear range proficiency test on the Roche Cobas 501 biochemical

  12. Quantification of rice blast disease progressions through Taqman real-time PCR.

    Science.gov (United States)

    Su'udi, Mukhamad; Kim, Jinyeong; Park, Jong-Mi; Bae, Shin-Chul; Kim, Donghern; Kim, Yong-Hwan; Ahn, Il-Pyung

    2013-09-01

    Rice blast caused by Magnaporthe oryzae is a major disease in the paddy field and also a representative model system in the investigation of plant-microbe interactions. This study was undertaken to provide the quantitative evaluation method that specifically determines the amount of M. oryzae proliferation in planta. Real-time PCR was used as the detection strategy in combination with the primer pair and Taqman probe specific to MHP1, a unigene encoding HYDROPHOBIN that is indispensable for normal virulence expression. Based on the crossing point values from the PCR reactions containing a series of increasing concentration of cloned amplicon or fungal genomic DNA, correlation among the template's copy number or its amount and amplification pattern was calculated. Reliability of this equation was further confirmed using the DNA samples from the rice leaves infected with compatible or incompatible strains of M. oryzae. The primer pair used in the Taqman real-time PCR reaction can recognize the existence of fungal DNA as low as 1 pg. In sum, our quantitative evaluation system is applicable and reliable in the blast diagnosis and also in the estimation of objective blast disease progression.

  13. Assessment of Legionella pneumophila in recreational spring water with quantitative PCR (Taqman) assay.

    Science.gov (United States)

    Shen, Shu-Min; Chou, Ming-Yuan; Hsu, Bing-Mu; Ji, Wen-Tsai; Hsu, Tsui-Kang; Tsai, Hsiu-Feng; Huang, Yu-Li; Chiu, Yi-Chou; Kao, Erl-Shyh; Kao, Po-Min; Fan, Cheng-Wei

    2015-07-01

    Legionella spp. are common in various natural and man-made aquatic environments. Recreational hot spring is frequently reported as an infection hotspot because of various factors such as temperature and humidity. Although polymerase chain reaction (PCR) had been used for detecting Legionella, several inhibitors such as humic substances, calcium, and melanin in the recreational spring water may interfere with the reaction thus resulting in risk underestimation. The purpose of this study was to compare the efficiencies of conventional and Taqman quantitative PCR (qPCR) on detecting Legionella pneumophila in spring facilities and in receiving water. In the results, Taqman PCR had much better efficiency on specifying the pathogen in both river and spring samples. L. pneumophila was detected in all of the 27 river water samples and 45 of the 48 hot spring water samples. The estimated L. pneumophela concentrations ranged between 1.0 × 10(2) and 3.3 × 10(5) cells/l in river water and 72.1-5.7 × 10(6) cells/l in hot spring water. Total coliforms and turbidity were significantly correlated with concentrations of L. pneumophila in positive water samples. Significant difference was also found in water temperature between the presence/absence of L. pneumophila. Our results suggest that conventional PCR may be not enough for detecting L. pneumophila particularly in the aquatic environments full of reaction inhibitors.

  14. Rapid Detection of Filoviruses by Real-time TaqMan Polymerase Chain Reaction Assays

    Institute of Scientific and Technical Information of China (English)

    Yi Huang; Hongping Wei; Yunpeng Wang; Zhengli Shi; Herve Raoul; Zhiming Yuan

    2012-01-01

    Ebola virus (EBOV) and Marburg virus (MARV) are causative agents of severe hemorrhagic fever with high mortality rates in humans and non-human primates and there is currently no licensed vaccine or therapeutics.To date,there is no specific laboratory diagnostic test in China,while there is a national need to provide differential diagnosis during outbreaks and for instituting acceptable quarantine procedures.In this study,the TaqMan RT-PCR assays targeting the nucleoprotein genes of the Zaire Ebolavirus (ZEBOV) and MARV were developed and their sensitivities and specificities were investigated.Our results indicated that the assays were able to make reliable diagnosis over a wide range of virus copies from 103 to 109,corresponding to the threshold of a standard RNA transcript.The results showed that there were about 1010 RNA copies per milliliter of virus culture supernatant,equivalent to 10,000 RNA molecules per infectious virion,suggesting the presence of many non-infectious particles.These data indicated that the TaqMan RT-PCR assays developed in this study will be suitable for future surveillance and specific diagnosis of ZEBOV and MARV in China.

  15. Comparison of the Analytical Performance Between cobas EGFR Assay and PCR-Clamp Method in the Detection of EGFR Mutations in Japanese Non-Small Cell Lung Cancer Patients.

    Science.gov (United States)

    Ai, Tomohiko; Yuri, Maiko; Tabe, Yoko; Kakimoto, Atsushi; Morishita, Soji; Tsuchiya, Koji; Takamochi, Kazuya; Kodama, Yuzo; Takahashi, Fumiyuki; Shigeki, Misawa; Horii, Takashi; Suzuki, Kenji; Takahashi, Kazuhisa; Miida, Takashi; Ohsaka, Akimichi

    2017-05-01

    EGFR, a tyrosine-kinase, plays an important role in the progression of lung cancer. Since genetic abnormality of EGFR alters the effects of tyrosine-kinase inhibitors targeting EGFR, molecular analyses of EGFR have recently gained more attention in the treatment of lung cancer. However, several different techniques are available and which method is superior has not been determined. In this study, we compared two recently developed PCR-based techniques, PCR-clamp method and cobas EGFR assay. Ninety-four surgical samples and 58 biopsy samples from patients suffering from non-small cell lung cancers (NSCLCs) were included in the study. Samples with positive and negative genetic abnormalities, 66 and 28 respectively, were chosen for PCR-Clamp methods. Those same samples were reanalyzed with cobas EGFR assay. The concordance between PCR-Clamp and cobas EGFR methods was 95.7%. PCR-Clamp failed to detect four mutations that were detected with cobas EGFR assay. These two methods were further tested by analyzing 58 random biopsy samples. The concordance for the biopsy samples was 93.1%, and PCR-Clamp, again, failed to detect three mutations that were detected with cobas EGFR assay. Our results showed both methods detected most of the known EGFR mutations and the concordance was similar to those previously reported in different ethnicities. However, in our study, PCR-Clamp method failed to detect a total of seven mutations that were detected with cobas EGFR assay. Thus, we concluded that cobas EGFR assay is an easier and more accurate screening assay than PCR-Clamp method in detecting EGFR genetic abnormalities.

  16. A clinical evaluation of the Cobas Fara clinical chemistry analyzer for some routine serum enzymes and glucose.

    Science.gov (United States)

    Moses, G C; Lightle, G O; Tuckerman, J F; Henderson, A R

    1987-11-01

    The authors evaluated the Cobas FARA centrifugal analyzer with respect to pipetting precision and accuracy, instrument temperature, spectrophotometric response, and analytic performance for the assay of five serum enzymes and glucose. Spectrophotometric response, temperature response, pipetting precision, and accuracy were satisfactory. However, sufficient time must be allowed for cuvet contents to reach a stable temperature before measurements are made. Total day-to-day imprecision (within plus between run) was less than 5% (coefficient of variation) for aspartate and alanine aminotransferases (AST; Enzyme Commission classification number [EC] EC 2.6.1.1; and ALT; EC 2.6.1.2); alkaline phosphatase (AP; EC 3.1.3.1); gamma-glutamyltransferase (GGT; EC 2.3.1.2); lactate dehydrogenase (LD; EC 1.1.1.17); creatine kinase (CK; EC 2.7.3.1); and glucose assays. Results compare well with those obtained with other current clinical chemistry analyzers; correlation coefficients were greater than 0.993. Sample-to-sample carryover was negligible, and method linearity was satisfactory for all tests.

  17. Taqman MGB探针冻融稳定性研究%Study on Freeze-thaw Cycles on Taqman MGB Stability

    Institute of Scientific and Technical Information of China (English)

    臧超; 王晶; 李亮; 隋志伟; 余笑波; 黎朋

    2011-01-01

    Taqman MGB探针的稳定性是实时荧光定量PCR准确定量的关键因素之一.然而在实验过程中,常常因为运输、实验室保存或使用条件的变化,造成探针的反复冻融,影响实验结果.采用高效液相色谱技术定量研究了反复冻融的MGB探针的稳定性;并比较分析了冻融探针对qPCR的影响程度.结果表明:MGB探针可以耐受30次以内的反复冻融,随着探针浓度的降低,探针反复冻融后qPCR量值波动区间增大,因此,为保证结果稳定性,qPCR实验使用的MGB探针应尽量减少反复冻融次数.

  18. Real-time PCR TaqMan assay for detecting Trichophyton tonsurans, a causative agent of tinea capitis, from hairbrushes.

    Science.gov (United States)

    Sugita, T; Shiraki, Y; Hiruma, M

    2006-09-01

    Tinea capitis caused by Trichophyton tonsurans is currently an epidemic in the United States, Europe, and Japan, and the cultivation of this microorganism is necessary for a definitive diagnosis. We recently developed a real-time PCR TaqMan assay as a culture-independent method for the rapid detection of T. tonsurans from hairbrushes.

  19. Detection of Food Allergens by Taqman Real-Time PCR Methodology.

    Science.gov (United States)

    García, Aina; Madrid, Raquel; García, Teresa; Martín, Rosario; González, Isabel

    2017-01-01

    Real-time PCR (polymerase chain reaction) has shown to be a very effective technology for the detection of food allergens. The protocol described herein consists on a real-time PCR assay targeting the plant ITS (Internal Transcribed Spacer) region, using species-specific primers and hydrolysis probes (Taqman) dual labeled with a reporter fluorophore at the 5' end (6-carboxyfluorescein, FAM) and a quencher fluorophore at the 3' end (Blackberry, BBQ). The species-specific real-time PCR systems (primers/probe) described in this work allowed the detection of different nuts (peanut, hazelnut, pistachio, almond, cashew, macadamia, walnut and pecan), common allergens present in commercial food products, with a detection limit of 0.1 mg/kg.

  20. Identification of field caught Anopheles gambiae s.s. and Anopheles arabiensis by TaqMan single nucleotide polymorphism genotyping

    Directory of Open Access Journals (Sweden)

    Bayoh Nabie M

    2007-02-01

    Full Text Available Abstract Background Identification of Anopheles gambiae s.s. and Anopheles arabiensis from field-collected Anopheles gambiae s.l. is often necessary in basic and applied research, and in operational control programmes. The currently accepted method involves use of standard polymerase chain reaction amplification of ribosomal DNA (rDNA from the 3' 28S to 5' intergenic spacer region of the genome, and visual confirmation of amplicons of predicted size on agarose gels, after electrophoresis. This report describes development and evaluation of an automated, quantitative PCR method based upon TaqMan™ single nucleotide polymorphism (SNP genotyping. Methods Standard PCR, and TaqMan SNP genotyping with newly designed primers and fluorophore-labeled probes hybridizing to sequences of complementary rDNA specific for either An. gambiae s.s. or An. arabiensis, were conducted in three experiments involving field-collected An. gambiae s.l. from western Kenya, and defined laboratory strains. DNA extraction was from a single leg, sonicated for five minutes in buffer in wells of 96-well PCR plates. Results TaqMan SNP genotyping showed a reaction success rate, sensitivity, and species specificity comparable to that of standard PCR. In an extensive field study, only 29 of 3,041 (0.95% were determined to be hybrids by TaqMan (i.e., having rDNA sequences from both species, however, all but one were An. arabiensis by standard PCR, suggesting an acceptably low (ca. 1% error rate for TaqMan genotyping in mistakenly identifying species hybrids. Conclusion TaqMan SNP genotyping proved to be a sensitive and rapid method for identification of An. gambiae s.l. and An. arabiensis, with a high success rate, specific results, and congruence with the standard PCR method.

  1. Effect of glacial acetic acid treatment of cervical ThinPrep specimens on HPV DNA detection with the cobas 4800 HPV test.

    Science.gov (United States)

    McMenamin, M; McKenna, M

    2013-10-01

    Cytology laboratories in the UK routinely treat unsatisfactory cervical liquid-based cytology (LBC) specimens with glacial acetic acid (GAA) to reduce the unsatisfactory rate. However, there is limited published data on the effect of GAA reprocessing on the molecular detection of human papillomavirus (HPV). The aim of this study was to assess the impact of GAA treatment of cervical ThinPrep(®) samples on HPV detection with the cobas(®) 4800 HPV Test (Roche Molecular Systems, Pleasanton, CA, USA). Residual ThinPrep samples (n = 121) were selected to provide a range of typical cytology results and enrich the study samples for HPV positivity. Specimens were equally split into two fractions: one part treated with 10% GAA and the other part left untreated. All samples were HPV tested using the cobas 4800 HPV Test, which simultaneously detects a total of 14 high-risk HPV (hrHPV) genotypes and individually identifies HPV16 and HPV18. The HPV positive/negative status of tested samples determined the level of agreement between treated and untreated fractions; one sample failed owing to detection of a clot by the instrument during pipetting, leaving 120 samples in the study. Statistical analysis was performed using an unweighted kappa. Analysis of overall HPV positivity showed 97.5% (117/120) agreement between the treated and untreated fractions with a kappa value of 0.95. There were 63/65 (96.9%) concordant HPV positive and 54/55 (98.2%) concordant HPV negative results. In addition to the three discordant results for overall HPV positivity, there were three HPV type-specific discrepancies giving a total of 114/120 concordant HPV results (95% agreement). Glacial acetic acid (GAA) treatment of cervical ThinPrep specimens does not have significant adverse affects on HPV detection with the cobas 4800 HPV Test. © 2013 John Wiley & Sons Ltd.

  2. Comparison of the Becton Dickinson strand displacement amplification and Cobas Amplicor Roche PCR for the detection of Chlamydia trachomatis: pooling versus individual tests

    DEFF Research Database (Denmark)

    Bang, D; Angelsø, Lene; Schirakow, Bente

    2003-01-01

    The objective of the study was to examine the influence of pooling Chlamydia trachomatis specimens. We compared Becton Dickinson ProbeTec strand displacement amplification (SDA) with Cobas Amplicor Roche (PCR). With PCR as the standard, SDA performed equally well in single-sample testing....... For pooled PCR samples (compared to individual PCR), we found a sensitivity of 100% and a specificity of 98.9%. For pooled SDA tests (compared to individual SDA), we found a sensitivity of 86.5% and a specificity of 98.9%. Our conclusion is that 2-sucrose phosphate buffer (2-SP) can be used for individual...

  3. Evaluation of KIMS immunoassays on a cobas c 501 analyzer for drugs of abuse and ethyl glucuronide testing in urine for forensic abstinence control.

    Science.gov (United States)

    Neukamm, Merja A; Bahrami, Arsham; Auwärter, Volker; Mehne, Felix M P; Höss, Eva

    2016-12-26

    For the medico-psychological assessment (MPA) during driving licence re-granting in Germany, abstinence control including urine samples is required. In these programmes, even small amounts of markers for drug or alcohol abuse have to be detected. Thus, the concentrations of the target compounds are very low, and, in consequence, the sensitivity of the applied screening method has to be much higher than for clinical use. Modified drugs of abuse and ethyl glucuronide immunoassays on a Roche cobas c 501 analyzer were evaluated for precision, accuracy, onboard calibration stability, cross reactivity, sensitivity, and specificity using authentic urine samples. Precision (intra-day and inter-day relative standard deviation (RSD) and accuracy (bias) at three concentrations were 12% or lower for all parameters. The calibrations remained stable (deviations amphetamines (21 days). Satisfactory cross reactivity was determined for the relevant analytes and also for several new psychoactive substances (NPS). The sensitivity was 100% for all parameters except methadone metabolite EDDP (92%) and fully met the sensitivity criteria for MPA urine testing. The presented kinetic interaction of microparticles in a solution (KIMS) immunoassays on a cobas c 501 thus provide a new method to reliably detect drug or alcohol consumption in abstinence control programmes requiring high sensitivity. Copyright © 2016 John Wiley & Sons, Ltd.

  4. Sensibilidad del equipo Cobas AmpliScreenTM HIV-1 Test, v1.5, para la detección de HIV-1

    Directory of Open Access Journals (Sweden)

    Lucía P Gomez

    Full Text Available Las técnicas de amplificación de ácidos nucleicos (NAT se incorporaron en los bancos de sangre para reducir el riesgo residual de transmisión de infecciones por vía transfusional. La cocirculación de distintas variantes del HIV-1 en Argentina indica la necesidad de evaluar la sensibilidad de los ensayos serológicos y moleculares disponibles para su detección. En este trabajo se evaluó la sensibilidad del equipo COBAS AmpliScreenTM HIV-1 Test, versión 1.5 (Roche, para detectar ARN viral en plasmas de individuos infectados con HIV-1 de Argentina. Los resultados demuestran que esta técnica tiene una alta sensibilidad para detectar ARN de HIV-1 en las condiciones ensayadas: para ensayo de mini-pooles (pooles = 50 copias de ARN/ml, la sensibilidad fue = 92 %, y para procedimiento estándar (plasmas = 207 copias de ARN/ml, la sensibilidad fue 100 %. Además, la técnica COBAS AmpliScreenTM HIV-1 Test, versión 1.5 (Roche, es adecuada para la detección de las variantes de HIV-1 prevalentes.

  5. TaqMan real-time PCR for detection and quantitation of squash leaf curl virus in cucurbits.

    Science.gov (United States)

    Kuan, Cheng-Ping; Huang, Hung-Chang; Chang, Chia-Che; Lu, Yi-Lin

    2012-02-01

    A real-time PCR assay based on the TaqMan chemistry was developed for reliable detection and quantitation of the squash leaf curl virus (SLCV) in melon and squash plants. This method was highly specific to SLCV and it was about one thousand times more sensitive than the conventional PCR method. The protocol of the real-time PCR established in this study enabled detection of as little as 10(2) copies of SLCV DNA with CP gene as the target. This TaqMan real-time PCR assay for detection and quantitation of SLCV would be a useful tool for application in quarantine and certification of SLCV in cucurbits as well as in the research of disease resistance and epidemiology.

  6. [Detection of human enteroviruses with real-time PCR assay using TaqMan fluorescent probe].

    Science.gov (United States)

    Leś, Katarzyna; Przybylski, Maciej; Dzieciatkowski, Tomasz; Młynarczyk, Grazyna

    2010-01-01

    Infections with human enteroviruses are common worldwide and cause a wide range of signs and symptoms. Nowadays in current diagnostics procedures older virological methods, such virus isolation in a cell cultures and seroneutralisation assay, are replaced with molecular biology tests. The aim of the study was development of real-time PCR assay for detection of human adenoviruses. DNA isolated from MK2 cell line infected with nineteen different enterovirus strains was used for development of a qualitative real-time PCR assay using primers targeting a conserved region of the 5'UTR region and a specific TaqMan probe. The analytical sensitivity of real-time PCR assay was tested using serial dilutions of Coxackie A9 cDNA in range between 10 degrees and 10(-8). For comparison typical end-point detected RT-PCR for enterovirus detection with the same cDNA dilutions was made. The sensitivity of novel method was about ten thousand-fold higher than older one. The conclusion is that real-time PCR is very advisable in diagnostics of diseases caused with enteroviruses. The high level of sensitivity, specificity, accuracy, and rapidity provided by this assay are favorable for the use in the detection of enteroviral RNA in clinical specimens, especially from neuroinfections.

  7. Performance of a Taqman Assay for Improved Detection and Quantification of Human Rhinovirus Viral Load

    Science.gov (United States)

    Ng, Kim Tien; Chook, Jack Bee; Oong, Xiang Yong; Chan, Yoke Fun; Chan, Kok Gan; Hanafi, Nik Sherina; Pang, Yong Kek; Kamarulzaman, Adeeba; Tee, Kok Keng

    2016-01-01

    Human rhinovirus (HRV) is the major aetiology of respiratory tract infections. HRV viral load assays are available but limitations that affect accurate quantification exist. We developed a one-step Taqman assay using oligonucleotides designed based on a comprehensive list of global HRV sequences. The new oligonucleotides targeting the 5′-UTR region showed high PCR efficiency (E = 99.6%, R2 = 0.996), with quantifiable viral load as low as 2 viral copies/μl. Assay evaluation using an External Quality Assessment (EQA) panel yielded a detection rate of 90%. When tested on 315 human enterovirus-positive specimens comprising at least 84 genetically distinct HRV types/serotypes (determined by the VP4/VP2 gene phylogenetic analysis), the assay detected all HRV species and types, as well as other non-polio enteroviruses. A commercial quantification kit, which failed to detect any of the EQA specimens, produced a detection rate of 13.3% (42/315) among the clinical specimens. Using the improved assay, we showed that HRV sheds in the upper respiratory tract for more than a week following acute infection. We also showed that HRV-C had a significantly higher viral load at 2–7 days after the onset of symptoms (p = 0.001). The availability of such assay is important to facilitate disease management, antiviral development, and infection control. PMID:27721388

  8. SNP genotyping using TaqMan technology: the CYP2D6*17 assay conundrum.

    Science.gov (United States)

    Gaedigk, Andrea; Freeman, Natalie; Hartshorne, Toinette; Riffel, Amanda K; Irwin, David; Bishop, Jeffrey R; Stein, Mark A; Newcorn, Jeffrey H; Jaime, Lazara Karelia Montané; Cherner, Mariana; Leeder, J Steven

    2015-03-19

    CYP2D6 contributes to the metabolism of many clinically used drugs and is increasingly tested to individualize drug therapy. The CYP2D6 gene is challenging to genotype due to the highly complex nature of its gene locus. TaqMan technology is widely used in the clinical and research settings for genotype analysis due to assay reliability, low cost, and the availability of commercially available assays. The assay identifying 1023C>T (rs28371706) defining a reduced function (CYP2D6*17) and several nonfunctional alleles, produced a small number of unexpected diplotype calls in three independent sets of samples, i.e. calls suggested the presence of a CYP2D6*4 subvariant containing 1023C>T. Gene resequencing did not reveal any unknown SNPs in the primer or probe binding sites in any of the samples, but all affected samples featured a trio of SNPs on their CYP2D6*4 allele between one of the PCR primer and probe binding sites. While the phenomenon was ultimately overcome by an alternate assay utilizing a PCR primer excluding the SNP trio, the mechanism causing this phenomenon remains elusive. This rare and unexpected event underscores the importance of assay validation in samples representing a variety of genotypes, but also vigilance of assay performance in highly polymorphic genes such as CYP2D6.

  9. TaqmanMGB探针冻融稳定性研究

    Institute of Scientific and Technical Information of China (English)

    臧超; 王晶; 李亮; 隋志伟; 余笑波; 黎朋

    2011-01-01

    TaqmanMGB探针的稳定性是实时荧光定量PCR准确定量的关键因素之一。然而在实验过程中,常常因为运输、实验室保存或使用条件的变化,造成探针的反复冻融,影响实验结果。采用高效液相色谱技术定量研究了反复冻融的MGB探针的稳定性;并比较分析了冻融探针对qPCR的影响程度。结果表明:MGB探针可以耐受30次以内的反复冻融,随着探针浓度的降低,探针反复冻融后qPCR量值波动区间增大,因此,为保证结果稳定性,qPCR实验使用的MGB探针应尽量减少反复冻融次数。

  10. A quantitative PCR (TaqMan assay for pathogenic Leptospira spp

    Directory of Open Access Journals (Sweden)

    Symonds Meegan L

    2002-07-01

    Full Text Available Abstract Background Leptospirosis is an emerging infectious disease. The differential diagnosis of leptospirosis is difficult due to the varied and often "flu like" symptoms which may result in a missed or delayed diagnosis. There are over 230 known serovars in the genus Leptospira. Confirmatory serological diagnosis of leptospirosis is usually made using the microscopic agglutination test (MAT which relies on the use of live cultures as the source of antigen, often performed using a panel of antigens representative of local serovars. Other techniques, such as the enzyme linked immunosorbent assay (ELISA and slide agglutination test (SAT, can detect different classes of antibody but may be subject to false positive reactions and require confirmation of these results by the MAT. Methods The polymerase chain reaction (PCR has been used to detect a large number of microorganisms, including those of clinical significance. The sensitivity of PCR often precludes the need for isolation and culture, thus making it ideal for the rapid detection of organisms involved in acute infections. We employed real-time (quantitative PCR using TaqMan chemistry to detect leptospires in clinical and environmental samples. Results and Conclusions The PCR assay can be applied to either blood or urine samples and does not rely on the isolation and culture of the organism. Capability exists for automation and high throughput testing in a clinical laboratory. It is specific for Leptospira and may discriminate pathogenic and non-pathogenic species. The limit of detection is as low as two cells.

  11. PolyA RT-PCR-based quantification of microRNA by using universal TaqMan probe.

    Science.gov (United States)

    Luo, Xin; Zhang, Jin; Wang, Huijun; Du, Yingying; Yang, Lu; Zheng, Fengyun; Ma, Duan

    2012-04-01

    Quantification of microRNAs (miRNAs) in tissues under normal and pathological conditions is important for elucidating miRNA functions. Based on a PolyA RT-PCR method we have described (J Zhang et al. Biochem Biophys Res Commun 2008 377:136-140), a modified miRNA quantification method was developed and validated using a universal TaqMan probe complementary to the reverse transcript primer. This method effectively detects miRNA expression in cell lines and tissues. The TaqMan probe is more accurate and reliable than the SYBR Green method since it was free from primer dimers. A series of miRNAs were tested in five different mouse tissues: the method differentiated different miRNAs of the same family. This universal TaqMan probe-based PolyA RT-PCR method showed its advantages in precision, simplicity and high-throughput capability compared with other miRNA-detecting methods.

  12. Performance of cobas® 4800 and m2000 real-time™ assays for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in rectal and self-collected vaginal specimen

    NARCIS (Netherlands)

    Geelen, Tanja H; Rossen, John W; Beerens, Antoine M; Poort, Linda; Morré, Servaas A; Ritmeester, Wilma S; van Kruchten, Harry E; van de Pas, Masja M; Savelkoul, Paul H M

    2013-01-01

    A prospective, multicenter trial was designed to compare the performance characteristics of the cobas® 4800 (Roche Diagnostics, Indianapolis, IN, USA) and m2000 real-time™ (Abbott Molecular Inc., Des Plaines, IL, USA) assays for detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG)

  13. Comparison of SYBR Green and TaqMan methods in quantitative real-time polymerase chain reaction analysis of four adenosine receptor subtypes

    Directory of Open Access Journals (Sweden)

    Mohamadhasan Tajadini

    2014-01-01

    Full Text Available Background: Real-time polymerase chain reaction (PCR is based on the revolutionary method of PCR. This technique is the result of PCR enormous sensitivity and real-time monitoring combination. In quantitative gene expression analysis, two methods have more popularity, SYBR Green and TaqMan, SYBR Green is relatively cost benefit and easy to use and technically based on binding the fluorescent dye to double-stranded deoxyribonucleic acid (dsDNA where TaqMan method has more expensive and based on dual labeled oligonucleotide and exonuclease activity of Taq polymerase enzyme. Specificity is the most important concern with the usage of any non-specific dsDNA-binding Dyes such as SYBR Green whiles more specificity showed by labeled oligonucleotide method such as TaqMan. In this study, we compared two common RT PCR methods, TaqMan and SYBR Green in measurement gene expression profile of adenosine receptors. Materials and Methods: Gene expression profiles of A1, A2A, A2B and A3 Adenosine receptors were analyzed by optimized TaqMan and SYBR Green quantitative RT PCR in breast cancer tissues. Primary expression data was normalizing by B. actin reference gene. Results: Efficiencies were calculated more than 95% for TaqMan and SYBR Green methods in all genes. The correlations between means of normalized data of each gene in two methods were positive and significant (P < 0.05. Conclusion: Data analysis showed that with the use of high performance primer and by use proper protocols and material we can make precise data by SYBR Green as TaqMan method. In other word by optimization of SYBR Green method, its performance and quality could be comparable to TaqMan method.

  14. Cobas 4800 HPV detection in the cervical, vaginal and urine samples of women with high-grade CIN before and after treatment.

    Science.gov (United States)

    Stanczuk, Grazyna A; Currie, Heather; Baxter, Gwen; Foster, Adele; Gibson, Lindsay; Graham, Catriona; Cuschieri, Kate

    2015-07-01

    To assess the performance of a clinically validated human papillomavirus (HPV) test (the Cobas 4800 HPV test) in urine and self-taken vaginal specimens within a colposcopy population and to assess HPV prevalence before and after treatment across the different biospecimens. A total of 100 women attending a colposcopy clinic provided three biospecimens (a clinician-taken liquid-based cytology sample (LBC), a self-taken vaginal sample and a urine sample) for HPV testing. HPV prevalence and concordance was compared across the biospecimens and clinical performance relative to the detection of cervical intraepithelial neoplasia (CIN)2+ and CIN3+ was assessed. A total of 39 women retuned at 6 months for a post-treatment follow-up appointment, and HPV concordance in all biospecimens was measured relative to their original HPV status. 65 cases of CIN2+ were detected in the baseline population; sensitivity for CIN2+ was 92% (82 to 97) for the vaginal and the LBC sample and 80.0 (68% to 88%) for the urine sample. In the follow-up (post treatment) population, women were twice as likely to be HPV positive in their urine or vaginal sample compared with the equivalent LBC sample. Vaginal and LBC samples showed very similar performance for the detection of CIN2+ in this population using the Cobas HPV test; further validation of these findings in screening contexts will be of value. Self-taken samples may have less utility in a 'test of cure' setting-given the higher prevalence of HPV relative to LBC. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  15. 多通道Taqman-探针荧光定量PCR鉴定MRSA方法的建立%Establishment of Muti-channel Taqman-Probe Fluorescence Quantitative PCR Identification MRSA Method

    Institute of Scientific and Technical Information of China (English)

    陈昌国; 李艳君; 郭建巍; 陈秋圆; 刘敏; 马志家; 郝秀红; 赵强元

    2016-01-01

    Objective To establish the method of identifying MRSA with Taqman-fluorescence quantitative PCR basing on mecA/nuc/fem B three gene combined detecting.Methods Taking the coagulase positive MRSA,which isolated from the clinical samples and confirmed by VITEK 2 compact microbial analyzer,as the research obj ect,designed mecA/nuc/fem B specific PCR primers and Taqman fluorescent probe by bio-software PrimerPremier 5 and Designer Beacon 7,FAM,HEX and ROX markers were used to label the fluorescent probe at 5’,and the end of 3’was labeled with BHQ1,detected by fluo-rescence quantitative PCR instrment.Results ①1 g/dl gel electrophoresis results showed that the primer’s specificity of mec A/nuc/fem B were good,and molecular weight of the amplification band consistent with the expected molecular weight and no non-specific amplification band.②Three genes were obtained specific amplification in a single tube single channel and single tube multiple channel detection in PCR,and the three gene amplification effect in a single tube single tube single chan-nel and multichannel PCR similar.Conclusion Successfully established a method of multi channel Taqman-probe fluores-cence quantitative PCR identification of MRSA,mec A/nuc/fem B combined detection can effectively differentiate coagulase negative and positive MRSA,improve the accuracy of identification.%目的建立基于mec A/nuc/fem B三基因联合的 Taqman-探针荧光定量 PCR鉴定耐甲氧西林金黄色葡萄球菌(MRSA)的方法。方法以常规检验标本中分离和采用 VITEK 2 Compact微生物分析仪鉴定为凝固酶阳性的 MRSA为研究对象,通过PrimerPremier5.0和Beacon Designer 7软件设计针对mec A/nuc/fem B特异性PCR引物及Taqman荧光探针,荧光探针5’端分别采用 FAM,HEX及 ROX标记,3’端采用BHQ1标记,在荧光定量PCR仪进行检测。结果①1 g/dl凝胶电泳结果显示mec A/nuc/fem B三个基因引物特异性较好,扩增出的

  16. Development and evaluation of TaqMan real-time PCR assay for detection of beak and feather disease virus.

    Science.gov (United States)

    Černíková, Lenka; Vitásková, Eliška; Nagy, Alexander

    2017-03-02

    Psittacine beak and feather disease (PBFD) is one of the most significant viral diseases in psittacine birds. The aim of the presented study was to develop a highly specific and sensitive TaqMan real-time PCR assay for universal detection of beak and feather disease virus (BFDV). Primers and a hydrolysis probe were selected on the highly conserved regions belonging to the ORF1 of the BFDV genome which were identified by aligning 814 genomic sequences downloaded from the GenBank database. The evaluation of the reaction parameters suggested a reaction efficiency of 97.1%, with consistent detection of 10(1) virus copies/μl of nucleic acid extract. The low values of standard deviation and coefficient of variation indicate a high degree of reproducibility and repeatability. The diagnostic applicability of the assay was proven on 36 BFDV positive and 107 negative specimens of psittacine origin representing 28 species. The assay showed a 100% ability to detect distinct genetic variants of the virus. Our data suggest that the presented TaqMan real-time PCR represents a specific, sensitive and reliable assay facilitating the molecular detection of BFDV.

  17. Comparison of the Cobas 4800 HPV and HPV 9G DNA Chip Tests for Detection of High-Risk Human Papillomavirus in Cervical Specimens of Women with Consecutive Positive HPV Tests But Negative Pap Smears.

    Science.gov (United States)

    Jun, Sun-Young; Park, Eun Su; Kim, Jiyoung; Kang, Jun; Lee, Jae Jun; Bae, Yoonjin; Kim, Sang-Il; Maeng, Lee-So

    2015-01-01

    Detecting high-risk (HR) HPV is important for clinical management of women with persistent HPV-positive and Pap-negative results. The Cobas 4800 HPV test is the first FDA-approved HPV DNA test that can be used alone as a first-line screening tool. The HPV 9G DNA chip test is a PCR-based DNA microarray assay. We evaluated the patients of consecutive HPV-positivity on HPV 9G DNA chip test without cytologic abnormalities. We then compared the performances of HPV 9G DNA chip and the Cobas 4800 HPV tests for detecting HR HPV with each other and confirmed HPV genotyping using direct sequencing. All 214 liquid-based cytology specimens were collected from 100 women with consecutive HPV-positive and Pap-negative results on the HPV 9G DNA chip test between May 2012 and Dec 2013, but only 180 specimens were available for comparing HPV test results. The HPV 9G DNA chip and the Cobas 4800 HPV tests agreed with each other in 81.7% of the samples, and the concordance rate was greater than 97.2% for detecting HPV-16 or -18. For HR genotypes other than HPV types 16 and 18, the two tests agreed for 81.1% of the samples. The sensitivity of both assays for detecting HR HPV was 100%, regardless of HR genotypes. The HPV 9G DNA chip test may be as effective as the Cobas 4800 HPV test in detecting HR HPV, and has a similar ability to identify HPV-16 and -18.

  18. Application and development of a TaqMan real-time PCR for detecting infectious spleen and kidney necrosis virus in Siniperca chuatsi.

    Science.gov (United States)

    Lin, Qiang; Fu, Xiaozhe; Liu, Lihui; Liang, Hongru; Guo, Huizhi; Yin, Shuwen; Kumaresan, Venkatesh; Huang, Zhibin; Li, Ningqiu

    2017-06-01

    Infectious spleen and kidney necrosis virus (ISKNV) is one of the major epidemiological agents that had caused great economic loss in Chinese perch (Siniperca chuatsi). In this study, a specific TaqMan real-time PCR was developed using a pair of primers and a TaqMan probe specific to the ORF007 gene of ISKNV to rapidly detect ISKNV copies in Chinese perch samples. This assay was optimized to produce linearity from 8.75 × 10(8) to 8.75 × 10(1) copies in standard curve with an efficiency of 98% and a R(2) value of 0.9999. Moreover, the minimum detection limit of this assay was 10,000 times more sensitive than that of conventional PCR method. The coefficients of variation of intra- and inter-assay repeatability were less than 2.4% and 3.3%, respectively. The viral distribution in different tissues of diseased Chinese perch was evaluated by TaqMan real-time PCR method and the highest level of viral copies was detected in spleen. Among the 76 diseased Chinese perch clinical samples, 35 and 29 were positive samples based on the TaqMan real-time PCR and conventional PCR methods, respectively, indicating that the TaqMan real-time PCR was more sensitive than conventional PCR. Therefore, the TaqMan real-time PCR should be a useful tool for the early surveillance and quantitation of ISKNV. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. 使用Taqman PCR技术建立人类血小板抗原-1~5、15分型体系%Establishment of HPA-1-to-5 and HPA-15 genotyping systems by Taqman PCR

    Institute of Scientific and Technical Information of China (English)

    沈彤; 赵玉林; 刘熔增; 刘达庄

    2011-01-01

    Objective To investigate gene frequencies of HPA-l-to-5 and HPA-15 from apheresis platelet donors in Shanghai,and evaluate a new genotyping technique. Methods A total of 500 platelet aphresis donors were genotyped for HPA-\\-to-5 and HPA-15 antigen systems by means of Taqman PCR,and 100 samples were selected randomly for comparison by PCR-SSP. Results The gene frequencies of HPA-la, HPA-1 b, HPA-2a, HPA-2b, HPA-3a, HPA-U, HPAAa, HPAAb, HPA-5a,HPA-5b,HPA-15a and HPA-I5b identified using Taqman PCR were 0.999,0.001,0. 953,0.047,0.582,0.418, 0.999,0.001,0.988,0. 012,0. 524 and 0. 476,respectively. In one case for typing HPA-5 allele.the result obtained by Taqman PCR was different compared with PCR-SSP. Conclusion The allele gene frequencies of EPA systems are not significantly different between Shanghai area and other regions in China,meanwhile,the results are comparable to the frequency distribution of HPA in Chinese Hans and fit Hardy-Weinberg equilibrium. Difference observed in the distribution of HPA-5 may be the result of nonspecific amplification of PCR-SSP according to sequencing confirmation. Taqman PCR technique with high specificity and timesaving advantages has good application prospect in typing for HPA systems,which is available as a significant complement to existing methods.%目的 了解上海地区单采血小板献血人群HPA-1~5、15多态性分布,分析评估新的分型技术.方法 利用TaqMan PCR技术对500份上海地区单采血小板供者标本进行HPA-1~5、15抗原系统等位基因分型,并随机抽取100份标本使用PCR-SSP技术进行比对.结果 HPA各等位基因频率分别为HPA-1a:0.999,HPA-1b:0.001,HPA-2a:0.953,HPA-2b:0.047,HPA-3a:0.582,HPA-3b:0.418,HPA-4a:0.999,HPA-4b:0.001,HPA-5a:0.988,HPA-5b:0.012,HPA-15a:0.524,HPA-15b:0.476;有1份标本HPA-5等位基因与SSP检测结果产生差异.结论 上海地区HPA各等位基因频率与国内各地区人群分布无明显差异,与中国汉族人群HPA分布情况基

  20. CYP2D7 sequence variation interferes with TaqMan CYP2D6*15 and *35 genotyping

    Directory of Open Access Journals (Sweden)

    Amanda K Riffel

    2016-01-01

    Full Text Available TaqMan™ genotyping assays are widely used to genotype CYP2D6, which encodes a major drug metabolizing enzyme. Assay design for CYP2D6 can be challenging owing to the presence of two pseudogenes, CYP2D7 and CYP2D8, structural and copy number variation and numerous single nucleotide polymorphisms (SNPs some of which reflect the wild-type sequence of the CYP2D7 pseudogene. The aim of this study was to identify the mechanism causing false positive CYP2D6*15 calls and remediate those by redesigning and validating alternative TaqMan genotype assays. Among 13,866 DNA samples genotyped by the CompanionDx® lab on the OpenArray platform, 70 samples were identified as heterozygotes for 137Tins, the key SNP of CYP2D6*15. However, only 15 samples were confirmed when tested with the Luminex xTAG CYP2D6 Kit and sequencing of CYP2D6-specific long range (XL-PCR products. Genotype and gene resequencing of CYP2D6 and CYP2D7-specific XL-PCR products revealed a CC>GT dinucleotide SNP in exon 1 of CYP2D7 that reverts the sequence to CYP2D6 and allows a TaqMan assay PCR primer to bind. Because CYP2D7 also carries a Tins, a false-positive mutation signal is generated. This CYP2D7 SNP was also responsible for generating false-positive signals for rs769258 (CYP2D6*35 which is also located in exon 1. Although alternative CYP2D6*15 and *35 assays resolved the issue, we discovered a novel CYP2D6*15 subvariant in one sample that carries additional SNPs preventing detection with the alternate assay. The frequency of CYP2D6*15 was 0.1% in this ethnically diverse U.S. population sample. In addition, we also discovered linkage between the CYP2D7 CC>GT dinucleotide SNP and the 77G>A (rs28371696 SNP of CYP2D6*43. The frequency of this tentatively functional allele was 0.2%. Taken together, these findings emphasize that regardless of how careful genotyping assays are designed and evaluated before being commercially marketed, rare or unknown SNPs underneath primer and/or probe

  1. Method comparison of the Ortho Vitros Fusion 5,1 chemistry analyzer and the Roche COBAS Integra 400 for urine drug screen testing in the emergency department.

    Science.gov (United States)

    Johnson-Davis, Kamisha L; Thompson, Catherine D; Clark, Chantry J; McMillin, Gwen A; Lehman, Christopher M

    2012-06-01

    Exposure to drugs and toxins is a major cause for the rising number of emergency department visits each year. Immunoassays are commonly used in the emergency department to provide rapid turnaround time for acute care. The purpose of this study was to compare two automated immunoassay chemistry analyzers to determine which platform produced the fewest number of false positive/negative results. Residual patient urine samples were were collected for each of the following drugs/drug classes: cocaine (n = 40), opiates (n = 45), and amphetamines (n = 54) and confirmed either positive or negative by mass spectrometry. Split sample analyses of these specimens were performed on both the Roche COBAS INTEGRA 400 plus and Ortho Vitros 5,1 FS instruments. The results from the two chemistry analyzers were compared to confirmed results. Both immunoassays were prone to false positive results for cocaine and false negative results for opiates and amphetamines. The Vitros Fusion analyzer generated fewer false positive and false negative results for opiate and amphetamine testing than the Roche Integra, but the platforms performed comparably for cocaine.

  2. A multiplex real-time polymerase chain reaction (TaqMan) assay for the simultaneous detection of Meloidogyne chitwoodi and M-fallax

    NARCIS (Netherlands)

    Zijlstra, C.; Hoof, van R.A.

    2006-01-01

    This study describes a multiplex real-time polymerase chain reaction (PCR) approach for the simultaneous detection of Meloidogyne chitwoodi and M. fallax in a single assay. The approach uses three fluorogenic minor groove binding (MGB) TaqMan probes: one FAM-labeled to detect M. chitwoodi, one VIC-l

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

    Science.gov (United States)

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

    2008-05-01

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

  4. Detection of elephant endotheliotropic herpesvirus type 1 in asymptomatic elephants using TaqMan real-time PCR.

    Science.gov (United States)

    Hardman, K; Dastjerdi, A; Gurrala, R; Routh, A; Banks, M; Steinbach, F; Bouts, T

    2012-02-25

    This study assessed the feasibility of identifying asymptomatic viral shedders using a novel TaqMan real-time PCR on trunk washes and swabs from the conjunctiva, palate and vulva of elephants. Six elephants from a UK collection were sampled weekly over a period of 11 weeks for this study. The herd prevalence of elephant endotheliotropic herpesvirus-1 (EEHV-1) was 100 per cent by PCR. The virus DNA was detected in all the sampling sites; however, the prevalence of virus DNA in the conjunctiva swabs was higher. In addition, Asian elephants from two continental European collections were sampled once and one animal tested positive on a trunk wash. The virus from this animal was phylogenetically typed as EEHV-1A based on 231 nucleotides of the terminase gene.

  5. Development of a real-time PCR method (Taqman) for rapid identification and quantification of Prorocentrum donghaiense

    Science.gov (United States)

    Yuan, Jian; Mi, Tiezhu; Zhen, Yu; Yu, Zhigang

    2012-09-01

    Prorocentrum donghaiense is a dinoflagellate that is widely distributed in the East China Sea and has become increasingly involved in Harmful Algal Blooms (HABs). Therefore, it is necessary to study this dinoflagellate to monitor HABs. In this study, 13 pairs of primers specific to P. donghaiense (within its internal transcribed spacer (ITS) regions) were designed for SYBR Green I real-time PCR. As the SYBR Green I real-time PCR could not identify P. donghaiense in a specific manner, a Taqman real-time PCR method was developed by designing a set of specific primers and a Taqman probe. A 10-fold serial dilution of recombinant plasmid containing ITS regions of P. donghaiense was prepared as standard samples and the standard curve was established. Additionally, we quantified the genomic DNA in P. donghaiense cells and utilized this DNA to prepare another 10-fold serial dilution of standard sample and accordingly set up the standard curve. The mathematic correlation between the cell number and its corresponding plasmid copy number was also established. In order to test the efficiency of the real-time PCR method, laboratory samples and P. donghaiense HAB field samples were employed for identification and quantitative analysis. As to laboratory samples, as few as 102 cells of P. donghaiense could be quantified precisely utilizing both centrifugation and filtration techniques. The quantification results from field samples by real-time PCR were highly similar to those by light microscopy. In conclusion, the real-time PCR could be applied to identify and quantify P. donghaiense in HABs.

  6. Development of a Real-Time PCR Method (Taqman) for Rapid Identification and Quantification of Prorocentrum donghaiense

    Institute of Scientific and Technical Information of China (English)

    YUAN Jian; MI Tiezhu; ZHEN Yu; YU Zhigang

    2012-01-01

    Prorocentrum donghaiense is a dinoflagellate that is widely distributed in the East China Sea and has become increasingly involved in Harmful Algal Blooms(HABs).Therefore,it is necessary to study this dinoflagellate to monitor HABs.In this study,13 pairs of primers specific to P.donghaiense(within its internal transcribed spacer(ITS)regions)were designed for SYBR Green Ⅰ real-time PCR.As the SYBR Green Ⅰ real-time PCR could not identify P donghaiense in a specific manner,a Taqman real-time PCR method was developed by designing a set of specific primers and a Taqman probe.A 10-fold serial dilution of recombinant plasmid containing ITS regions of P.donghaiense was prepared as standard samples and the standard curve was established.Additionally,we quantified the genomic DNA in P.donghaiense cells and utilized this DNA to prepare another 10-fold serial dilution of standard sample and accordingly set up the standard curve.The mathematic correlation between the cell number and its corresponding plasmid copy number was also established.In order to test the efficiency of the real-time PCR method,laboratory samples and P.donghaiense HAB field samples were employed for identification and quantitative analysis.As to laboratory samples,as few as 102 cells of P.donghaiense could be quantified precisely utilizing both centrifugation and filtration techniques.The quantification results from field samples by real-time PCR were highly similar to those by light microscopy.In conclusion,the real-time PCR could be applied to identify and quantify P.donghaiense in HABs.

  7. 罗氏 Co bas c701全自动生化分析仪性能评价%Performance evaluation of Roche Cobas c 701 fully automatic biochemical analyzer

    Institute of Scientific and Technical Information of China (English)

    邓小玲; 侯玉磊; 陈特; 毕小云

    2015-01-01

    Objective To assess the performance of Roche Cobas c701 fully automatic biochemical analyzer. Methods According to EP15‐A2 from Clinical and Laboratory Standards Institute, the electrolyte (potassium, sodi‐um and chloride) and covers all the wavelengths of nine projects (alanine transaminase, aspartate transaminase, alka‐line phosphatase, gamma‐glutamine transaminase, creatinine and urea nitrogen, glucose, total protein, three acyl glyc‐erin) were measured by Roche Cobas c701 analyzer and original reagents. The precisions and accuracies of all parame‐ters were verified. Results In the 2 levels of tested parameters, the standard deviation of repeatability (Sr )was ≤the manufacture′s standard deviation of repeatability (σr ), and the standard deviation of prescision (St ) was≤the manu‐facture′s standard prescision (σt ), the prescision was acceptable and similar to what the manufacter declared. Correla‐tions between theoretic value and actual value were good (regression coefficient was :0. 999 4-1. 000 0). The bias of all parameters was acceptable (within the prescribed scope of CLIA′88)with Roche cobas c701analyzer, compared with the external quality assessment of the ministry of health clinical inspection center. Conclusion The repeatabili‐ty, precision and accuracy of the parameters by Roche Cobas c701 reach the performance that the manufacturer de‐clares.%目的:对罗氏Cobas c701全自动生化分析仪进行性能评价。方法按照美国临床和实验室标准化协会EP15‐A2文件的要求,通过电解质(钾、钠、氯)和涵盖各波长的9个项目(丙氨酸氨基转移酶、天门冬氨酸氨基转移酶、碱性磷酸酶、γ‐谷氨酰转移酶、肌酐、尿素氮、葡萄糖、总蛋白、三酰甘油)对仪器的精密度、准确度、线性范围等进行验证。结果所有检测项目的重复性标准差(Sr)≤厂家声明的标准差(σr)、精密度的标准差(St)≤σt ,均

  8. Effect of the hemoglobin-based oxygen carrier HBOC-201 on laboratory instrumentation: cobas integra, chiron blood gas analyzer 840, Sysmex SE-9000 and BCT.

    Science.gov (United States)

    Wolthuis, A; Peek, D; Scholten, R; Moreira, P; Gawryl, M; Clark, T; Westerhuis, L

    1999-01-01

    As part of a clinical trial, we evaluated the effects of the hemoglobin-based oxygen-carrier (HBOC) HBOC-201 (an ultrapurified, stroma-free bovine hemoglobin product, Biopure, Cambridge, MA, USA) on our routine clinical chemistry analyzer (Cobas Integra, F. Hoffmann-La Roche Ltd, Basel, Switzerland ), blood gas analyzer (Chiron 840, Chiron Diagnostics Corporation, East Walpole, MA, USA), routine hemocytometry analyzer (Sysmex SE-9000, TOA Medical Electronics Co Ltd., Kobe, Japan), hemostasis analyzer (BCT, Dade-Behring, Marburg, Germany) and bloodbanking system (Dia-Med-ID Micro Typing System, DiaMed AG, Cressier, Switzerland). The maximum tested concentration of HBOC-201 was 65 g/l. Of the 27 routine clinical chemistry tests challenged with HBOC-201, bilirubin-direct, creatine kinase MB-fraction (CK-MB), creatine kinase (CK), gamma-glutamyltransferase (GGT), magnesium and uric acid were influenced by even low concentrations of HBOC-201. These tests were excluded from use on the plasma of patients treated with HBOC-201. Since the non-availability of the cardiac marker CK-MB may lead to problems in acute situations, we introduced the qualitative Trop T-test (Boehringer Mannheim), which was not influenced. The applicability of another nine tests was limited by the concentration of the HBOC-201 in the patients' plasma. No interference of HBOC-201 in routine hemocytometry, hemostasis-analysis and red-blood cell agglutination detection (blood-bank tests) was observed. Although immediate patient-care was not compromised, routine use of hemoglobin-based oxygen carriers will have a strong impact on logistical management. The development of robust laboratory tests free from the interference of the pigmented oxygen carriers should therefore precede its introduction into routine transfusion medicine.

  9. Serum ASAT, ALAT, ALP, LD, GT, and CK determined in the Cobas-Bio centrifugal analyser by the methods of the Scandinavian Committee on Enzymes.

    Science.gov (United States)

    Izquierdo, J M; Sotorrío, P; Alvarez-Uría, J; Estrada, J M; Quirós, A

    1982-04-01

    The recommended methods of the Scandinavian Committee on Enzymes [4, 5, 6, 7, 8] have been applied to the Cobas-Bio centrifugal analyser. Reagents and serum volumes were scaled down and final molarities were kept equal. Serum volumes in microliters were as follows: ASAT 30, ALAT 30, ALP 3, LD 5, GT 20, and CK 10. Including the dead space of the sample cup, the volume needed to perform all six tests was 113 microliter. Within-run and between-run precision (CV%) were as follows: ASAT 1.32 and 1.95, ALAT 1.68 and 2.93, ALP 1.56 and 3.10, LD 1.63 and 4.44, GT 0.81 and 2.23, and CK 1.02 and 1.94. Mean deviations (%) from target values of two commercial sera were as follows: ASAT -0.3 and -0.4, ALAT -0.4 and -2.2, ALP -1.8 and -7.3, LD 0.4 and -6.2, GT 13.9 and -10.7, and CK -4.9 and -1.3. Results of all the methods correlated well with those obtained with their respective manual methods. Analytical time for 28 samples of each analyte was 10 min, apart from CK which was 14 min. Reagent cost per sample was 0.6, 0.9, 0.1, 0.3, 0.9, and 26 US cents respectively. All reagents were prepared in the laboratory, except those for CK which were bought from J.T. Baker (Phillipsburgh, NJ, USA). In conclusion, the methods keep the features of the manual methods but they are more precise and practicable, much faster and cheaper, and use minimal amounts of sera more convenient for paediatric work.

  10. Neospora caninum DNA detection by TaqMan real-time PCR assay in experimentally infected pregnant heifers.

    Science.gov (United States)

    Pereira, Gabriel Ribas; Vogel, Fernanda Silveira Flores; Bohrer, Rodrigo Camponogara; da Nóbrega, Janduí Escarião; Ilha, Gustavo Freitas; da Rosa, Paulo Roberto Antunes; Glanzner, Werner Giehl; Camillo, Giovana; Braunig, Patricia; de Oliveira, João Francisco Coelho; Gonçalves, Paulo Bayard Dias

    2014-01-31

    Neosporosis has been considered the main cause of abortion between the first and the second trimester of pregnancy in cattle. Therefore, the objective of this study was to identify the presence of Neospora caninum DNA obtained from experimental models based on the evaluation of different areas of the fetal nervous system and organs from heifers previously inoculated with NC-1 after or before insemination. This study was performed with Hereford × Nelore (n=29) heifers and all animals were considered free of diseases at the beginning of the experiment. All animals were bred by fixed-time artificial insemination (TAI) and allocated as follows: (a) seronegative heifers subjected to TAI (TAI, n=9), (b) heifers infected with N. caninun 60 days prior to TAI (NC-1+TAI, n=9), and (c) heifers submitted to TAI and infected with N. caninum 60 days later (TAI+NC-1, n=11). The pregnancy was confirmed by transrectal ultrasonography 35 days after TAI and evaluated every 30 days until the end of gestation. Fetuses were collected surgically at 170 days of gestation, and immediately necropsied to remove tissues aseptically. Samples of the central nervous system (CNS), heart, kidney, lung, liver, skeletal muscle and caruncle were collected for DNA extraction. Days of gestation at abortion and interval from abortion to first insemination were examined by Student's t-test. At 35 days of gestation the pregnancy rates in the group NC-1+TAI (4/9, 44.4%) was lower than in the control group (8/9, 88.8%, P<0.05). At 60 days, the pregnancy rates in the NC-1+TAI group (0/4, 0%) was lower compared to TAI+NC-1 (5/7, 71.4%) and control (6/8, 75.0%) groups (P<0.05). Animals from the group NC-1+TAI were re-inseminated 60 days after the first TAI. After pregnancy losses throughout the study, 5 animals (TAI), 3 animals (NC-1+TAI) and 5 animals (TAI+NC-1) maintained pregnancy until 170 days of gestation. TaqMan RT-PCR demonstrated the presence of N. caninum DNA in the medulla and right posterior

  11. Taqman MGB探针快速定量检测VHSV方法的研究%Absolute quantitative real-time RT-PCR assay for rapid detection of viral hemorrhagic septicemia virus (VHSV) with Taqman MGB probe

    Institute of Scientific and Technical Information of China (English)

    许建明; 张念之; 蒋一男; 张利峰; 夏春

    2010-01-01

    为建立准确实时地定量检测病毒性出血性败血症病毒(VHSV),在VHSV-N基因保守区设计了Taqman MGB探针与引物对,随后,采用体外转录技术获得了VHSV-N基因RNA,并以此为绝对定量标准品,建立了绝对定量(AQ)检测VHSV的实时荧光RT-PCR法(AQ-RT-PCR方法),并与世界动物卫生组织(OIE)推荐的普通RT-PCR法进行了比较.此荧光RT-PCR法特异性好,与其他鱼类弹状病毒无交叉反应.检测线性范围为10~(10)~10~2拷贝/反应,灵法度达10~2 拷贝/反应.此检测灵敏度比OIE推举的RT-PCR法高出5个数量级,比嵌套RT-PCR高出1个数量级.此法是出入境检疫VHSV的有效方法.

  12. Differentiation of the traditional Chinese medicinal plants Euphorbia humifusa and E. maculata from adulterants by TaqMan real-time polymerase chain reaction.

    Science.gov (United States)

    Xue, Heng-Gang; Wang, Hong; Li, De-Zhu; Xue, Chun-Ying; Wang, Qing-Zhong

    2008-02-01

    DNA sequence analysis of the rDNA internal transcribed spacer 1 (ITS1) and TaqMan real-time polymerase chain reaction were exploited for their applications in the differentiation of the traditional chinese medicinal plants euphorbia humifusa and e. maculata from three related adulterants e. hypericifolia, E. atoto and E. prostrata. The data demonstrated that variations in the ITS1 regions were very low at the intra-species level but extremely high at the inter-species level, so that they could be easily distinguished at the DNA level. The sequence difference allowed an effective and reliable differentiation of E. humifusa and E. maculata from the adulterants by TaqMan real-time PCR.

  13. Identification and quantification of genetically modified Moonshade carnation lines using conventional and TaqMan real-time polymerase chain reaction methods.

    Science.gov (United States)

    Li, Peng; Jia, Junwei; Bai, Lan; Pan, Aihu; Tang, Xueming

    2013-07-01

    Genetically modified carnation (Dianthus caryophyllus L.) Moonshade was approved for planting and commercialization in several countries from 2004. Developing methods for analyzing Moonshade is necessary for implementing genetically modified organism labeling regulations. In this study, the 5'-transgene integration sequence was isolated using thermal asymmetric interlaced (TAIL)-PCR. Based upon the 5'-transgene integration sequence, conventional and TaqMan real-time PCR assays were established. The relative limit of detection for the conventional PCR assay was 0.05 % for Moonshade using 100 ng total carnation genomic DNA, corresponding to approximately 79 copies of the carnation haploid genome, and the limits of detection and quantification of the TaqMan real-time PCR assay were estimated to be 51 and 254 copies of haploid carnation genomic DNA, respectively. These results are useful for identifying and quantifying Moonshade and its derivatives.

  14. Novel TaqMan real-time polymerase chain reaction assay for verifying the authenticity of meat and commercial meat products from game birds.

    Science.gov (United States)

    Rojas, María; González, Isabel; Pavón, Miguel Angel; Pegels, Nicolette; Lago, Adriana; Hernández, Pablo E; García, Teresa; Martín, Rosario

    2010-06-01

    Species-specific real-time polymerase chain reaction (PCR) assays using TaqMan probes have been developed for verifying the labeling of meat and commercial meat products from game birds, including quail, pheasant, partridge, guinea fowl, pigeon, Eurasian woodcock and song thrush. The method combines the use of species-specific primers and TaqMan probes that amplify small fragments (amplicons <150 base pairs) of the mitochondrial 12S rRNA gene, and an endogenous control primer pair that amplifies a 141-bp fragment of the nuclear 18S rRNA gene from eukaryotic DNA. Analysis of experimental raw and heat-treated binary mixtures as well as of commercial meat products from the target species demonstrated the suitability of the assay for the detection of the target DNAs.

  15. Detection of seasonal H3N2 influenza A virus by type-specific TaqMan minor groove binder probe assay

    Science.gov (United States)

    Wang, Ruixue; Schwartzman, Louis M.; Memoli, Matthew J.; Taubenberger, Jeffery K.

    2011-01-01

    Despite the emergence of the pandemic H1N1 influenza A virus in 2009, seasonal H3N2 viruses continue to co-circulate in the population, and may even predominate in the coming influenza season. We describe a specific minor groove binder Taqman assay for H3N2 viruses with a detection limit of 16.5 standard DNA copies. PMID:21429691

  16. Comparison of nested-multiplex, Taqman & SYBR Green real-time PCR in diagnosis of amoebic liver abscess in a tertiary health care institute in India

    Directory of Open Access Journals (Sweden)

    K P Dinoop

    2016-01-01

    Interpretation & conclusions: Taqman real-time PCR targeting the 18S rRNA had the highest positivity rate evaluated in this study. Both nested multiplex and SYBR Green real-time PCR assays utilized were evaluated to give accurate results. Real-time PCR assays can be used as the gold standard in rapid and reliable diagnosis, and appropriate management of amoebiasis, replacing the conventional molecular methods.

  17. Establishment of TaqMan Real-time Quantitative PCR Assay for Foreign Gene Copy Numbers in Transgenic Soybean

    Institute of Scientific and Technical Information of China (English)

    Qiu You-wen; Gao Xue-jun; Qi Bang-ruo; Li Lu; Zhen Zhen

    2012-01-01

    TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in non-GMO soybeans as the endogenous reference standard, the gradient dilution method was used to separately calculate Ct value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies, and then to calculate the copies of samples through substituting thus-obtained Ct into the standard curve equation. The standard curve equation of endogenous reference gene was y =–3.422x+35.201, R2=0.998; the standard curve equation of exogenous gene was y =–3.495x+35.303, R2=0.999. The sample copies was got by putting Ct value into the standard curve equation, and it was the ratio of exogenous gene and reference gene. We found that CaMV35S gene in transgenic soy was single copy.

  18. Quantitation of mule duck in goose foie gras using TaqMan real-time Polymerase Chain Reaction.

    Science.gov (United States)

    Rodríguez, Miguel A; García, Teresa; González, Isabel; Asensio, Luis; Hernández, Pablo E; Martín, Rosario

    2004-03-24

    A real-time quantitative Polymerase Chain Reaction (PCR) method has been developed for the quantitation of mule duck (Anas platyrhynchos x Cairina moschata) in binary duck/goose foie gras mixtures. The method combines the use of real-time PCR with duck-specific and endogenous control "duck + goose" primers to measure duck content and total foie gras content, respectively. Both PCR systems (duck-specific and duck + goose) were designed on the mitochondrial 12S ribosomal RNA gene (rRNA). The duck-specific system amplifies a 96 bp fragment from duck DNA, whereas the duck + goose system amplifies a 120 bp fragment from duck and goose DNA. The method measures PCR product accumulation through a FAM-labeled fluorogenic probe (TaqMan). The C(t) (threshold cycle) values obtained from the duck + goose system are used to normalize the ones obtained from the duck-specific system. Analysis of experimental duck/goose foie gras binary mixtures demonstrated the suitability of the assay for the detection and quantitation of duck in the range of 1-25%. This genetic marker can be very useful to avoid mislabeling or fraudulent species substitution of goose by duck in foie gras.

  19. Development a diagnostic pan-dermatophyte TaqMan probe real-time PCR assay based on beta tubulin gene.

    Science.gov (United States)

    Mirhendi, Hossein; Motamedi, Marjan; Makimura, Koichi; Satoh, Kazuo

    2016-08-01

    Early differentiation of dermatophytosis from other cutaneous mycoses is essential to avoid inaccurate therapy. DNA-based techniques including real-time PCR have increasingly been considered for detection of fungal elements in clinical specimens. In this study, after partial sequence analysis of beta tubulin (BT2) gene in 13 common and rare pathogenic dermatophyte species, a pan-dermatophyte primer and probe set was designed in a TaqMan probe-based PCR format. The sensitivity and specificity of the system was tested with 22 reference strains of dermatophytes, 234 positive clinical specimens, 32 DNA samples extracted from normal nails, several fungi other than dermatophytes and human DNAs. Analytical detection limit of the designed PCR on serially diluted DNAs of prepared recombinant plasmid indicated that only five molecules per sample are the minimum number for reliable detection by the assay. A total of 226 out of 234 (96.5%) DNAs extracted from clinical samples, but none of the 32 nail samples, from healthy volunteers were positive in PCR. The real-time PCR targeted beta tubulin gene established in this study could be a sensitive diagnostic tool which is significantly faster than the conventional culture method and should be useful in the clinical settings, in large-scale epidemiological studies and in clinical trials of antifungal therapy.

  20. Detection of Bordetella avium by TaqMan real-time PCR in tracheal swabs from wildlife birds.

    Science.gov (United States)

    Stenzel, T; Pestka, D; Tykałowski, B; Śmiałek, M; Koncicki, A; Bancerz-Kisiel, A

    2017-03-28

    Bordetella avium, the causing agent of bordetellosis, a highly contagious infection of the respiratory tract in young poultry, causes significant losses in poultry farming throughout the world. Wildlife birds can be a reservoir of various pathogens that infect farm animals. For this reason the studies were conducted to estimate the prevalence of Bordetella avium in wildlife birds in Poland. Tracheal swab samples were collected from 650 birds representing 27 species. The bacterial DNA was isolated directly from the swabs and screened for Bordetella avium by TaqMan real-time PCR. The assay specificity was evaluated by testing DNA isolated from 8 other bacteria that can be present in avian respiratory tract, and there was no amplification from non-Bordetella avium agents. Test sensitivity was determined by preparing standard tenfold serial dilutions of DNA isolated from positive control. The assay revealed to be sensitive, with detection limit of approximately 4.07x10^2 copies of Bordetella avium DNA. The genetic material of Bordetella avium was found in 54.54% of common pheasants, in 9.09% of Eurasian coots, in 3.22% of black-headed gulls and in 2.77% of mallard ducks. The results of this study point to low prevalence of Bordetella avium infections in wildlife birds. The results also show that described molecular assay proved to be suitable for the rapid diagnosis of bordetellosis in the routine diagnostic laboratory.

  1. Identification of hantavirus infection by Western blot assay and TaqMan PCR in patients hospitalized with acute kidney injury.

    Science.gov (United States)

    Oldal, Miklós; Németh, Viktória; Madai, Mónika; Kemenesi, Gábor; Dallos, Bianka; Péterfi, Zoltán; Sebők, Judit; Wittmann, István; Bányai, Krisztián; Jakab, Ferenc

    2014-06-01

    Hantaviruses, one of the causative agents of viral hemorrhagic fevers, represent a considerable healthcare threat. In Hungary, Dobrava-Belgrade virus (DOBV) and Puumala virus (PUUV) are the main circulating hantavirus species, responsible for the clinical picture known as hemorrhagic fever with renal syndrome, a disease that may be accompanied by acute kidney injury (AKI), requiring hospitalization with occasionally prolonged recovery phase. A total of 20 patient sera were collected over a 2-year period from persons hospitalized with AKI, displaying clinical signs and laboratory findings directly suggestive for hantavirus infection. Samples were tested using an immunoblot assay, based on complete viral nucleocapsid proteins to detect patients' IgM and IgG antibodies against DOBV and PUUV. In parallel, all specimens were also tested by 1-step real-time TaqMan reverse-transcriptase polymerase chain reaction to confirm infection and to determine the causative hantavirus genotype. We present here the first Hungarian clinical study spanning across 2 years and dedicated specifically to assess acute kidney injuries, in the context of hantavirus prevalence.

  2. Taqman real-time PCR detects Avipoxvirus DNA in blood of Hawai'i 'amakihi (Hemignathus virens).

    Science.gov (United States)

    Farias, Margaret E M; LaPointe, Dennis A; Atkinson, Carter T; Czerwonka, Christopher; Shrestha, Rajesh; Jarvi, Susan I

    2010-05-27

    Avipoxvirus sp. is a significant threat to endemic bird populations on several groups of islands worldwide, including Hawai'i, the Galapagos Islands, and the Canary Islands. Accurate identification and genotyping of Avipoxvirus is critical to the study of this disease and how it interacts with other pathogens, but currently available methods rely on invasive sampling of pox-like lesions and may be especially harmful in smaller birds. Here, we present a nested TaqMan Real-Time PCR for the detection of the Avipoxvirus 4b core protein gene in archived blood samples from Hawaiian birds. The method was successful in amplifying Avipoxvirus DNA from packed blood cells of one of seven Hawaiian honeycreepers with confirmed Avipoxvirus infections and 13 of 28 Hawai'i 'amakihi (Hemignathus virens) with suspected Avipoxvirus infections based on the presence of pox-like lesions. Mixed genotype infections have not previously been documented in Hawai'i but were observed in two individuals in this study. We anticipate that this method will be applicable to other closely related strains of Avipoxvirus and will become an important and useful tool in global studies of the epidemiology of Avipoxvirus.

  3. Taqman real-time PCR detects Avipoxvirus DNA in blood of Hawai'i 'amakihi (Hemignathus virens.

    Directory of Open Access Journals (Sweden)

    Margaret E M Farias

    Full Text Available BACKGROUND: Avipoxvirus sp. is a significant threat to endemic bird populations on several groups of islands worldwide, including Hawai'i, the Galapagos Islands, and the Canary Islands. Accurate identification and genotyping of Avipoxvirus is critical to the study of this disease and how it interacts with other pathogens, but currently available methods rely on invasive sampling of pox-like lesions and may be especially harmful in smaller birds. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present a nested TaqMan Real-Time PCR for the detection of the Avipoxvirus 4b core protein gene in archived blood samples from Hawaiian birds. The method was successful in amplifying Avipoxvirus DNA from packed blood cells of one of seven Hawaiian honeycreepers with confirmed Avipoxvirus infections and 13 of 28 Hawai'i 'amakihi (Hemignathus virens with suspected Avipoxvirus infections based on the presence of pox-like lesions. Mixed genotype infections have not previously been documented in Hawai'i but were observed in two individuals in this study. CONCLUSIONS/SIGNIFICANCE: We anticipate that this method will be applicable to other closely related strains of Avipoxvirus and will become an important and useful tool in global studies of the epidemiology of Avipoxvirus.

  4. Evaluation of point mutation detection in Mycobacterium tuberculosis with isoniazid resistance using real-time PCR and TaqMan probe assay.

    Science.gov (United States)

    Riahi, F; Derakhshan, M; Mosavat, A; Soleimanpour, S; Rezaee, S A

    2015-03-01

    Rapid methods for diagnosis of Mycobacterium tuberculosis (Mtb) drug resistance and choosing appropriate antibiotic treatment are pivotal. Thirty isoniazid (INH)-resistant and 30 INH-susceptible Mtb isolates were evaluated using minimum inhibitory concentration (MIC) method followed by multiplex real-time PCR (RT-PCR). Amplification refractory mutation system (ARMS) for detection of mutation in 315 codon of katG gene and single-nucleotide polymorphism (SNP) for detection of mutation in -15 (C>T) in the regulatory zone of mabA-inhA were carried out using the TaqMan method. Primers and probe were used for IS6110 region of Mtb as an internal amplification control. The sensitivity and specificity of the RT-PCR TaqMan probe for detection of Mtb complex were 100 %. Detection of INH-resistant Mtb using the ARMS method for KatG had 69 % sensitivity and 100 % specificity. The sensitivity and specificity of SNP in mabA-inhA fragment for detection of INH-resistant Mtb were 53 and 100 %, respectively. Furthermore, considering both regions, the sensitivity of RT-PCR has increased to 75 %. This study revealed that the qPCR-TaqMan method can be used as a standard tool for diagnosis of Mtb. Moreover, ARMS and SNP RT-PCR TaqMan methods can be used as rapid screening methods for detection of INH-resistant Mtb.

  5. Calibration of quantitative real-time TaqMan PCR by correlation with hyphal biomass and ITS copies in mycelia of Piloderma croceum.

    Science.gov (United States)

    Raidl, S; Bonfigli, R; Agerer, R

    2005-11-01

    DNA-based quantification methods such as real-time TaqMan PCR allow a rapid and highly sensitive species-specific quantification of isolated fungal DNA material, but most quantification systems are only able to measure relative amounts of biomass or biomass changes during different treatments. In this experiment, an already established DNA quantification system for the ectomycorrhizal fungus Piloderma croceum, based on the ITS region of ribosomal DNA, was calibrated to absolute biomass to obtain a direct correlation between mycelial biomass and isolated ITS copies. Thin layers of sterile mycelia were cultured on slides. The mycelial biomass was calculated from measurements of the total hyphal length using image analysis, followed by determination of the mycelial volume, and multiplied by the specific weight of hyphae obtained from literature data. Using the very same mycelium, the number of ITS copies was quantified by TaqMan PCR. The mean value of 1047 (+/- 185) copies per mm hypha results in possible data for a direct conversion: one billion (10 (9)) ITS copies corresponded to 0.79 mg hyphal dry weight. For the ribosomal ITS multi-copy genes, the number of ITS copies could be calculated to approx. 152 (+/- 26) copies per dikaryotic cell. These conversion data now allow determination of the mycelial biomass of Piloderma croceum using real-time TaqMan PCR, a prerequisite for competition experiments with Piloderma croceum.

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

    OpenAIRE

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

    2011-01-01

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

  7. Establishment and application of a TaqMan real-time PCR assay for the detection of encephalomyo-carditis virus%脑心肌炎病毒 TaqMan real-time PCR检测方法的建立及应用

    Institute of Scientific and Technical Information of China (English)

    张海霞; 冯若飞; 王丹; 凡静静; 谢晶莹; 马忠仁; 冯玉萍

    2013-01-01

    Objective To establish a TaqMan real-time PCR assay for the detection of encephalo-myocarditis virus ( EMCV) .Methods Based on the conservative region of 3D gene of EMCV published in GenBank , a pair of primers and one TaqMan probe were designed and synthesized .Then a TaqMan real-time PCR assay was set up and the reactive system was optimized .The sensitivity and specificity of the assay was evaluated respectively .The TaqMan real-time PCR assay was then carried out to detect 98 randomly selected swine serum samples and the results were compared with those by using ELISA .Results The Ct value of the templates had a good linear relationship with the log starting quantity , with a correlation coefficient of 0.995.The TaqMan real-time PCR assay was only specific for EMCV and its sensitivity was 100 times higher than that of the ordinary PCR .The coincidence rate between the established assay and the ELISA assay was 98.0%in the detection of 98 blood samples.Conclusion The TaqMan real-time PCR assay for the detec-tion of EMCV was successfully established with advantages of high sensitivity and good specificity .It could be used for detection of EMCV and quantitative analysis .%目的建立脑心肌炎病毒( EMCV) TaqMan real-time PCR检测方法。方法根据GenBank中公布的EMCV 3D基因保守区段设计并合成1对引物和1条TaqMan 探针,建立EMCV TaqMan real-time PCR检测方法,且对体系进行优化;对该法进行灵敏性、特异性验证;采用建立的方法对98份猪血清样本进行检测,并与ELISA结果进行比较。结果建立的EMCV TaqMan real-time PCR检测方法线性关系较好,以质粒标准品构建的标准曲线相关系数R2为0.995;灵敏性比普通PCR高100倍,且仅能特异性检出 EMCV;对猪血清样本的检测与 ELISA 法检测结果符合率为98.0%。结论已建立了EMCV TaqMan real-time PCR检测方法,该法灵敏性高、特异性好,可用于EMCV的检测及定量分析。

  8. Establishment and application on TaqMan MGB probe real-time PCR for rapid detection of brucellosis%Taqman MGB探针实时荧光定量PCR检测布鲁菌病的研究

    Institute of Scientific and Technical Information of China (English)

    刘艳红; 王清

    2013-01-01

    OBJECTIVE To standardize a TaqMan MGB probe real-time PCR for screening and detection on Brucella DNA in blood and evaluate its methodology. METHODS TaqMan MGB probe was designed according to the sequence of IS711 gene.The PCR reaction system was optimized strictly.Used clinical and laboratory standards institute (CLSI) evaluation program to evaluate the Brucella DNA quantitative methods' linear range, sensitivity, specificity and diagnostic accuracy. RESULTS Linear was in the rang 4.0×102-4.0×108 copies/ml, variation within and between groups ranged from1.8% to 6.5% and 2.6% to 9.1% respectively, lower limit was 400 copies/ml of the quantitative method of Brucella DNA.And there was a good linearity with Cr value (Ct = -1.391 1 Ln (x) +41.65, r = -0.995 8). In a test of 157 samples, the positive coincident rate of clinical brucellosis, chronic brucellosis, clinically suspected and risk people was 90.3%, 40%, 6.7%, 12.2% respectively, and the negative coincident rate was 100%. CONCLUSION These results show that the real-time PCR assay is far more sensitive than conventional cultures, which makes this technique a very useful tool for the diagnosis of brucellosis.%目的 建立血液标本布鲁菌DNA荧光定量检测方法,探讨其临床应用价值.方法 用PUCm-T载体和PCR纯化产物连接,转染DH5a菌,筛选阳性菌落,提取质粒,制作外标准品;在布鲁菌基因组IS711序列设计一对引物和TaqMan MGB探针,严格优化反应物的组成和扩增条件,并对该方法进行评价.结果 建立的布鲁菌DNA荧光定量PCR方法最低检出率为400拷贝/ml;批内误差为1.8%~6.5%,批间误差为2.6%~9.1%;在4.0×102~4.0×108拷贝/ml之间与Ct值具有很好的线性(Ct =-1.391 1 Ln (x) +41.65,r=-0.995 8);具有较好的稳定性.在布鲁菌临床监测中发现,157份血液标本,用荧光定量PCR检测与临床检查结果(临床阳性、慢性期患者、症状可疑、阳性畜周围人群与重点职业人

  9. Cobas601与ARCHITECTi2000SR两种化学发光免疫检测系统的比对试验结果分析

    Institute of Scientific and Technical Information of China (English)

    赵子瑜

    2014-01-01

    目的:探讨两种Cobas601与ARCHITEC-Ti2000SR化学发光免疫检测系统结果的差别性和相关性。方法:每天常规标本先用ARCHITECTi2000SR系统检测后,选择在线性范围内的针对各项目不同浓度的标本,再用Co-bas601系统检测,每个试验项目选择20个不同浓度,2个月完成,并同时每天在两套系统上做高、中、低质控,然后选择ARCHITECTi2000SR作为参考仪器进行数据比对分析。结果:不同检测系统对佰乐不同浓度质控物的甲功项目(T3、FT3、T4,FT4,TSH,Anti-Tg,Anti-Tpo)、肿瘤项目(AFP,CEA, CA125、CA199、CA153、β-HCG、Ferritin、F-PSA、T-PSA)、性激素(Test、E2、LH、FSH、Prog、PRL)、胰岛素、C肽等24个项目测定结果日间CV及总CV均小于t±15%的日间CV允许范围。24个项目各自20个不同浓度在两个检测系统测定结果除了CA199无阳性及结果相关,Anti-Tg有阳性相关,无结果相关外,其余22个项目均有阳性和结果相关。结论:通过比对试验,可以发现不同检测系统之间的差异,保证同一实验室在检测同一项目时结果的延续性。

  10. Multilocus sequence typing of Mycoplasma hyorhinis strains identified by a real-time TaqMan PCR assay.

    Science.gov (United States)

    Tocqueville, Véronique; Ferré, Séverine; Nguyen, Ngoc Hong Phuc; Kempf, Isabelle; Marois-Créhan, Corinne

    2014-05-01

    A real-time TaqMan PCR assay based on the gene encoding the protein p37 was developed to detect Mycoplasma hyorhinis. Its specificity was validated with 29 epidemiologically unrelated M. hyorhinis strains (28 field strains and one reference strain) and other mycoplasma species or with other microorganisms commonly found in pigs. The estimated detection limit of this qPCR assay was 125 microorganism equivalents/μl. The same 29 epidemiologically unrelated M. hyorhinis strains and four previously fully sequenced strains were typed by two portable typing methods, the sequencing of the p37 gene and a multilocus sequence typing (MLST) scheme. The first method revealed 18 distinct nucleotide sequences and insufficient discriminatory power (0.934). The MLST scheme was developed with the sequenced genomes of the M. hyorhinis strains HUB-1, GDL-1, MCLD, and SK76 and based on the genes dnaA, rpoB, gyrB, gltX, adk, and gmk. In total, 2,304 bp of sequence was analyzed for each strain. MLST was capable of subdividing the 33 strains into 29 distinct sequence types. The discriminatory power of the method was >0.95, which is the threshold value for interpreting typing results with confidence (D=0.989). Population analysis showed that recombination in M. hyorhinis occurs and that strains are diverse but with a certain clonality (one unique clonal complex was identified). The new qPCR assay and the robust MLST scheme are available for the acquisition of new knowledge on M. hyorhinis epidemiology. A web-accessible database has been set up for the M. hyorhinis MLST scheme at http://pubmlst.org/mhyorhinis/.

  11. The potential of TaqMan Array Cards for detection of multiple biological agents by real-time PCR.

    Directory of Open Access Journals (Sweden)

    Phillip A Rachwal

    Full Text Available The TaqMan Array Card architecture, normally used for gene expression studies, was evaluated for its potential to detect multiple bacterial agents by real-time PCR. Ten PCR assays targeting five biological agents (Bacillus anthracis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, and Yersinia pestis were incorporated onto Array Cards. A comparison of PCR performance of each PCR in Array Card and singleplex format was conducted using DNA extracted from pure bacterial cultures. When 100 fg of agent DNA was added to Array Card channels the following levels of agent detection (where at least one agent PCR replicate returned a positive result were observed: Y. pestis 100%, B. mallei & F. tularensis 93%; B. anthracis 71%; B. pseudomallei 43%. For B. mallei & pseudomallei detection the BPM2 PCR, which detects both species, outperformed PCR assays specific to each organism indicating identification of the respective species would not be reproducible at the 100 fg level. Near 100% levels of detection were observed when 100 fg of DNA was added to each PCR in singleplex format with singleplex PCRs also returning sporadic positives at the 10 fg per PCR level. Before evaluating the use of Array Cards for the testing of environmental and clinical sample types, with potential levels of background DNA and PCR inhibitors, users would therefore have to accept a 10-fold reduction in sensitivity of PCR assays on the Array Card format, in order to benefit for the capacity to test multiple samples for multiple agents. A two PCR per agent strategy would allow the testing of 7 samples for the presence of 11 biological agents or 3 samples for 23 biological agents per card (with negative control channels.

  12. The predictive value of selected serum microRNAs for acute GVHD by TaqMan MicroRNA arrays.

    Science.gov (United States)

    Zhang, Chunyan; Bai, Nan; Huang, Wenrong; Zhang, Pengjun; Luo, Yuan; Men, Shasha; Wen, Ting; Tong, Hongli; Wang, Shuhong; Tian, Ya-Ping

    2016-10-01

    Currently, the diagnosis of acute graft-versus-host disease (aGVHD) is mainly based on clinical symptoms and biopsy results. This study was designed to further explore new no noninvasive biomarkers for aGVHD prediction/diagnosis. We profiled miRNAs in serum pools from patients with aGVHD (grades II-IV) (n = 9) and non-aGVHD controls (n = 9) by real-time qPCR-based TaqMan MicroRNA arrays. Then, predictive models were established using related miRNAs (n = 38) and verified by a double-blind trial (n = 54). We found that miR-411 was significantly down regulated when aGVHD developed and recovered when aGVHD was controlled, which demonstrated that miR-411 has potential as an indicator for aGVHD monitoring. We developed and validated a predictive model and a diagnostic model for aGVHD. The predictive model included two miRNAs (miR-26b and miR-374a), which could predict an increased risk for aGVHD 1 or 2 weeks in advance, with an AUC, Positive Predictive Value (PPV), and Negative Predictive Value (NPV) of 0.722, 76.19 %, and 69.70 %, respectively. The diagnostic model included three miRNAs (miR-28-5p, miR-489, and miR-671-3p) with an AUC, PPV, and NPV of 0.841, 85.71 % and 83.33 %, respectively. Our results show that circulating miRNAs (miR-26b and miR-374a, miR-28-5p, miR-489 and miR-671-3p) may serve as biomarkers for the prediction and diagnosis of grades II-IV aGVHD.

  13. Development of SYBR Green and TaqMan quantitative real-time PCR assays for hepatopancreatic parvovirus (HPV) infecting Penaeus monodon in India.

    Science.gov (United States)

    Yadav, Reena; Paria, Anutosh; Mankame, Smruti; Makesh, M; Chaudhari, Aparna; Rajendran, K V

    2015-12-01

    Hepatopancreatic parvovirus (HPV) infects Penaeus monodon and causes mortality in the larval stages. Further, it has been implicated in the growth retardation in cultured P. monodon. Though different geographical isolates of HPV show large sequence variations, a sensitive PCR assay specific to Indian isolate has not yet been reported. Here, we developed a sensitive SYBR Green-based and TaqMan real-time PCR for the detection and quantification of the virus. A 441-bp PCR amplicon was cloned in pTZ57 R/T vector and the plasmid copy number was estimated. A 10-fold serial dilution of the plasmid DNA from 1 × 10(9) copies to 1 copy was prepared and used as the standard. The primers were tested initially using the standard on a conventional PCR format to determine the linearity of detection. The standards were further tested on real-time PCR format using SYBR Green and TaqMan chemistry and standard curves were generated based on the Ct values from three well replicates for each dilution. The assays were found to be sensitive, specific and reproducible with a wide dynamic range (1 × 10(9) to 10 copies) with coefficient of regression (R(2)) > 0.99, calculated average slope -3.196 for SYBR Green assay whereas, for TaqMan assay it was >0.99 and -3.367, respectively. The intra- and inter-assay variance of the Ct values ranged from 0.26% to 0.94% and 0.12% to 0.81%, respectively, for SYBR Green assay, and the inter-assay variance of the Ct values for TaqMan assay ranged from 0.07% to 1.93%. The specificity of the assays was proved by testing other DNA viruses of shrimp such as WSSV, IHHNV and MBV. Standardized assays were further tested to detect and quantify HPV in the post-larvae of P. monodon. The result was further compared with conventional PCR to test the reproducibility of the test. The assay was also used to screen Litopeneaus vannamei, Macrobrachium rosenbergii and Scylla serrata for HPV. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Phylogeny of the genus Synchytrium and the development of TaqMan PCR assay for sensitive detection of Synchytrium endobioticum in soil.

    Science.gov (United States)

    Smith, Donna S; Rocheleau, Hélène; Chapados, Julie T; Abbott, Cathryn; Ribero, Sharon; Redhead, Scott A; Lévesque, C André; De Boer, Solke H

    2014-04-01

    Potato wart, caused by the fungal pathogen Synchytrium endobioticum, is a serious disease with the potential to cause significant economic damage. The small subunit (SSU) and internal transcribed spacer (ITS) ribosomal DNA (rDNA) were sequenced for several Synchytrium spp., showing a high rate of variability for both of these markers among the different species and monophyly of the genus within phylum Chytridiomycota. The intergenic nontranscribed spacer (IGS) of rDNA was sequenced for different pathotypes and showed no intraspecific variation within S. endobioticum, similar to the other rDNA markers from this study. To facilitate screening for the pathogen in soil, three TaqMan polymerase chain reaction (PCR) assays were developed from SSU, ITS, and IGS rDNA sequences to detect S. endobioticum sporangia in the chloroform-flotation fraction of sieved soil extracts. In the screening portion of the method, a first TaqMan assay targeting the SSU rDNA was developed with positive results that were further confirmed with amplicon melt analysis. A synthetic reaction control cloned into a plasmid was incorporated into the procedure, facilitating the validation of negative results. The presence of the reaction control did not adversely affect the efficiency of the SSU target amplification. A second TaqMan assay targeting the ITS-1 region was developed as a confirmatory test. There was 100% accordance between the SSU and ITS-1 TaqMan assays. Utilizing these two assays in tandem achieved good specificity for S. endobioticum, generating negative results with the cloned SSU and ITS-1 regions from all 14 other Synchytrium spp. considered. Spike recovery experiments indicated that these assays, targeting the SSU and ITS-1 rDNA regions, developed from a phylogeny dataset of the genus, could reliably detect a single sporangium in the chloroform flotation fraction of a soil extract. Good correlation between microscopic detection of sporangia and PCR results in both positive and

  15. Asymmetric real-time PCR and multiplex melting curve analysis with TaqMan probes for detecting PIK3CA mutations

    Directory of Open Access Journals (Sweden)

    Irina V. Botezatu

    2015-12-01

    Full Text Available The data in this article are related to the research article entitled “Optimization of melting analysis with TaqMan probes for detection of KRAS, NRAS, and BRAF mutations” Botezatu et al. [1]. Somatic mutations in the PIK3CA gene (“hot spots” in exons 9 and 20 are found in many human cancers, and their presence can determine prognosis and a treatment strategy. An effective method of mutation scanning PIK3CA in clinical laboratories is DNA Melting Analysis (DMA (Vorkas et al., 2010; Simi et al., 2008 [2,3]. It was demonstrated recently that the TaqMan probes which have been long used in Real Time PCR may also be utilized in DMA (Huang et al., 2011 [4]. After optimization of this method Botezatu et al. [1], it was used for multiplex scanning PIK3CA hotspot mutations in formalin-fixed paraffin-embedded (FFPE samples from patients with colorectal and lung cancer.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

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

  17. Development of an on-site rapid real-time polymerase chain reaction system and the characterization of suitable DNA polymerases for TaqMan probe technology.

    Science.gov (United States)

    Furutani, Shunsuke; Naruishi, Nahoko; Hagihara, Yoshihisa; Nagai, Hidenori

    2016-08-01

    On-site quantitative analyses of microorganisms (including viruses) by the polymerase chain reaction (PCR) system are significantly influencing medical and biological research. We have developed a remarkably rapid and portable real-time PCR system that is based on microfluidic approaches. Real-time PCR using TaqMan probes consists of a complex reaction. Therefore, in a rapid real-time PCR, the optimum DNA polymerase must be estimated by using actual real-time PCR conditions. In this study, we compared the performance of three DNA polymerases in actual PCR conditions using our rapid real-time PCR system. Although KAPA2G Fast HS DNA Polymerase has the highest enzymatic activity among them, SpeedSTAR HS DNA Polymerase exhibited better performance to rapidly increase the fluorescence signal in an actual real-time PCR using TaqMan probes. Furthermore, we achieved rapid detection of Escherichia coli in 7 min by using SpeedSTAR HS DNA Polymerase with the same sensitivity as that of a conventional thermal cycler.

  18. Bat white-nose syndrome: a real-time TaqMan polymerase chain reaction test targeting the intergenic spacer region of Geomyces destructanstructans.

    Science.gov (United States)

    Muller, Laura K.; Lorch, Jeffrey M.; Lindner, Daniel L.; O'Connor, Michael; Gargas, Andrea; Blehert, David S.

    2013-01-01

    The fungus Geomyces destructans is the causative agent of white-nose syndrome (WNS), a disease that has killed millions of North American hibernating bats. We describe a real-time TaqMan PCR test that detects DNA from G. destructans by targeting a portion of the multicopy intergenic spacer region of the rRNA gene complex. The test is highly sensitive, consistently detecting as little as 3.3 fg of genomic DNA from G. destructans. The real-time PCR test specifically amplified genomic DNA from G. destructans but did not amplify target sequence from 54 closely related fungal isolates (including 43 Geomyces spp. isolates) associated with bats. The test was further qualified by analyzing DNA extracted from 91 bat wing skin samples, and PCR results matched histopathology findings. These data indicate the real-time TaqMan PCR method described herein is a sensitive, specific, and rapid test to detect DNA from G. destructans and provides a valuable tool for WNS diagnostics and research.

  19. Assessing the utility of three TaqMan probes for the diagnosis of tuberculosis and resistance to rifampin and isoniazid in Veracruz, México.

    Science.gov (United States)

    Zenteno-Cuevas, Roberto; Cuevas-Cordoba, Betzaida; Enciso, Antonio; Enciso, Leonor; Cuellar, Aremy

    2012-03-01

    Mutations at codons 526 and 531 in the rpoB gene and at 315 in the katG gene are considered diagnostic markers for resistance to rifampin and isoniazid in tuberculosis. The aim of this study was to design and evaluate three TaqMan probes for the identification of these mutations in 138 respiratory samples positive for acid-fast bacilli, and 32 clinical isolates from a region with considerable levels of drug resistance. The specificities of the probes for the diagnosis of resistance to both drugs were 100%; however, the sensitivities were calculated to be 50% for isoniazid and 56% for rifampin. DNA sequencing of rpoB and katG; and the spoligotyping assay of the clinical isolates, confirmed the diversity of the mutations and the presence of 11 spoligotypes with a shared international type and eight unique spoligotypes. Analysis of the respiratory samples identified 22 (16%) as drug-resistant and 4 (3%) as multidrug-resistant tuberculosis. The diagnostic value of the TaqMan probes was compromised by the diversity of mutations found in the clinical isolates. This highlights the need for better understanding of the molecular mechanisms responsible for drug resistance prior to the use of molecular probes, especially in regions with significant levels of drug-resistant tuberculosis.

  20. Detection and measurement of benzimidazole resistance alleles in Haemonchus contortus using real-time PCR with locked nucleic acid Taqman probes.

    Science.gov (United States)

    Walsh, Thomas K; Donnan, Alison A; Jackson, Frank; Skuce, Philip; Wolstenholme, Adrian J

    2007-03-31

    Benzimidazole resistance is a common problem in parasitic nematodes of ruminants and early detection is vital if its spread is to be monitored and controlled. Real time PCR offers a fast and reliable method for rapid detection and measurement of resistance allele frequencies. In Haemonchus contortus a single nucleotide polymorphism at codon 200 of the beta-tubulin gene (TTC to TAC), causing a phenylalanine to tyrosine amino acid substitution, has been shown to be involved in many cases of resistance. Locked nucleic acid (LNA) Taqman probes have been used in this work to detect and measure the frequency of resistance alleles in individual and multiple H. contortus. Detection of resistant genotypes using LNA Taqman probes in individual H. contortus is simpler and more reliable than with previously described assays. Measurement of the frequency of resistant alleles in populations of H. contortus was achieved by using the cycle threshold (C(t)) values and a standard curve derived from populations with known allele frequencies. Results using the LNA probes on individual and multiple worms gave similar results to the allele specific PCR. The sensitivity of the LNA assay on multiple nematodes allowed reliable detection of > or = 10% resistance allele frequency. Using the final fluorescence method, it was possible to differentiate populations with approximately 0, 5 and 10% resistance allele frequencies.

  1. Quantitative duplex TaqMan real-time polymerase chain reaction for the assessment of the etiologic agent of epizootic bovine abortion.

    Science.gov (United States)

    Brooks, Roxann S; Blanchard, Myra T; Anderson, Mark L; Hall, Mark R; Stott, Jeffery L

    2011-11-01

    Epizootic bovine abortion (EBA), also commonly known as "foothill abortion," is a late-term abortion primarily in beef cattle with significant economic impacts in California, Nevada, and Oregon. The causative agent is a novel deltaproteobacterium (aoEBA) closely related to the order Myxococcales and vectored by the soft-shelled tick Ornithodoros coriaceus. Historically, diagnosis has relied upon the pathologic examination of the fetus and the presence of elevated fetal serum immunoglobulins. Identification of the etiologic agent, a unique deltaproteobacterium, permitted the development of a quantitative duplex real-time polymerase chain reaction (qPCR) using a unique 90-bp sequence of aoEBA 16S ribosomal RNA gene in conjunction with an 88-bp sequence of the bovine β-actin gene. Reaction efficiencies were 100.9% for the 16S aoEBA gene and 93.1% for the bovine β-actin gene. Application of the duplex TaqMan to a set of aoEBA-infected fetal bovine necropsy tissues demonstrated the assay to be robust in quantitatively identifying the aoEBA bacteria and establishing host-tissue pathogen load. Consistent with previously reported immunohistochemical data, organized lymphoid tissue generally carried the heaviest bacterial load as compared to non-lymphoid tissue. The newly developed duplex TaqMan assay will facilitate diagnosis in difficult cases and provide an invaluable tool for delineating the pathogenesis of EBA.

  2. TaqMan MGB探针实时荧光定量PCR快速检测布鲁氏菌%Development of a TaqMan MGB-probe based real-time fluorescencequantitative PCR assay for rapid detection of Brucella

    Institute of Scientific and Technical Information of China (English)

    高正琴; 邢进; 冯育芳; 岳秉飞; 贺争鸣

    2011-01-01

    The new generation TaqMan Minor Groove Binding (MGB) probe approach was used to develop the specific and sensitive real time fluorescence quantitative PCR (RTFQ-PCR) assay for rapid detecting Brucella in our study. The specific primers and probe for TaqMan MGB-probe based RTFQ-PCR were designed based on 16S rRNA sequence of genus Brucella. A TaqMan MGB-probe based RTFQ-PCR assay was established, and its specificity, sensitivity and stability were assessed. Then, the established TaqMan MGB-probe based RTFQ-PCR assay was applied to detect Brucella in 773 animal specimens during 2008 - 2010, and compared with conventional PCR assay. The specificity of this established TaqMan MGB-probe based RTFQ-PCR was high and there were no cross-reactivity with Yersinia enterocolitica , Yersinia pseudotuberculosis, Salmonella enterica, Escherichia colt, Pseudomonasaeruginosa , Campylobacter jejuni, and Clostridium piliforme. The correlation coefficient and slope value of standard curve were 0. 999 and -3. 301 respectively and the efficiency of TaqMan MGB-probe based RTFQ-PCR was 100.872%. The TaqMan MGB-probe based RTFQ-PCR assay was able to accurately detect Brucella DNA from brucellosis-positive specimens. The detection limit for this assay was 9. 3 copies, and the sensitivity of this assay was 100-fold higher than conventional PCR assay. The TaqMan MGB-probe based RTFQ-PCR was preformed to detect Brucella in 773 animal specimens, and a total of 53 specimens were positive for Brucella. However, there was only 37 specimens were positive by conventional PCR. The results showed that TaqMan MGB-probe based RTFQ-PCR for Brucella was more sensitive than conventional PCR assay, and it could detect Brucella DNA from animal specimens directly, and detection time is only 2 hours. To the knowledge of the authors, this is the first TaqMan MGB-probe based RTFQ-PCR assay for the direct detection of Brucella in animal specimens. The technique appears to be sufficiently adaptable to meet the

  3. Comparison of the Xpert MTB/RIF test with an IS6110-TaqMan real-time PCR assay for direct detection of Mycobacterium tuberculosis in respiratory and nonrespiratory specimens.

    Science.gov (United States)

    Armand, Sylvie; Vanhuls, Pascale; Delcroix, Guy; Courcol, René; Lemaître, Nadine

    2011-05-01

    The sensitivities of the Xpert MTB/RIF test and an in-house IS6110-based real-time PCR using TaqMan probes (IS6110-TaqMan assay) for the detection of Mycobacterium tuberculosis complex (MTBC) DNA were compared by use of 117 clinical specimens (97 culture positive and 20 culture negative for MTBC) that were frozen in sediment. The 97 clinical specimens included 60 respiratory and 37 nonrespiratory specimens distributed into 36 smear-positive and 61 smear-negative specimens. Among the 97 culture-positive specimens, 4 had rifampin-resistant isolates. Both methods were highly specific and exhibited excellent sensitivity (100%) with smear-positive specimens. The sensitivity of the Xpert MTB/RIF test with the whole smear-negative specimens was more reduced than that of the IS6110-TaqMan assay (48 versus 69%, P = 0.005). Both methods exhibited similar sensitivities with smear-negative respiratory specimens, but the Xpert MTB/RIF test had lower sensitivity with smear-negative nonrespiratory specimens than the IS6110-TaqMan assay (37 versus 71%, P = 0.013). Finally, the sensitivities of the Xpert MTB/RIF test and the IS6110-TaqMan assay were 79% and 84%, respectively, with respiratory specimens and 53% and 78%, respectively (P = 0.013), with nonrespiratory specimens. The Xpert MTB/RIF test correctly detected the rifampin resistance in smear-positive specimens but not in the one smear-negative specimen. The Xpert MTB/RIF test is a simple rapid method well adapted to a routine laboratory that appeared to be as sensitive as the IS6110-TaqMan assay with respiratory specimens but less sensitive with paucibacillary specimens, such as smear-negative nonrespiratory specimens.

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

    Directory of Open Access Journals (Sweden)

    Damodar Paudel

    2011-01-01

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

  5. Rapid quantification of hepatitis B virus DNA by real-time PCR using efficient TaqMan probe and extraction of virus DNA

    Institute of Scientific and Technical Information of China (English)

    Yan-Qin Lu; Jin-Xiang Han; Peng Qi; Wei Xu; Yan-Hui Zu; Bo Zhu

    2006-01-01

    AIM: To rapidly quantify hepatitis B virus (HBV) DNA by real-time PCR using efficient TaqMan probe and extraction methods of virus DNA.METHODS: Three standards were prepared by cloning PCR products which targeted S, C and X region of HBV genome into pGEM-T vector respectively. A pair of primers and matched TaqMan probe were selected by comparing the copy number and the Ct values of HBV serum samples derived from the three different standard curves using certain serum DNA. Then the efficiency of six HBV DNA extraction methods including guanidinium isothiocyanate, proteinase K, NaI, NaOH lysis, alkaline lysis and simple boiling was analyzed in sample A, B and C by real-time PCR. Meanwhile, 8 clinical HBV serum samples were quantified.RESULTS: The copy number of the same HBV serum sample originated from the standard curve of S, C and Xregions was 5.7 x 104/mL, 6.3 x 102/mL and 1.6 x 103/mL respectively. The relative Ct value was 26.6, 31.8 and 29.5 respectively. Therefore, primers and matched probe from S region were chosen for further optimization of six extraction methods. The copy number of HBV serum samples A, B and C was 3.49 x 109/mL, 2.08 x 106/mL and 4.40 x 107/mL respectively, the relative Ct value was 19.9, 30 and 26.2 in the method of NaOH lysis,which was the efficientest among six methods. Simple boiling showed a slightly lower efficiency than NaOH lysis. Guanidinium isothiocyanate, proteinase K and NaI displayed that the copy number of HBV serum sample A,B and C was around 105/mL, meanwhile the Ct value was about 30. Alkaline failed to quantify the copy number of three HBV serum samples. Standard deviation (SD) and coefficient variation (CV) were very low in all 8 clinical HBV serum samples, showing that quantification of HBV DNA in triplicate was reliable and accurate.CONCLUSION: Real-time PCR based on optimized primers and TaqMan probe from S region in combination with NaOH lysis is a simple, rapid and accurate method for quantification of HBV serum DNA.

  6. Is real-time PCR better than conventional PCR for Mycobacterium tuberculosis complex detection in clinical samples?

    Science.gov (United States)

    Tortoli, Enrico; Urbano, Pasquale; Marcelli, Fiorella; Simonetti, Tullia M; Cirillo, Daniela M

    2012-08-01

    Cobas Amplicor MTB and later Cobas TaqMan MTB were used to test a very large series of consecutive specimens received for tuberculosis diagnosis. Performance parameters were estimated and compared overall and for separate specimen categories. Both systems showed excellent specificity, and that of TaqMan was the higher. The sensitivities were similar but satisfactory only with respiratory specimens and smear-positive samples.

  7. Multicenter comparison study of both analytical and clinical performance across 4 Roche HCV RNA assays utilizing different platforms.

    Science.gov (United States)

    Vermehren, Johannes; Stelzl, Evelyn; Maasoumy, Benjamin; Michel-Treil, Veronique; Berkowski, Caterina; Marins, Ed G; Paxinos, Ellen E; Marino, Enrique; Wedemeyer, Heiner; Sarrazin, Christoph; Kessler, Harald H

    2017-01-25

    Antiviral treatment efficacy for chronic HCV infection is determined based on measurement of HCV RNA at specific time points throughout therapy by highly sensitive and accurate HCV RNA assays. This study evaluated the performance of two recently-developed real-time PCR HCV RNA assays, the cobas HCV for use on the cobas 6800/8800 systems (cobas 6800/8800 HCV) and the cobas HCV for use on the cobas 4800 system (cobas 4800 HCV) in comparison to two established assays, the COBAS AmpliPrep/COBAS TaqMan HCV Quantitative Test, version 2.0 (CAP/CTM v2) and the COBAS TaqMan HCV Test, version 2.0 for use with the High Pure System (HPS/CTM v2). Limit of detection (LOD) and linearity at lower concentrations (5-1000 IU/mL) were assessed for cobas 6800/8800 HCV and cobas 4800 HCV using WHO standard traceable panels representing HCV genotypes (GT) 1-4. Pairwise assay comparisons were also performed using 245 clinical samples representing HCV GT 1-4.cobas 6800/8800 HCV and cobas 4800 HCV were linear at low HCV RNA concentrations (<0.3 log10 IU/mL difference between expected and observed results) with LOD of 8.2 IU/mL and 11.7 IU/mL, respectively, for GT1. The new assays showed excellent agreement with CAP/CTM v2 and HPS/CTM v2 results in samples with quantifiable viral load. Concordance across the 6 million IU/mL cutoff was high among all four assays (90-94%). In conclusion, both cobas 6800/8800 HCV and cobas 4800 HCV tests are sensitive and linear, and correlate well with established Roche assays used in clinical practice.

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

    Science.gov (United States)

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

    2017-06-01

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

  9. TaqMan real-time PCR assays to assess arbuscular mycorrhizal responses to field manipulation of grassland biodiversity: effects of soil characteristics, plant species richness, and functional traits.

    Science.gov (United States)

    König, Stephan; Wubet, Tesfaye; Dormann, Carsten F; Hempel, Stefan; Renker, Carsten; Buscot, François

    2010-06-01

    Large-scale (temporal and/or spatial) molecular investigations of the diversity and distribution of arbuscular mycorrhizal fungi (AMF) require considerable sampling efforts and high-throughput analysis. To facilitate such efforts, we have developed a TaqMan real-time PCR assay to detect and identify AMF in environmental samples. First, we screened the diversity in clone libraries, generated by nested PCR, of the nuclear ribosomal DNA internal transcribed spacer (ITS) of AMF in environmental samples. We then generated probes and forward primers based on the detected sequences, enabling AMF sequence type-specific detection in TaqMan multiplex real-time PCR assays. In comparisons to conventional clone library screening and Sanger sequencing, the TaqMan assay approach provided similar accuracy but higher sensitivity with cost and time savings. The TaqMan assays were applied to analyze the AMF community composition within plots of a large-scale plant biodiversity manipulation experiment, the Jena Experiment, primarily designed to investigate the interactive effects of plant biodiversity on element cycling and trophic interactions. The results show that environmental variables hierarchically shape AMF communities and that the sequence type spectrum is strongly affected by previous land use and disturbance, which appears to favor disturbance-tolerant members of the genus Glomus. The AMF species richness of disturbance-associated communities can be largely explained by richness of plant species and plant functional groups, while plant productivity and soil parameters appear to have only weak effects on the AMF community.

  10. Detection of Jaagsiekte sheep retrovirus in apparently healthy sheep by real-time TaqMan PCR in comparison with histopathological findings

    Directory of Open Access Journals (Sweden)

    Bahari Aliasghar

    2016-03-01

    Full Text Available Introduction: The aim of this study was to use TaqMan real-time PCR technique to investigate Jaagsiekte sheep retrovirus (JSRV proviral DNA in whole blood samples of sheep, and compare the results to those of histopathological examinations. Material and Methods: Eighty blood samples from clinically healthy sheep were randomly collected before the animals were slaughtered. Ten tissue samples from each lung and associated caudal mediastinal lymph node were taken. Results: Fifteen (18.75% blood samples were found to contain proviral DNA, and 11 (13.75% corresponding lung samples showed microscopic changes consistent with ovine pulmonary adenocarcinoma. None of the samples displayed metastases to the caudal mediastinal lymph nodes. The prominent pattern of neoplastic nodules consisted of acinar (alveolar form. Conclusion: The results indicated the higher sensitivity of real-time PCR compared to histopathological examinations in detection of ovine pulmonary adenocarcinoma.

  11. Improved Safety for Molecular Diagnosis of Classical Rabies Viruses by Use of a TaqMan Real-Time Reverse Transcription-PCR "Double Check" Strategy

    DEFF Research Database (Denmark)

    Hoffmann, B.; Freuling, C. M.; Wakeley, P. R.

    2010-01-01

    by a combined assay that detected all samples as positive. In addition, the introduction of labeled positive controls (LPC) increased the diagnostic safety of the single as well as the combined assay. Based on the newly developed, alternative assay for the detection of rabies virus and the application of LPCs......To improve the diagnosis of classical rabies virus with molecular methods, a validated, ready-to-use, real-time reverse transcription-PCR (RT-PCR) assay was developed. In a first step, primers and 6-carboxyfluorescien-labeled TaqMan probes specific for rabies virus were selected from the consensus...... sequence of the nucleoprotein gene of 203 different rabies virus sequences derived from GenBank. The selected primer-probe combination was highly specific and sensitive. During validation using a sample set of rabies virus strains from the virus archives of the Friedrich-Loeffler-Institut (FLI; Germany...

  12. 应用TaqMan荧光定量PCR检测牛分枝杆菌%Detection of Mycobacterium bovis with TaqMan real-time PCR

    Institute of Scientific and Technical Information of China (English)

    王春雨; 王振国; 刘金华; 宋战昀; 周亮; 高宏伟; 王全凯

    2011-01-01

    为建立快速检测牛分枝杆菌(M.bovis)的TaqMan荧光定量PCR方法,本研究以GenBank登录的M.bovis特有229 bp基因为研究对象,设计并合成引物及探针.该方法具有较好的特异性,与标准质控菌株呈阳性反应,与其他微生物样品呈阴性反应;灵敏性最低检测值可达1 pg/mL;对20阳性临床样品进行荧光定量PCR检测,均为阳性;而对培养为阴性的20份临床样品进行检测,6份为阳性.该研究结果表明,建立的方法特异性强,敏感性高,稳定性好,能够用于M.bovis的鉴别检测,对牛分枝杆菌病的快速检测和早期诊断具有重要意义.%A TaqMan real-time PCR assay was developed for detection of Mycobacterium bovis infection in cattle based on primers and TaqMan probe derived from M. bovis sequence in GenBank. The specific test showed that the assay had positive results for detection of M. bovis strains and negative for other bacteria. This assay could detect single bacteria. Comparing with other methed on 20 PPD positive clinical samples which were negative by bacteria isolation, six samples were positive detected by the real-time PCR. Indicating the real-time PCR is a rapid and specific assay for detection of M. bovis infection and could be used in the early diagnosis of bovine tuberculosis.

  13. TaqMan real-time PCR assays for single-nucleotide polymorphisms which identify Francisella tularensis and its subspecies and subpopulations.

    Directory of Open Access Journals (Sweden)

    Dawn N Birdsell

    Full Text Available Francisella tularensis, the etiologic agent of tularemia and a Class A Select Agent, is divided into three subspecies and multiple subpopulations that differ in virulence and geographic distribution. Given these differences, there is a need to rapidly and accurately determine if a strain is F. tularensis and, if it is, assign it to subspecies and subpopulation. We designed TaqMan real-time PCR genotyping assays using eleven single nucleotide polymorphisms (SNPs that were potentially specific to closely related groups within the genus Francisella, including numerous subpopulations within F. tularensis species. We performed extensive validation studies to test the specificity of these SNPs to particular populations by screening the assays across a set of 565 genetically and geographically diverse F. tularensis isolates and an additional 21 genetic near-neighbor (outgroup isolates. All eleven assays correctly determined the genetic groups of all 565 F. tularensis isolates. One assay differentiates F. tularensis, F. novicida, and F. hispaniensis from the more genetically distant F. philomiragia and Francisella-like endosymbionts. Another assay differentiates F. tularensis isolates from near neighbors. The remaining nine assays classify F. tularensis-confirmed isolates into F. tularensis subspecies and subpopulations. The genotyping accuracy of these nine assays diminished when tested on outgroup isolates (i.e. non F. tularensis, therefore a hierarchical approach of assay usage is recommended wherein the F. tularensis-specific assay is used before the nine downstream assays. Among F. tularensis isolates, all eleven assays were highly sensitive, consistently amplifying very low concentrations of DNA. Altogether, these eleven TaqMan real-time PCR assays represent a highly accurate, rapid, and sensitive means of identifying the species, subspecies, and subpopulation of any F. tularensis isolate if used in a step-wise hierarchical scheme. These assays

  14. Universal detection of hepatitis E virus by two real-time PCR assays: TaqMan and Primer-Probe Energy Transfer.

    Science.gov (United States)

    Gyarmati, Péter; Mohammed, Nahla; Norder, Helene; Blomberg, Jonas; Belák, Sándor; Widén, Frederik

    2007-12-01

    Hepatitis E virus (HEV) is a major cause of food- and waterborne diseases in countries with poor sanitation. Furthermore, travellers to such countries are also at risk of contracting the virus. Noteworthily, during the last decade an increasing number of non-travel-related cases were recorded even in countries with high sanitary standards. An alternative, direct route of infection, from animals to humans (zoonotic transmission) is suspected to be the cause of recent cases of hepatitis E. In order to provide rapid and sensitive methods for detecting the virus in various hosts, two real-time PCR methods were developed and compared: a TaqMan and Primer-Probe Energy Transfer (PriProET) assay. These highly sensitive novel methods provide valuable diagnostic tools to investigate zoonotic transmission, to detect the virus in the food chain and in research related to the potential of hepatitis E virus to cross the species barrier. The results show that the two novel PCR assays are robust, highly sensitive and specific for broad range detection of the four genotypes of HEV. Compared to PriProET, the TaqMan assay appears to perform slightly better, with higher fluorescence values for positive samples. However, the PriProET has the benefit of better tolerating the point mutations in the target nucleic acids. Thus, it provides a more powerful tool to detect new virus variants. These new molecular diagnostic assays are practical tools that can be employed in the area of public health, for disease diagnosis and for tracking outbreaks. In basic research the methods provide new tools to study HEV biology, including virus-host interactions and transmission between various host species.

  15. Utility of IgM ELISA, TaqMan real-time PCR, reverse transcription PCR, and RT-LAMP assay for the diagnosis of Chikungunya fever.

    Science.gov (United States)

    Reddy, Vijayalakshmi; Ravi, Vasanthapuram; Desai, Anita; Parida, Manmohan; Powers, Ann M; Johnson, Barbara W

    2012-11-01

    Chikungunya fever a re-emerging infection with expanding geographical boundaries, can mimic symptoms of other infections like dengue, malaria which makes the definitive diagnosis of the infection important. The present study compares the utility of four laboratory diagnostic methods viz. IgM capture ELISA, an in house reverse transcription PCR for the diagnosis of Chikungunya fever, TaqMan real-time PCR, and a one step reverse transcription-loop mediated isothermal amplification assay (RT-LAMP). Out of the 70 serum samples tested, 29 (41%) were positive for Chikungunya IgM antibody by ELISA and 50 (71%) samples were positive by one of the three molecular assays. CHIKV specific nucleic acid was detected in 33/70 (47%) by reverse transcription PCR, 46/70 (66%) by TaqMan real-time PCR, and 43/70 (62%) by RT-LAMP assay. A majority of the samples (62/70; 89%) were positive by at least one of the four assays used in the study. The molecular assays were more sensitive for diagnosis in the early stages of illness (2-5 days post onset) when antibodies were not detectable. In the later stages of illness, the IgM ELISA is a more sensitive diagnostic test. In conclusion we recommend that the IgM ELISA be used as an initial screening test followed one of the molecular assays in samples that are collected in the early phase of illness and negative for CHIKV IgM antibodies. Such as approach would enable rapid confirmation of the diagnosis and implementation of public health measures especially during outbreaks. Copyright © 2012 Wiley Periodicals, Inc.

  16. Development of a TaqMan Allelic Discrimination Assay for detection of Single Nucleotides Polymorphisms associated with anti-malarial drug resistance

    Directory of Open Access Journals (Sweden)

    Kamau Edwin

    2012-01-01

    Full Text Available Abstract Background Anti-malarial drug resistance poses a threat to current global efforts towards control and elimination of malaria. Several methods are used in monitoring anti-malarial drug resistance. Molecular markers such as single nucleotide polymorphism (SNP for example are increasingly being used to identify genetic mutations related to anti-malarial drug resistance. Several methods are currently being used in analysis of SNP associated with anti-malarial drug resistance and although each one of these methods has unique strengths and shortcoming, there is still need to improve and/or develop new methods that will close the gap found in the current methods. Methods TaqMan Allelic Discrimination assays for detection of SNPs associated with anti-malarial drug resistance were designed for analysis on Applied Biosystems PCR platform. These assays were designed by submitting SNP sequences associated with anti-malarial drug resistance to Applied Biosystems website. Eleven SNPs associated with resistance to anti-malarial drugs were selected and tested. The performance of each SNP assay was tested by creating plasmid DNAs carrying codons of interests and analysing them for analysis. To test the sensitivity and specificity of each SNP assay, 12 clinical samples were sequenced at codons of interest and used in the analysis. Plasmid DNAs were used to establish the Limit of Detection (LoD for each assay. Results Data from genetic profiles of the Plasmodium falciparum laboratory strains and sequence data from 12 clinical samples was used as the reference method with which the performance of the SNP assays were compared to. The sensitivity and specificity of each SNP assay was establish at 100%. LoD for each assay was established at 2 GE, equivalent to less than 1 parasite/μL. SNP assays performed well in detecting mixed infection and analysis of clinical samples. Conclusion TaqMan Allelic Discrimination assay provides a good alternative tool in

  17. Quantification of CD147/basigin Splicing Variants by Taqman qPCR in Epithelium Ovarian Cancer%Taqman qPCR检测CD147/Basigin剪接变异体在人上皮性卵巢癌中的表达

    Institute of Scientific and Technical Information of China (English)

    赵淑华; 杨红; 陈必良; 姚念玲; 康卫卫

    2013-01-01

    目的:应用Taqman qPCR技术检测CD147/basigin剪接变异体在人上皮性卵巢癌组织与正常卵巢组织中的表达差异.方法:运用半定量RT-PCR技术检测CD 147/basigin剪接变异体在上皮性卵巢癌细胞系中的表达;Taqman qPCR检测CD 147/basigin剪接变异体在人上皮性卵巢癌细胞系中的表达分布;进一步通过收集32例上皮性卵巢癌组织与26例正常卵巢组织,提取组织RNA,反转录cDNA,Taqman qPCR检测CD147/basigin剪接变异体mRNA在上皮性卵巢癌组织与正常卵巢组织中的表达差异.结果:半定量RT-PCR结果显示basigin-2,basigin-3和basigin-4在上皮性卵巢癌细胞系中均有表达,主要以basigin-2为主;Taqman qPCR检测到三种剪接变异体在不同卵巢癌细胞系中表达不同,basigin-2在卵巢癌细胞系中较basigin-3,basigin-4表达较高,basigin-4较basigin-3略高;Basigin-2剪接变异体在高转移Ho-8910pm细胞中表达较高,在低转移HO-8910细胞中表达较低.组织Taqman qPCR检测basigin-2和basigin-4在上皮性卵巢癌组织中的表达水平显著高于正常卵巢组织(P值分别为<0.0001和0.0261),basigin-3的表达水平略有升高(P=0.2616),但无统计学意义.结论:三种剪接变异体在卵巢癌组织中较正常卵巢组织表达上调.CD147/basigin-2在高转移卵巢癌细胞系HO-8910pm中高表达,在低转移卵巢癌细胞系HO-8910中低表达,且表达强度与上皮性卵巢癌的转移相关;探讨CD 147/basigin-2在上皮性卵巢癌中的高表达,为卵巢癌的进一步治疗开辟一新途径.

  18. 登革病毒Taqman双重荧光PCR分型研究%Typing of dengue virus Taqman with double real-time PCR

    Institute of Scientific and Technical Information of China (English)

    董瑞玲; 甄胜西; 孙杰; 李微; 王佃鹏; 徐媛; 朱玉兰

    2011-01-01

    目的 建立鉴定登革病毒型别的双重实时Taqman PCR反应体系,以准确快速鉴定登革病毒型别.方法 根据GenBank上已发表的登革病毒四个型别的全基因序列,进行对比分析,分别设计登革病毒的四个型别引物和探针,登革Ⅰ、Ⅲ型探针用FAM-TAMARA标记,登革Ⅱ、Ⅳ型探针用JOE-TAMARA标记.经过条件优化后,建立检测登革病毒Ⅰ/Ⅱ型和Ⅲ/Ⅳ型的两套双重实时荧光RT-PCR方法,扩增四型登革病毒RNA、登革病毒阴性样本和登革病毒RNA稀释样本,检测方法的特异性、重复性和检测限性.结果 通过设计筛选序列和优化反应条件,建立登革病毒Ⅰ、Ⅱ型和登革病毒Ⅲ、Ⅳ型的双重荧光PCR反应体系,通过试验证明,所建立的方法具有良好的特异性、重复性和检测限性,能准确快速地对登革病毒进行分型.结论 建立了一种快速双重荧光PCR方法能同时对登革病毒进行分型和鉴定.%Objective To establishing a multiplex real-time Taqman PCR method to quickly and correctly identify dengue virus type. Methods According to the gene sequences of the four dengue types from the GenBank.four series of dengue virus type-specified primers and probes were designed. Dengue virus I and E's probes were labelled with FAM-TAMARA,while dengue virus II and IV's probes were labelled with JOE-TAMARA. The reaction condition was optimized. Two multiplex real-time PCR methods were established to identify dengue virus I and II and dengue virus III and IV accordingly. Four type dengue virus RNA,dengue virus negative samples,dengue virus RNA diluted samples were tested to identify the specificity,reproducibility and sensitivity of the method. Results The dengue virus I and II(dengue virus HI and IV)multiplex typing real-time PCR method could quicky identify dengue virus type witjh good specificity,reproducibility and sensitivity. Conclusion A multiplex real-time Taqman PCR method were established that can quickly

  19. A new molecular approach to help conclude drowning as a cause of death: simultaneous detection of eight bacterioplankton species using real-time PCR assays with TaqMan probes.

    Science.gov (United States)

    Uchiyama, Taketo; Kakizaki, Eiji; Kozawa, Shuji; Nishida, Sho; Imamura, Nahoko; Yukawa, Nobuhiro

    2012-10-10

    We developed a novel tool for concluding drowning as a cause of death. We designed nine primer pairs to detect representative freshwater or marine bacterioplankton (aquatic bacteria) and then used real-time PCR with TaqMan probes to rapidly and specifically detect them. We previously cultured the genus Aeromonas, which is a representative freshwater bacterial species, in blood samples from 94% of victims who drowned in freshwater and the genera Vibrio and/or Photobacterium that are representative marine bacteria in 88% of victims who drowned in seawater. Based on these results, we simultaneously detected eight species of bacterioplankton (Aeromonas hydrophila, A. salmonicida; Vibrio fischeri, V. harveyi, V. parahaemolyticus; Photobacterium damselae, P. leiognathi, P. phosphoreum) using three sets of triplex real-time PCR assays and TaqMan probes labelled with fluorophores (FAM, NED, Cy5). We assayed 266 specimens (109 blood, 157 tissues) from 43 victims, including 32 who had drowned in rivers, ditches, wells, sea or around estuaries. All lung samples of these 32 victims were TaqMan PCR-positive including the lung periphery into which water does not readily enter postmortem. On the other hand, findings in blood and/or closed organs (kidney or liver) were PCR-positive in 84% of the drowned victims (except for those who drowned in baths) although the conventional test detected diatoms in closed organs in only 44% of the victims. Thus, the results of the PCR assay reinforced those of diatom tests when only a few diatoms were detectable in organs due to the low density of diatoms in the water where they were found. Multiplex TaqMan PCR assays for bacterioplankton were rapid, less laborious and high-throughput as well as sensitive and specific. Therefore, these assays would be useful for routine forensic screening tests to estimate the amount and type of aspirated water.

  20. Development of an mlrA Gene-Directed TaqMan PCR Assay for Quantitative Assessment of Microcystin-Degrading Bacteria within Water Treatment Plant Sand Filter Biofilms▿

    Science.gov (United States)

    Hoefel, Daniel; Adriansen, Caroline M. M.; Bouyssou, Magali A. C.; Saint, Christopher P.; Newcombe, Gayle; Ho, Lionel

    2009-01-01

    We report for the first time a quantitative mlrA gene-directed TaqMan PCR assay for the rapid detection of microcystin-degrading bacteria. This was applied, in combination with 16S ribosomal DNA-directed quantitative PCR and denaturing gradient gel electrophoresis, to study virgin sand filter column biofilm development and to correlate mlrA gene abundance with microcystin removal efficiency. PMID:19502429

  1. Application of TaqMan qPCR for the detection and monitoring of Naegleria species in reservoirs used as a source for drinking water.

    Science.gov (United States)

    Kao, Po-Min; Hsu, Bing-Mu; Hsu, Tsui-Kang; Chiu, Yi-Chou; Chang, Chung-Liang; Ji, Wen-Tsai; Huang, Shih-Wei; Fan, Cheng-Wei

    2014-10-01

    Naegleria spp. can be found in the natural aquatic environments. Naegleria fowleri can cause fatal infections in the central nervous system in humans and animals, and the most important source of infection is through direct water contact. In this study, PCR of 5.8S ribosomal RNA (rRNA) gene and internal transcribed spacer (ITS) region was performed in order to identify Naegleria isolates and quantify the Naegleria spp. by TaqMan real-time quantitative PCR in reservoir water samples. The occurrence of Naegleria spp. was investigated in 57 water samples from reservoirs with culture and PCR positive in 2 of them (3.5%), respectively. The total detection rate was 7.0% (4/ 57) for Naegleria spp. The identified species included Naegleria spp., Naegleria canariensis, and Naegleria clarki. N. fowleri was not found in Taiwan's reservoirs used for drinking purposes. The concentrations of Naegleria spp. in detected positive reservoir water samples were in the range of 599 and 3.1 × 10(3) cells/L. The presence or absence of Naegleria spp. within the reservoir water samples showed significant difference with the levels of water temperature. The presence of Naegleria spp. in reservoirs considered a potential public health threat if pathogenic species exist in reservoirs.

  2. Comparison of the ABI 7700 System (TaqMan) and Competitive PCR for Quantification of IS6110 DNA in Sputum during Treatment of Tuberculosis

    Science.gov (United States)

    Desjardin, L. e.; Chen, Y.; Perkins, M. D.; Teixeira, L.; Cave, M. D.; Eisenach, K. D.

    1998-01-01

    Mycobacterium tuberculosis can persist in sputum for long periods of time after the initiation of antituberculosis chemotherapy. The purpose of this study was to determine whether quantitative estimates of M. tuberculosis DNA in sputum correlate with the numbers of viable bacilli and thus measure the therapeutic response of patients during treatment. Two methods of M. tuberculosis DNA quantification were examined by using DNA isolated from sputum specimens serially collected during the course of chemotherapy. A competitive PCR assay was compared to an automated system of real-time quantification with the ABI Prism 7700 Sequence Detection System (TaqMan). The ABI 7700 system uses standard PCR in conjunction with a fluorogenic probe in which the intensity of fluorescence is proportional to the amount of target DNA present. The results showed that both PCR systems are reproducible and accurate. The amounts of M. tuberculosis DNA quantified in sputum corresponded well with the numbers of acid-fast bacilli (AFB) counted by microscopy. Before initiation of antituberculosis therapy, measures of AFB, M. tuberculosis DNA, and cultivable bacilli were similar, suggesting that quantification of DNA is a good method for measuring the initial bacillary load. However, the rate of disappearance of both AFB and M. tuberculosis DNA did not correlate with the decline in cultivable bacilli in the specimen; therefore, these tests are not appropriate for monitoring treatment efficacy. PMID:9650945

  3. Establishment and Application of a TaqMan Real-Time Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Rubella Virus RNA

    Institute of Scientific and Technical Information of China (English)

    Li-Hong ZHAO; Yu-Yan MA; Hong WANG; Shu-Ping ZHAO; Wei-Ming ZHAO; Hua LI; Lei-Yi WANG

    2006-01-01

    The aim of this study was to establish and apply a real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) for rubella virus (RV) RNA. First, the primer and TaqMan probe concentrations, as well as reaction temperatures were optimized to establish an efficient real-time quantitative RT-PCR assay for RV RNA. Next, an RV-specific PCR amplicon was made as an external standard to estimate the linearity, amplification efficiency, analytical sensitivity and reproducibility of the real time quantitative assay. Finally, the assay was applied to quantify RVRNA in clinical samples for rubella diagnosis.The RV-specific PCR amplicon was prepared for evaluation of the assay at 503 bp, and its original concentration was 2.75×109 copies/μl. The real time quantitative assay was shown to have good linearity (R2=0.9920), high amplification efficiency (E=1.91), high sensitivity (275 copies/ml), and high reproducibility (variation coefficient range, from 1.25% to 3.58%). Compared with the gold standard, the specificity and sensitivity of the assay in clinical samples was 96.4% and 86.4%, respectively. Therefore, the established quantitative RT-PCR method is a simple, rapid, less-labored, quantitative, highly specific and sensitive assay for RV RNA.

  4. Rapid and Quantitative Detection of Leifsonia xyli subsp. xyli in Sugarcane Stalk Juice Using a Real-Time Fluorescent (TaqMan) PCR Assay

    Science.gov (United States)

    Fu, Hua-Ying; Sun, Sheng-Ren; Wang, Jin-Da; Ahmad, Kashif; Wang, Heng-Bo; Chen, Ru-Kai

    2016-01-01

    Ratoon stunting disease (RSD) of sugarcane, one of the most important diseases seriously affecting the productivity of sugarcane crops, was caused by the bacterial agent Leifsonia xyli subsp. xyli (Lxx). A TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR) assay was established in this study for the quantification of Lxx detection in sugarcane stalk juice. A pair of PCR primers (Pat1-QF/Pat1-QR) and a fluorogenic probe (Pat1-QP) targeting the Part1 gene of Lxx were used for the qPCR assay. The assay had a detection limit of 100 copies of plasmid DNA and 100 fg of Lxx genomic DNA, which was 100-fold more sensitive than the conventional PCR. Fifty (28.7%) of 174 stalk juice samples from two field trials were tested to be positive by qPCR assay, whereas, by conventional PCR, only 12.1% (21/174) were tested to be positive with a published primer pair CxxITSf#5/CxxITSr#5 and 15.5% (27/174) were tested to be positive with a newly designed primer pair Pat1-F2/Pat1-R2. The new qPCR assay can be used as an alternative to current diagnostic methods for Lxx, especially when dealing with certificating a large number of healthy cane seedlings and determining disease incidence accurately in commercial fields.

  5. Rapid and Quantitative Detection of Leifsonia xyli subsp. xyli in Sugarcane Stalk Juice Using a Real-Time Fluorescent (TaqMan PCR Assay

    Directory of Open Access Journals (Sweden)

    Hua-Ying Fu

    2016-01-01

    Full Text Available Ratoon stunting disease (RSD of sugarcane, one of the most important diseases seriously affecting the productivity of sugarcane crops, was caused by the bacterial agent Leifsonia xyli subsp. xyli (Lxx. A TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR assay was established in this study for the quantification of Lxx detection in sugarcane stalk juice. A pair of PCR primers (Pat1-QF/Pat1-QR and a fluorogenic probe (Pat1-QP targeting the Part1 gene of Lxx were used for the qPCR assay. The assay had a detection limit of 100 copies of plasmid DNA and 100 fg of Lxx genomic DNA, which was 100-fold more sensitive than the conventional PCR. Fifty (28.7% of 174 stalk juice samples from two field trials were tested to be positive by qPCR assay, whereas, by conventional PCR, only 12.1% (21/174 were tested to be positive with a published primer pair CxxITSf#5/CxxITSr#5 and 15.5% (27/174 were tested to be positive with a newly designed primer pair Pat1-F2/Pat1-R2. The new qPCR assay can be used as an alternative to current diagnostic methods for Lxx, especially when dealing with certificating a large number of healthy cane seedlings and determining disease incidence accurately in commercial fields.

  6. Development of a Taqman real-time PCR assay for rapid detection and quantification of Vibrio tapetis in extrapallial fluids of clams

    Directory of Open Access Journals (Sweden)

    Adeline Bidault

    2015-12-01

    Full Text Available The Gram-negative bacterium Vibrio tapetis is known as the causative agent of Brown Ring Disease (BRD in the Manila clam Venerupis (=Ruditapes philippinarum. This bivalve is the second most important species produced in aquaculture and has a high commercial value. In spite of the development of several molecular methods, no survey has been yet achieved to rapidly quantify the bacterium in the clam. In this study, we developed a Taqman real-time PCR assay targeting virB4 gene for accurate and quantitative identification of V. tapetis strains pathogenic to clams. Sensitivity and reproducibility of the method were assessed using either filtered sea water or extrapallial fluids of clam injected with the CECT4600T V. tapetis strain. Quantification curves of V. tapetis strain seeded in filtered seawater (FSW or extrapallial fluids (EF samples were equivalent showing reliable qPCR efficacies. With this protocol, we were able to specifically detect V. tapetis strains down to 1.125 101 bacteria per mL of EF or FSW, taking into account the dilution factor used for appropriate template DNA preparation. This qPCR assay allowed us to monitor V. tapetis load both experimentally or naturally infected Manila clams. This technique will be particularly useful for monitoring the kinetics of massive infections by V. tapetis and for designing appropriate control measures for aquaculture purposes.

  7. Development of a TaqMan Probe-Based Insulated Isothermal Polymerase Chain Reaction (iiPCR) Assay for Detection of Fusarium oxysporum f. sp. cubense Race 4

    Science.gov (United States)

    Lin, Yi-Jia; Chang, Tsai-De; Hong, Li-Ling; Chen, Tzu-Yu; Chang, Pi-Fang Linda

    2016-01-01

    This study developed a novel and inexpensive detection method based on a TaqMan probe-based insulated isothermal polymerase chain reaction (iiPCR) method for the rapid detection of Panama disease caused by Fusarium oxysporum f. sp. cubense (Foc) race 4, which is currently among the most serious fungal vascular diseases worldwide. By using the portable POCKIT™ device with the novel primer set iiFoc-1/iiFoc-2, the Foc race 4 iiPCR assay (including DNA amplification and signal monitoring) could be completed within one hour. The developed Foc race 4 iiPCR assay is thus a user-friendly and efficient platform designed specifically for the detection of Foc race 4. The detection limit of this optimized Foc iiPCR system was estimated to be 1 copy of the target standard DNA as well as 1 fg of the Foc genomic DNA. This approach can serve as a rapid detection method for in planta detection of Foc race 4 in field-infected banana. It was concluded that this molecular detection procedure based on iiPCR has good potential for use as an efficient detection method. PMID:27448242

  8. Development of a TaqMan Probe-Based Insulated Isothermal Polymerase Chain Reaction (iiPCR Assay for Detection of Fusarium oxysporum f. sp. cubense Race 4.

    Directory of Open Access Journals (Sweden)

    Ying-Hong Lin

    Full Text Available This study developed a novel and inexpensive detection method based on a TaqMan probe-based insulated isothermal polymerase chain reaction (iiPCR method for the rapid detection of Panama disease caused by Fusarium oxysporum f. sp. cubense (Foc race 4, which is currently among the most serious fungal vascular diseases worldwide. By using the portable POCKIT™ device with the novel primer set iiFoc-1/iiFoc-2, the Foc race 4 iiPCR assay (including DNA amplification and signal monitoring could be completed within one hour. The developed Foc race 4 iiPCR assay is thus a user-friendly and efficient platform designed specifically for the detection of Foc race 4. The detection limit of this optimized Foc iiPCR system was estimated to be 1 copy of the target standard DNA as well as 1 fg of the Foc genomic DNA. This approach can serve as a rapid detection method for in planta detection of Foc race 4 in field-infected banana. It was concluded that this molecular detection procedure based on iiPCR has good potential for use as an efficient detection method.

  9. Development and validation of a range of endogenous controls to support the implementation of practical Taqman real-time PCR-based surveillance for fish diseases within aquaculture.

    Science.gov (United States)

    Bland, F; McIntosh, R; Bain, N; Snow, M

    2012-06-01

    The use of Taqman real-time PCR-based technology has recently become more frequent in the detection of pathogens in the aquaculture industry. This interest has necessitated the development of robust and reliable pathogen-detection assays. The development of a range of endogenous control assays to be run alongside these diagnostic assays works to further increase confidence in the latter. This study describes the design of a range of endogenous control assays based on the elongation factor 1-α (EF1-α) gene specific to a range of fish species including Atlantic salmon, Salmo salar; rainbow trout, Oncorhynchus mykiss; brown trout, Salmo trutta; cod, Gadus morhua; haddock, Melanogrammus aeglefinus; saithe, Pollachius virens; whiting, Merlangius merlangus; Norway pout, Trisopterus esmarkii; carp (family Cyprinidae), roach, Rutilus rutilus; European eel, Anguilla anguilla; and herring, Clupea harengus, as well as a number of fish cell lines. Evidence is provided of the validation of these assays for specific species, a range of tissue types and cell lines as well as an example of the potential uses of these assays.

  10. TaqMan DNA technology confirms likely overestimation of cod (Gadus morhua L.) egg abundance in the Irish Sea: implications for the assessment of the cod stock and mapping of spawning areas using egg-based methods.

    Science.gov (United States)

    Fox, C J; Taylor, M I; Pereyra, R; Villasana, M I; Rico, C

    2005-03-01

    Recent substantial declines in northeastern Atlantic cod stocks necessitate improved biological knowledge and the development of techniques to complement standard stock assessment methods (which largely depend on accurate commercial catch data). In 2003, an ichthyoplankton survey was undertaken in the Irish Sea and subsamples of 'cod-like' eggs were analysed using a TaqMan multiplex, PCR (polymerase chain reaction) assay (with specific probes for cod, haddock and whiting). The TaqMan method was readily applied to the large number of samples (n = 2770) generated during the survey and when combined with a manual DNA extraction protocol had a low failure rate of 6%. Of the early stage 'cod-like' eggs (1.2-1.75 mm diameter) positively identified: 34% were cod, 8% haddock and 58% whiting. As previous stock estimates based on egg surveys for Irish Sea cod assumed that the majority of 'cod-like' eggs were from cod, the TaqMan results confirm that there was probably substantial contamination by eggs of whiting and haddock that would have inflated estimates of the stock biomass.

  11. Frequencies of HNA-1, HNA-3, HNA-4, and HNA-5 in the Danish and Zambian populations determined using a novel TaqMan real time polymerase chain reaction method.

    Science.gov (United States)

    Nielsen, K R; Koelbaek, M D; Varming, K; Baech, J; Steffensen, R

    2012-09-01

    In this study, we report a novel real time polymerase chain reaction (Q-PCR) method using TaqMan probes for human neutrophil antigens (HNA)-1, -3, -4, and -5 genotyping. The method was validated in a Caucasian Danish population, a Zambian population, and in clinical samples using three different methods: an in-house polymerase chain reaction with sequence-specific primers (PCR-SSP) method, a commercial available PCR-SSP kit and a novel Q-PCR method. We observed no discrepancy in the genotype frequencies determined by the PCR-SSP methods and the TaqMan assay in the populations studied. In tests of a family of Nigerian origin and in samples carrying the rare SLC44A2*1:2 genotype, different results were produced by the commercial PCR-SSP kit and the real-time TaqMan assay. The TaqMan-based genotyping method was rapid and reproducible, allowing high-throughput HNA-1, -3, -4, and -5 genotyping.

  12. High resolution TaqMan real-time PCR approach to detect hazelnut DNA encoding for ITS rDNA in foods.

    Science.gov (United States)

    López-Calleja, Inés María; de la Cruz, Silvia; Pegels, Nicolette; González, Isabel; García, Teresa; Martín, Rosario

    2013-12-01

    A broad range of foods have been described as causing allergies, but the majority of allergic reactions can be ascribed to a limited number of food components. Recent extensive surveys showed how tree nuts, particularly hazelnut (Corylus avellana L.) seeds, rank amongst the most important sources of food allergy. In order to protect the allergic consumer, efficient and reliable methods are required for the detection of allergenic ingredients. For this purpose, we have developed a real-time polymerase chain reaction (PCR) for detection of hazelnut in commercial food products. In this way a specific hazelnut primer pair based on the ITS marker (70 bp) and a nuclease (TaqMan) probe labelled with FAM and BHQ were designed. Sensibility of real-time PCR was determined by analysis of raw and heat treated hazelnut-wheat flour mixtures with a range of detection of 0.1-100,000 ppm. Practical applicability of the real-time PCR assay developed for determining hazelnut in different food matrices was investigated by analyzing 179 commercial foodstuffs comprising snacks, biscuits, chocolates, bonbons, creams, nut bars, ice creams, precooked meals, breads, beverages, yogurts, cereals, meat products, rice cake and nougat. From the total of samples analyzed, 40 commercial food products that didn't declare hazelnut nor traces on the label were found to contain hazelnut. The real-time PCR method proposed herein due to its high sensitivity facilitates the detection of hazelnut traces in commercial food products and can also be useful for monitoring the effectiveness of cleaning processes and as consequence, can help to prevent the food allergic consumer from unintentional ingestion of hidden allergens.

  13. Detection of viable Salmonella in ice cream by TaqMan real-time polymerase chain reaction assay combining propidium monoazide

    Directory of Open Access Journals (Sweden)

    Yuexia Wang

    2015-09-01

    Full Text Available Real-time polymerase chain reaction (PCR allows rapid detection of Salmonella in frozen dairy products, but it might cause a false positive detection result because it might amplify DNA from dead target cells as well. In this study, Salmonella-free frozen ice cream was initially inoculated with heat-killed Salmonella Typhimurium cells and stored at −18°C. Bacterial DNA extracted from the sample was amplified using TaqMan probe-based real-time PCR targeting the invA gene. Our results indicated that DNA from the dead cells remained stable in frozen ice cream for at least 20 days, and could produce fluorescence signal for real-time PCR as well. To overcome this limitation, propidium monoazide (PMA was combined with real-time PCR. PMA treatment can effectively prevent PCR amplification from heat-killed Salmonella cells in frozen ice cream. The PMA real-time PCR assay can selectively detect viable Salmonella at as low as 103 CFU/mL. Combining 18 hours of pre-enrichment with the assay allows for the detection of viable Salmonella at 100 CFU/mL and avoiding the false-positive result of dead cells. The PMA real-time PCR assay provides an alternative specifically for detection of viable Salmonella in ice cream. However, when the PMA real-time PCR assay was evaluated in ice cream subjected to frozen storage, it obviously underestimated the contamination situation of viable Salmonella, which might lead to a false negative result. According to this result, the use of enrichment prior to PMA real-time PCR analysis remains as the more appropriate approach.

  14. Efficient diagnosis and treatment follow-up of human brucellosis by a novel quantitative TaqMan real-time PCR assay: a human clinical survey.

    Science.gov (United States)

    Sohrabi, Majid; Mohabati Mobarez, Ashraf; Khoramabadi, Nima; Hosseini Doust, Reza; Behmanesh, Mehrdad

    2014-12-01

    Rapid and effective diagnosis of brucellosis is a challenge for clinicians. Even when diagnosis is on time and therapy is initiated, meticulous follow-up appointments are crucial for ensuring the efficacy of the treatment. Due to shortcomings of serological methods, molecular diagnosis, especially real-time PCR, is becoming a main approach in laboratory diagnostics. Thus, the development of efficient procedures and standardization of the PCR tests will have a great impact on the precise detection and quantification of bacterial DNA loads, which is valuable for the medical management of brucellosis patients. We developed a new TaqMan real-time PCR directed to bcsp31, a shared gene of the brucellae. The bcsp31 gene fragment was cloned into pJET1.2. Recombinant pJET1.2-bcsp31 was linearized by HindIII digestion, and the product was used for the preparation of a standard curve. A panel of Brucella spp. and non-Brucella pathogens was tested. No bacterial genomes other than those of the brucellae were detected. According to the results, specificity of the method was 100%. In a clinical assessment, the positive-control group comprised 37 patients with microbiologically confirmed brucellosis, and 25 healthy individuals served as the negative-control group. By the end of the treatment period, there was a significant decrease in the DNA load of the 37 brucellosis patients, which persisted for the 4 weeks of monitoring after treatment, suggesting that our proposed method is an efficient monitoring tool. Serum samples prior to any treatment were collected from the 25 serologically suspicious patients and assessed by our method; 72% of these patients tested positive for brucellosis.

  15. Development of a TaqMan real-time PCR assay for quantification of airborne conidia of Botrytis squamosa and management of botrytis leaf blight of onion.

    Science.gov (United States)

    Carisse, O; Tremblay, D M; Lévesque, C A; Gindro, K; Ward, P; Houde, A

    2009-11-01

    The use of a DNA-based method for quantifying airborne inoculum of Botrytis squamosa, a damaging pathogen of onion, was investigated. A method for purifying DNA from conidia collected using rotating-arm samplers and quantifying it using a TaqMan real-time quantitative polymerase chain reaction (qPCR) assay is described. The sensitivity of the qPCR assay was high, with a detection limit of 2 conidia/rod. A linear relationship between numbers of conidia counted with a compound microscope and those determined with the qPCR assay was obtained. Receiver operating characteristic curve analysis was used to evaluate the reliability of the two methods of conidia quantification (microscope examination and qPCR assay) to predict the risk of disease being below or above a damage threshold (D(th)). In total, 142 field samples from commercial onion fields were analyzed. At damage thresholds of 5 or 10 lesions/leaf, conidia quantification with the qPCR assay was more reliable at predicting disease risk than conidia quantification based on microscope counts. The proportion of decisions where the disease was present and predicted was higher for the qPCR assay than for the microscope counts, with values of 0.95 and 0.89 compared with 0.79 and 0.81 for D(th) of 5 and 10 lesions/leaf, respectively. The proportion of decisions where the disease was present but not predicted was lower for the qPCR assay than for microscope counts, with values of 0.05 and 0.11 compared with 0.20 and 0.19 for D(th) of 5 and 10 lesions/leaf, respectively. The results demonstrated that this new qPCR assay was reliable for quantifying B. squamosa airborne inoculum in commercial onion fields and that molecular conidia quantification could be used as a component of a risk management system for Botrytis leaf blight.

  16. Performance evaluation of Xpert MTB/RIF in a moderate tuberculosis incidence compared with TaqMan MTB and TRCRapid M.TB.

    Science.gov (United States)

    Tsuyuguchi, Kazunari; Nagai, Hideaki; Ogawa, Kenji; Matsumoto, Tomoshige; Morimoto, Kozo; Takaki, Akiko; Mitarai, Satoshi

    2017-02-01

    Xpert MTB/RIF is an automated nucleic acid amplification test (NAT) that can detect the presence of Mycobacterium tuberculosis complex (MTC) in clinical specimens as well as rifampicin (RIF) resistance resulting from rpoB mutation. Despite its high sensitivity and specificity for diagnosing tuberculosis (TB) with or without RIF resistance, the clinical performance of the test is variable. In this study, we evaluated the performance of Xpert MTB/RIF in a setting of moderate TB burden and high medical resources. A total of 427 sputum specimens were obtained from 237 suspected TB cases. Of these, 159 were identified as active TB, while the other 78 were non-TB diseases. The overall sensitivity and specificity of MTC detection by Xpert MTB/RIF using culture results as a reference were 86.8% [95% confidence interval (CI): 81.8%-90.6%] and 96.8% (95% CI: 93.1%-98.5%), respectively. Among MTC-positive culture specimens, Xpert MTB/RIF positivity was 95.2% (95% CI: 91.2%-97.5%) in smear-positive and 44.7% (95% CI 30.1-60.3) in smear-negative specimens. Xpert MTB/RIF was similar to other NATs (TaqMan MTB and TRCRapid M.TB) in terms of performance. Xpert MTB/RIF detected 25 RIF-resistant isolates as compared to 22 with the mycobacterial growth indicator tube antimicrobial susceptibility testing system, yielding a sensitivity of 100% (95% CI: 85.1%-100%) and specificity of 98.3% (95% CI: 95.1%-99.4%). These results indicate that although sensitivity in smear-negative/culture-positive specimens was relatively low, Xpert MTB/RIF is a useful diagnostic tool for detecting TB and RIF resistance even in settings of moderate TB burden. Copyright © 2016. Published by Elsevier Ltd.

  17. Analytical Performance of a Multiplex Real-Time PCR Assay Using TaqMan Probes for Quantification of Trypanosoma cruzi Satellite DNA in Blood Samples

    Science.gov (United States)

    Abate, Teresa; Cayo, Nelly M.; Parrado, Rudy; Bello, Zoraida Diaz; Velazquez, Elsa; Muñoz-Calderon, Arturo; Juiz, Natalia A.; Basile, Joaquín; Garcia, Lineth; Riarte, Adelina; Nasser, Julio R.; Ocampo, Susana B.; Yadon, Zaida E.; Torrico, Faustino; de Noya, Belkisyole Alarcón; Ribeiro, Isabela; Schijman, Alejandro G.

    2013-01-01

    Background The analytical validation of sensitive, accurate and standardized Real-Time PCR methods for Trypanosoma cruzi quantification is crucial to provide a reliable laboratory tool for diagnosis of recent infections as well as for monitoring treatment efficacy. Methods/Principal Findings We have standardized and validated a multiplex Real-Time quantitative PCR assay (qPCR) based on TaqMan technology, aiming to quantify T. cruzi satellite DNA as well as an internal amplification control (IAC) in a single-tube reaction. IAC amplification allows rule out false negative PCR results due to inhibitory substances or loss of DNA during sample processing. The assay has a limit of detection (LOD) of 0.70 parasite equivalents/mL and a limit of quantification (LOQ) of 1.53 parasite equivalents/mL starting from non-boiled Guanidine EDTA blood spiked with T. cruzi CL-Brener stock. The method was evaluated with blood samples collected from Chagas disease patients experiencing different clinical stages and epidemiological scenarios: 1- Sixteen Venezuelan patients from an outbreak of oral transmission, 2- Sixty three Bolivian patients suffering chronic Chagas disease, 3- Thirty four Argentinean cases with chronic Chagas disease, 4- Twenty seven newborns to seropositive mothers, 5- A seronegative receptor who got infected after transplantation with a cadaveric kidney explanted from an infected subject. Conclusions/Significance The performing parameters of this assay encourage its application to early assessment of T. cruzi infection in cases in which serological methods are not informative, such as recent infections by oral contamination or congenital transmission or after transplantation with organs from seropositive donors, as well as for monitoring Chagas disease patients under etiological treatment. PMID:23350002

  18. Detection of viable Salmonella in ice cream by TaqMan real-time polymerase chain reaction assay combining propidium monoazide.

    Science.gov (United States)

    Wang, Yuexia; Yang, Ming; Liu, Shuchun; Chen, Wanyi; Suo, Biao

    2015-09-01

    Real-time polymerase chain reaction (PCR) allows rapid detection of Salmonella in frozen dairy products, but it might cause a false positive detection result because it might amplify DNA from dead target cells as well. In this study, Salmonella-free frozen ice cream was initially inoculated with heat-killed Salmonella Typhimurium cells and stored at -18°C. Bacterial DNA extracted from the sample was amplified using TaqMan probe-based real-time PCR targeting the invA gene. Our results indicated that DNA from the dead cells remained stable in frozen ice cream for at least 20 days, and could produce fluorescence signal for real-time PCR as well. To overcome this limitation, propidium monoazide (PMA) was combined with real-time PCR. PMA treatment can effectively prevent PCR amplification from heat-killed Salmonella cells in frozen ice cream. The PMA real-time PCR assay can selectively detect viable Salmonella at as low as 10(3) CFU/mL. Combining 18 hours of pre-enrichment with the assay allows for the detection of viable Salmonella at 10(0) CFU/mL and avoiding the false-positive result of dead cells. The PMA real-time PCR assay provides an alternative specifically for detection of viable Salmonella in ice cream. However, when the PMA real-time PCR assay was evaluated in ice cream subjected to frozen storage, it obviously underestimated the contamination situation of viable Salmonella, which might lead to a false negative result. According to this result, the use of enrichment prior to PMA real-time PCR analysis remains as the more appropriate approach. Copyright © 2015. Published by Elsevier B.V.

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

    Science.gov (United States)

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

    2010-05-18

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

  20. Analytical performance of a multiplex Real-Time PCR assay using TaqMan probes for quantification of Trypanosoma cruzi satellite DNA in blood samples.

    Directory of Open Access Journals (Sweden)

    Tomas Duffy

    Full Text Available BACKGROUND: The analytical validation of sensitive, accurate and standardized Real-Time PCR methods for Trypanosoma cruzi quantification is crucial to provide a reliable laboratory tool for diagnosis of recent infections as well as for monitoring treatment efficacy. METHODS/PRINCIPAL FINDINGS: We have standardized and validated a multiplex Real-Time quantitative PCR assay (qPCR based on TaqMan technology, aiming to quantify T. cruzi satellite DNA as well as an internal amplification control (IAC in a single-tube reaction. IAC amplification allows rule out false negative PCR results due to inhibitory substances or loss of DNA during sample processing. The assay has a limit of detection (LOD of 0.70 parasite equivalents/mL and a limit of quantification (LOQ of 1.53 parasite equivalents/mL starting from non-boiled Guanidine EDTA blood spiked with T. cruzi CL-Brener stock. The method was evaluated with blood samples collected from Chagas disease patients experiencing different clinical stages and epidemiological scenarios: 1- Sixteen Venezuelan patients from an outbreak of oral transmission, 2- Sixty three Bolivian patients suffering chronic Chagas disease, 3- Thirty four Argentinean cases with chronic Chagas disease, 4- Twenty seven newborns to seropositive mothers, 5- A seronegative receptor who got infected after transplantation with a cadaveric kidney explanted from an infected subject. CONCLUSIONS/SIGNIFICANCE: The performing parameters of this assay encourage its application to early assessment of T. cruzi infection in cases in which serological methods are not informative, such as recent infections by oral contamination or congenital transmission or after transplantation with organs from seropositive donors, as well as for monitoring Chagas disease patients under etiological treatment.

  1. Assessment of a Novel Automatic Real-Time PCR Assay on the Cobas 4800 Analyzer as a Screening Platform for Hepatitis C Virus Genotyping in Clinical Practice: Comparison with Massive Sequencing

    Science.gov (United States)

    Nieto-Aponte, Leonardo; Ruiz-Ripa, Alicia; Tabernero, David; Gonzalez, Carolina; Gregori, Josep; Vila, Marta; Asensio, Miriam; Garcia-Cehic, Damir; Ruiz, Gerardo; Chen, Qian; Ordeig, Laura; Llorens, Meritxell; Saez, Montserrat; Esteban, Juan I.; Esteban, Rafael; Buti, Maria; Pumarola, Tomas

    2016-01-01

    ABSTRACT The unequivocal identification of hepatitis C virus (HCV) subtypes 1a/1b and genotypes 2 to 6 is required for optimizing the effectiveness of interferon-free, direct-acting antiviral therapies. We compared the performance of a new real-time HCV genotyping assay used on the Cobas 4800 system (C4800) with that of high-resolution HCV subtyping (HRCS). In total, 502 samples were used, including 184 samples from chronic HCV patients (from routine laboratory activity during April 2016), 5 stored samples with double HCV genotype infections for testing the limitations of the method, and 313 samples from a screening protocol implemented in our hospital (from May to August 2016) based on the new method to further determine its genotyping accuracy. A total of 282 samples, including 171 from April 2016 (the 13 remaining had too low of a viral load for HRCS), 5 selected with double infections, and 106 from screening, were analyzed by both methods, and 220 were analyzed only by the C4800. The C4800 correctly subtyped 125 of 126 1a/1b samples, and the 1 remaining sample was reported as genotype 1. The C4800 correctly genotyped 38 of 45 non-1a/1b samples (classified by HRCS), and it reported the remaining 7 samples as indeterminate. One hundred two of 106 non-1a/1b genotype samples that were identified using the C4800 for screening were confirmed by HRCS. In the 4 remaining samples, 3 were correctly reported as genotype 1 (without defining the subtype) and 1 was reported as indeterminate. None of the samples were misgenotyped. Four of 7 samples with double HCV infections were correctly genotyped by the C4800. Excluding the 5 selected double-infected samples, the C4800 showed 95.7% concordant results for genotyping HCVs 2 to 6 and 1a/1b subtyping, and 99.2% concordance for subtyping 1a/1b single infections in clinical samples. To improve laboratory workflow, we propose using the C4800 as a first-line test for HCV genotyping and 1a/1b classification, followed by

  2. Multicenter comparison of Roche COBAS AMPLICOR MONITOR version 1.5, Organon Teknika NucliSens QT with Extractor, and Bayer Quantiplex version 3.0 for quantification of human immunodeficiency virus type 1 RNA in plasma.

    Science.gov (United States)

    Murphy, D G; Côté, L; Fauvel, M; René, P; Vincelette, J

    2000-11-01

    The performance and characteristics of Roche COBAS AMPLICOR HIV-1 MONITOR version 1.5 (CA MONITOR 1.5) UltraSensitive (usCA MONITOR 1. 5) and Standard (stCA MONITOR 1.5) procedures, Organon Teknika NucliSens HIV-1 RNA QT with Extractor (NucliSens), and Bayer Quantiplex HIV RNA version 3.0 (bDNA 3.0) were compared in a multicenter trial. Samples used in this study included 460 plasma specimens from human immunodeficiency virus (HIV) type 1 (HIV-1)-infected persons, 100 plasma specimens from HIV antibody (anti-HIV)-negative persons, and culture supernatants of HIV-1 subtype A to E isolates diluted in anti-HIV-negative plasma. Overall, bDNA 3.0 showed the least variation in RNA measures upon repeat testing. For the Roche assays, usCA MONITOR 1.5 displayed less variation in RNA measures than stCA MONITOR 1.5. NucliSens, at an input volume of 2 ml, showed the best sensitivity. Deming regression analysis indicated that the results of all three assays were significantly correlated (P < 0.0001). However, the mean difference in values between CA MONITOR 1.5 and bDNA 3.0 (0.274 log(10) RNA copies/ml; 95% confidence interval, 0.192 to 0.356) was significantly different from 0, indicating that CA MONITOR 1.5 values were regularly higher than bDNA 3.0 values. Upon testing of 100 anti-HIV-negative plasma specimens, usCA MONITOR 1.5 and NucliSens displayed 100% specificity, while bDNA 3.0 showed 98% specificity. NucliSens quantified 2 of 10 non-subtype B viral isolates at 1 log(10) lower than both CA MONITOR 1.5 and bDNA 3.0. For NucliSens, testing of specimens with greater than 1,000 RNA copies/ml at input volumes of 0.1, 0.2, and 2.0 ml did not affect the quality of results. Additional factors differing between assays included specimen throughput and volume requirements, limit of detection, ease of execution, instrument work space, and costs of disposal. These characteristics, along with assay performance, should be considered when one is selecting a viral load assay.

  3. Taqman MGB探针研究中国人群TNF-α基因启动子区单核苷酸多态性%Genotyping of Single Nucleotide Polymorphisms in TNF-α Promoter Region in Chinese Population Using Taqman MGB Probes

    Institute of Scientific and Technical Information of China (English)

    庾蕾; 庄志雄; 谢富焕; 黄海雄; 叶小明

    2006-01-01

    [目的]了解中国人群肿瘤坏死因子α(the tumour necrosis factor α,TNF-α)基因启动子区多态性.[方法]随机选取20名深圳地区汉族健康体检者,采用两对引物PCR扩增TNF-α基因启动子区(-1389nt~+125 nt),对PCR产物进行序列分析,寻找单核苷酸多态性位点(single nucleotide polymorphisms,SNPs).采用Taqman MGB探针建立了-857nt(C/T)位点的实时定量PCR(the real-time PCR)分型方法,并对中国汉族、壮族、布依族,水族及苗族群体共1 108份样本进行了基因分型.[结果]在启动子区(-1322nt~+67 nt),发现6个SNP位点,即-885(A/G)、-863(C/A)、-646(G/A)、-648(G/A)、-568(G/C)和-857(C/T,其中位点-646 nt(G→A)为新发现SNP.-885、-648及-568nt位点碱基虽然与Genbank不同,但测序的20个个体基因分型相同.中国人群-857nt(C/T)位点基因型频率分别为0.79(CC),0.19(CT)和0.02(TT),中国汉族、壮族、布依族,水族及苗族群体间无显著性差异.中国人群-857T等位基因频率为0.116,与文献报道的韩国人群相同,但比日本人群低.[结论]中国人群TNF-α基因启动子区单核苷酸多态性可能较保守.采用Taqman MGB探针实时定量PCR技术对SNPs进行基因分型简便、快速及准确,易于自动化,为大规模的疾病相关性研究提供了有效的工具.

  4. Cross-platform comparison of SYBR® Green real-time PCR with TaqMan PCR, microarrays and other gene expression measurement technologies evaluated in the MicroArray Quality Control (MAQC study

    Directory of Open Access Journals (Sweden)

    Dial Stacey L

    2008-07-01

    Full Text Available Abstract Background The MicroArray Quality Control (MAQC project evaluated the inter- and intra-platform reproducibility of seven microarray platforms and three quantitative gene expression assays in profiling the expression of two commercially available Reference RNA samples (Nat Biotechnol 24:1115-22, 2006. The tested microarrays were the platforms from Affymetrix, Agilent Technologies, Applied Biosystems, GE Healthcare, Illumina, Eppendorf and the National Cancer Institute, and quantitative gene expression assays included TaqMan® Gene Expression PCR Assay, Standardized (Sta RT-PCR™ and QuantiGene®. The data showed great consistency in gene expression measurements across different microarray platforms, different technologies and test sites. However, SYBR® Green real-time PCR, another common technique utilized by half of all real-time PCR users for gene expression measurement, was not addressed in the MAQC study. In the present study, we compared the performance of SYBR Green PCR with TaqMan PCR, microarrays and other quantitative technologies using the same two Reference RNA samples as the MAQC project. We assessed SYBR Green real-time PCR using commercially available RT2 Profiler™ PCR Arrays from SuperArray, containing primer pairs that have been experimentally validated to ensure gene-specificity and high amplification efficiency. Results The SYBR Green PCR Arrays exhibit good reproducibility among different users, PCR instruments and test sites. In addition, the SYBR Green PCR Arrays have the highest concordance with TaqMan PCR, and a high level of concordance with other quantitative methods and microarrays that were evaluated in this study in terms of fold-change correlation and overlap of lists of differentially expressed genes. Conclusion These data demonstrate that SYBR Green real-time PCR delivers highly comparable results in gene expression measurement with TaqMan PCR and other high-density microarrays.

  5. Detection and Typing of Herpes Simplex Virus (HSV) in Mucocutaneous Samples by TaqMan PCR Targeting a gB Segment Homologous for HSV Types 1 and 2

    OpenAIRE

    Namvar, Lilly; Olofsson, Sigvard; Bergström, Tomas; Lindh, Magnus

    2005-01-01

    Herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) are major causes of mucocutaneous lesions and severe infections of the central nervous system. Here a new semiautomated method for detecting and typing of HSV was used to analyze 479 mucocutaneous swab samples. After DNA extraction using a Magnapure LC robot, a 118-bp segment of the gB region was amplified by real-time PCR utilizing type-specific TaqMan probes to identify HSV-1 or HSV-2. HSV detection in a single well using probes labeled wit...

  6. Evaluation and comparison of SYBR Green I Real-Time PCR and TaqMan Real-Time PCR methods for quantitative assay of Listeria monocytogenes in nutrient broth and milk

    OpenAIRE

    Karatzas, Kimon Andreas G.

    2012-01-01

    Specific traditional plate count method and real-time PCR systems based on SYBR Green I and TaqMan technologies using a specific primer pair and probe for amplification of iap-gene were used for quantitative assay of Listeria monocytogenes in seven decimal serial dilution series of nutrient broth and milk samples containing 1.58 to 1.58×107 cfu /ml and the real-time PCR methods were compared with the plate count method with respect to accuracy and sensitivity. In this study, the plate count m...

  7. TaqMan MGB probe real- time fluorescence quantitative PCR for rapid detection of Mycoplasma%TaqMan MGB探针法实时荧光定量PCR快速检测支原体的研究

    Institute of Scientific and Technical Information of China (English)

    高正琴; 邢进; 冯育芳; 岳秉飞; 贺争鸣

    2011-01-01

    目的:建立特异、敏感、快速检测支原体的TaqMan MGB探针实时荧光定量PCR方法.方法:针对支原体16S rRNA基因的保守区设计特异性引物和探针,建立支原体TaqMan MGB探针实时荧光定量PCR方检测方法,验证方法的特异性、敏感性和稳定性.对2008~2010年期间在北京采集的680份小型猪、小鼠、大鼠样本中的支原体进行检测,同时进行分离培养和常规PCR检测.结果:建立的TaqMan MGB探针实时荧光定量PCR方法对支原体的检测具有高度的特异性,对空肠弯曲菌、支气管鲍特杆菌、肺炎克雷伯杆菌、侵肺巴斯德氏菌、大肠埃希菌、铜绿假单胞菌、肺炎链球菌、乙型溶血性链球菌均无交叉反应,检测的灵敏度达9.2拷贝.标准曲线显示各浓度范围内具有良好的线性关系,相关系数为0.999,斜率为-3.328,TaqMan MGB探针实时荧光定量PCR效率为100%.对680份动物样本进行检测,结果TaqMan MGB探针实时荧光定量PCR和常规PCR均能检出77份支原体阳性样本,但分离培养未能检出支原体阳性样本.结果显示,建立的TaqMan MGB探针实时荧光定量PCR方法比细菌分离培养方法更敏感,能够直接从动物样本中检出支原体DNA,检测时间仅为2h.结论:本研究建立了一种可靠、快速、灵敏的检测支原体的TaqMan MGB探针实时荧光定量PCR方法,并且成功应用于小型猪、小鼠、大鼠样本中支原体的检测.该技术为动物源性药品和生物制品中支原体的快速检测提供了实用的工具.%Objective: To develop a TaqMan MGB probe - based, sensitive and specific real - time fluorescence quantitative PCR assay for rapid detection of Mycoplasma. Methods: Primers and probes specific to 16S rRNA gene of Mycoplasma were designed. A TaqMan MGB probe - based, real - time fluorescence quantitative PCR was established. The specificity, sensitivity and stability of the assay were assessed. Then, the established TaqMan MGB

  8. Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples

    Science.gov (United States)

    Cura, Carolina I.; Duffy, Tomas; Lucero, Raúl H.; Bisio, Margarita; Péneau, Julie; Jimenez-Coello, Matilde; Calabuig, Eva; Gimenez, María J.; Valencia Ayala, Edward; Kjos, Sonia A.; Santalla, José; Mahaney, Susan M.; Cayo, Nelly M.; Nagel, Claudia; Barcán, Laura; Málaga Machaca, Edith S.; Acosta Viana, Karla Y.; Brutus, Laurent; Ocampo, Susana B.; Aznar, Christine; Cuba Cuba, Cesar A.; Gürtler, Ricardo E.; Ramsey, Janine M.; Ribeiro, Isabela; VandeBerg, John L.; Yadon, Zaida E.; Osuna, Antonio; Schijman, Alejandro G.

    2015-01-01

    Background Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI–TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR). Methods/Principal Findings The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm. Conclusions/Significance Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production. PMID:25993316

  9. Development of TaqMan real-time polymerase chain reaction for the detection of the newly emerging form of carbapenem resistance gene in clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii

    Directory of Open Access Journals (Sweden)

    V Manchanda

    2011-01-01

    Full Text Available Purpose: The newly emerging form of the so-called New Delhi Metallo-beta-lactamases (NDM-1 has been reported recently from patients worldwide and broadly thought as a potential source for the major global health problem. Thus, it is important to study the epidemiology of the so-called NDM-1 harbouring bacteria to prevent its further spread and to place effective control measures. The present study describes the use of the real-time polymerase chain reaction (PCR assay for the detection of the bla NDM-1 gene using TaqMan probes among clinical isolates. Materials and Methods: Clinical isolates of Escherichia coli (11 strains, Klebsiella pneumoniae (17 strains and Acinetobacter baumannii (six strains that were resistant to either of the carbapenems (meropenem or imipenem were included in the study. The presence of carbapenemases in such strains was confirmed using the modified Hodge test. A real-time PCR assay was optimized for the detection of NDM-1 using a cloned synthetic gene fragment followed by testing of the clinical isolates. The findings were further confirmed using PCR and gene sequencing. Results: TaqMan probe assay displayed a good detection limit with analytical sensitivity of the assay up to 10 copies of bla NDM-1 gene per reaction. The isolates of E. coli and K. pneumoniae revealed narrow range crossing point values (Cp values between (12-17 cycles (mean Cp value 14, indicating number of bla NDM-1 gene copies of 106-108. The wider range of Cp values (15-34 cycles with a higher mean Cp value (23.6 was observed in A. baumannii with number of bla NDM-1 gene copies of 103-108. Conclusions: The study demonstrates that real-time PCR assay based on TaqMan chemistry is a useful technique for the detection of bla NDM-1 harbouring clinical isolates of E. coli, K. pneumoniae and A. baumannii. The assay has great precision in measuring the number of bla NDM-1 gene copies per specimen of DNA.

  10. A型肉毒梭菌atx基因TaqMan探针荧光定量PCR检测%Detection of atx gene in Clostridium botulinum using TaqMan probe FQ-PCR

    Institute of Scientific and Technical Information of China (English)

    王春晖; 赵素慧; 韦耀; 周莹; 万成松

    2012-01-01

    目的 采用TaqMan探针荧光定量PCR方法对A型肉毒梭菌atx基因进行检测.方法 以atx基因为靶基因,设计引物和TaqMan探针,优化反应条件,制作定量标准曲线,进行灵敏度、特异性和重复性验证,建立A型肉毒梭菌TaqMan荧光定量PCR检测方法.结果 构建质粒pUC57 -△atx,标准曲线在103~107拷贝数之间有较好的线性关系,相关系数为0.997,灵敏度达到22个拷贝数,比普通PCR提高约100倍;能选择性检测A型肉毒梭菌,与其他5种食源性病原菌无交叉反应,结果与普通PCR一致;重复性试验表明,同一浓度的15个平行样品的变异系数为1.0%.结论 A型肉毒梭菌TaqMan探针荧光定量PCR方法具有灵敏度高、特异性强、重复性好等特点,可以快速、准确、定量地检测A型肉毒梭菌.%Objective To detect the atx gene of Clostridium botulinum(C. botulinum) type A neurotoxin(bont/A) using TaqMan probe fluorescence quantitative polymerase chain reaction( FQ-PCR). Methods By targeting the gene atx of bont/A,designing primer and TaqMan probe, optimizing the reaction conditions, making quantitative standard curve, and verifying sensitivity,specificity and repeatability of the method, we established a TaqMan FQ-PCR method to detect bont/A. Results Plasmid pUC57-Δatx was constructed successfully. The standard curve established had good linearity when gene quantity was between 103 - 107 copies,and the coefficient of correlation was 0. 997. The limit of detection for FQ-PCR was 22 copies. The sensitivity was about 100 times higher than that of ordinary PCR. The FQ-PCR method could selectively detect C. botulinum and there were no cross reactions with other five food-borne pathogen samples,consisting with the results of ordinary PCR. Repeatability tests showed that the coefficient of variation of 15 parallel samples in same concentration was only 1.0%. Conclusion A TaqMan probe FQ-PCR method with high sensitivity and specificity, and good

  11. Application of TaqMan fluorescent probe-based quantitative real-time PCR assay for the environmental survey of Legionella spp. and Legionella pneumophila in drinking water reservoirs in Taiwan.

    Science.gov (United States)

    Kao, Po-Min; Hsu, Bing-Mu; Hsu, Tsui-Kang; Ji, Wen-Tsai; Huang, Po-Hsiang; Hsueh, Chih-Jen; Chiang, Chuen-Sheue; Huang, Shih-Wei; Huang, Yu-Li

    2014-08-15

    In this study, TaqMan fluorescent quantitative real-time PCR was performed to quantify Legionella species in reservoirs. Water samples were collected from 19 main reservoirs in Taiwan, and 12 (63.2%) were found to contain Legionella spp. The identified species included uncultured Legionella spp., L. pneumophila, L. jordanis, and L. drancourtii. The concentrations of Legionella spp. and L. pneumophila in the water samples were in the range of 1.8×10(2)-2.6×10(3) and 1.6×10(2)-2.4×10(2) cells/L, respectively. The presence and absence of Legionella spp. in the reservoir differed significantly in pH values. These results highlight the importance that L. pneumophila, L. jordanis, and L. drancourtii are potential pathogens in the reservoirs. The presence of L. pneumophila in reservoirs may be a potential public health concern that must be further examined.

  12. Broad-range detection of arboviruses belonging to Simbu serogroup lineage 1 and specific detection of Akabane, Aino and Peaton viruses by newly developed multiple TaqMan assays.

    Science.gov (United States)

    Shirafuji, Hiroaki; Yazaki, Ryu; Shuto, Yozo; Yanase, Tohru; Kato, Tomoko; Ishikura, Youji; Sakaguchi, Zenjiro; Suzuki, Moemi; Yamakawa, Makoto

    2015-12-01

    TaqMan assays were developed for the broad-range detection of arboviruses belonging to Simbu serogroup lineage 1 in the genus Orthobunyavirus and also for the specific detection of three viruses in the lineage, Akabane, Aino and Peaton viruses (AKAV, AINOV and PEAV, respectively). A primer and probe set was designed for the broad-range detection of Simbu serogroup lineage 1 (Pan-Simbu1 set) mainly targeting AKAV, AINOV, PEAV, Sathuperi and Shamonda viruses (SATV and SHAV), and the forward and reverse primers of the Pan-Simbu1 set were also used for the specific detection of AKAV with another probe (AKAV-specific set). In addition, two more primer and probe sets were designed for AINOV- and PEAV-specific detection, respectively (AINOV- and PEAV-specific sets). All of the four primer and probe sets successfully detected targeted viruses, and thus broad-range and specific detection of all the targeted viruses can be achieved by using two multiplex assays and a single assay in a dual (two-color) assay format when another primer and probe set for a bovine β-actin control is also used. The assays had an analytical sensitivity of 10 copies/tube for AKAV, at least 100 copies/tube for AINOV, 100 copies/tube for PEAV, one copy/tube for SATV and at least 10 copies/tube for SHAV, respectively. Diagnostic sensitivity of the assays was tested with field-collected bovine samples, and the results suggested that the sensitivity was higher than that of a conventional RT-PCR. These data indicate that the newly developed TaqMan assays will be useful tools for the diagnosis and screening of field-collected samples for infections of AKAV and several other arboviruses belonging to the Simbu serogroup lineage 1.

  13. TaqMan real-time reverse transcription-PCR assay for universal detection and quantification of avian hepatitis E virus from clinical samples in the presence of a heterologous internal control RNA.

    Science.gov (United States)

    Troxler, Salome; Marek, Ana; Prokofieva, Irina; Bilic, Ivana; Hess, Michael

    2011-04-01

    Avian hepatitis E virus (HEV) isolates could be separated into at least three genotypes. In this study, the development of the first duplex TaqMan real-time reverse transcription-PCR (RT-PCR) assay for detection and quantification of avian HEV is presented. Primers and probes binding within relatively conserved open reading frame 3 (ORF3) were designed. Tenfold dilution series of in vitro-transcribed avian HEV RNA were used as the standard for quantification. A 712-bp region of the green fluorescent protein gene was transcribed in vitro and used as a heterologous internal control for both RNA isolation and real-time RT-PCR. The duplex real-time RT-PCR for avian HEV had an efficiency of 1.04, a regression squared value of 0.996, and a sensitivity of approximately 3.6 × 10(3) copies per reaction mixture when in vitro-transcribed RNA was used as the template. The presence of in vitro-transcribed heterologous internal control RNA did not affect amplification of avian HEV RNA compared to that achieved by the single assay. The sensitivity of the real-time RT-PCR assay was comparable to that of conventional RT-PCR, and it was shown to be highly specific, as tissues from uninfected chickens, mammalian HEVs, and other viral genomes did not produce positive signals. All tested field samples with virus belonging to different avian HEV genotypes were successfully detected with this new duplex TaqMan real-time RT-PCR assay.

  14. 应用Taqman荧光定量PCR快速检测宠物食品中的沙门氏菌%Taqman PCR-based Methods for Rapid Detection of Salmonella in Pet Food

    Institute of Scientific and Technical Information of China (English)

    贾俊涛; 崔鹤; 马云; 曾静; 姜英辉; 李正义

    2012-01-01

    [目的]建立快速检测宠物食品中沙门氏菌(Salmonella)的荧光定量PCR方法.[方法]根据Genbank中登录的沙门氏菌吸附和侵袭上皮细胞表面蛋白invA基因序列设计合成引物和探针,检测该方法的特异性和敏感性.[结果]该方法具有良好的特异性,对沙门氏菌的检测灵敏度可达到17 CFU/反应(25 μl/反应).应用该方法检测人工污染宠物食品,样品经18 h增菌后,结果均为阳性.[结论]该研究建立的Taqman荧光定量PCR方法可以快速、准确地检测宠物食品中的沙门氏菌.%[ Objective ] To establish a Taqman real-time PCR for detection of Salmonella in pet food. [ Method ] A pair of primers and a probe were designed based on published nucleotide sequence of invA gene encoding the invasion protein of Salmonella enterica. [ Result] The assay detects Salmonella specifically. The detection limit of the real-time PCR was 17 CEU/test (25 μl/test) for the positive strain. This method was effective to detect artificially contaminated pet food. [ Conclusion ] The results showed that Taqman PCR assay was rapid and accure for detection of Salmonella from infected pet food.

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

    Institute of Scientific and Technical Information of China (English)

    刘海燕; 赵丽丽; 牛银杰; 祝明皓; 刘胜旺; 陈洪岩

    2015-01-01

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

  16. TaqMan探针荧光定量PCR检测花生油中掺入棕榈油的研究%Determination of palm oil adulterated in peanut oil with the TaqMan probe -based RT- PCR method

    Institute of Scientific and Technical Information of China (English)

    周慧; 梁宇斌; 吴苏喜; 李晓明; 裴伟; 杨涛

    2011-01-01

    The method of Real - time fluorescence quantitative polymerase chain reaction ( RT - PCR) with TaqMan fluorescent probe was chosen to fast detect the amount of palm oil mixed in peanut oil. MT3 - B gene of palm was selected as target gene to detect palm oil from peanut oil. The primers of MT3 - B and TaqMan probe were designed, MT3 - B gene reconstructed plasmid was built as absolute quantitative criteria for quantitative RT - PCR to establish standard curve. Peanut oil blended with 1% -40% concentration gradient palm oil was extracted DNA to test palm content by RT - PCR. The result showed that the correlation coefficient (R1) of standard curve with logarithmic linear regression analysis was 0.996. When the adulteration of palm oil in peanut oil reached 5% volume, MT3 - B gene of 17.431 copies per milli-liter of mixed oil could be detected . The method showed good sensitivity, specificity and repeatability.%根据棕榈内源基因MT3 -B设计引物和TaqMan探针,采用基因重组技术构建用于检测棕榈基因MT3 -B的重组质粒作为绝对定量标准品,建立标准曲线,对花生油中掺入棕榈油1% ~40%梯度混合油品提取DNA进行棕榈成分定量检测.结果表明,重组质粒标准品荧光定量标准曲线对数线性回归分析相关系数(R2)为0.996;花生油中掺入棕榈油达到5%时,可检出每亳升混合油品中棕榈MT3 -B基因17.431 copies,检测的重复性和特异性好.

  17. 黄热病毒实时荧光定量PCR方法的建立%Establishment on TaqMan real-time fluorescent quantitative PCR for detecting yellow fever virus

    Institute of Scientific and Technical Information of China (English)

    祁倩; 劳小荣; 陈蔼珍; 熊建英; 周旋; 张其威; 朱利; 李凌; 万成松

    2013-01-01

    OBJECTIVE To establish a specific,sensitive method with real-time fluorescence quantitative PCR assay for the rapid detection of yellow fever virus (YFV).METHODS In order to test yellow fever virus for simple and fast real-time detection with high sensitivity and high specificity,we selected the gene plasmid of yellow fever virus as the positive template.Used TaqMan probe method,we produced standard curve.RESULTS The sensitivity of this way was 3.457 copies/μl.In the group and variation coefficients between groups were less than 5%.The results showed that the method had a good sensitivity and stability.According to the mix of experiment with dengue virus type 1 to 4 and Japanese encephalitis virus,we proved this method had a good specificity.CONCLUSION The TaqMan real-time fluorescent quantitative PCR assay is a rapid,sensitive and specific method for the quantitative detection of YFV ; it's applicable for the early clinical diagnosis of YFV and there are very good social and economic benefits.%目的 建立黄热病毒的实时荧光定量PCR检测技术,以实现黄热病毒感染早期的筛选和检测.方法 以带有黄热病毒基因质粒为阳性模板,采用TaqMan探针法,制作标准曲线,以实现对黄热病毒的简单快速的高灵敏度、高特异性实时检测.结果 该方法检测的最低拷贝数可以达到3.457 copies/μl.组内及组间变异系数均低于5%,证明该方法具有良好的敏感性及稳定性.以1~4型登革病毒和乙脑病毒基因组为模板,检测结果为阴性,证明该方法有良好的特异性.结论 该方法可快速、灵敏、特异、定量检测黄热病毒,能用于黄热病毒感染的早期诊断.

  18. New approaches for the standardization and validation of a real-time qPCR assay using TaqMan probes for quantification of yellow fever virus on clinical samples with high quality parameters

    Science.gov (United States)

    Fernandes-Monteiro, Alice G; Trindade, Gisela F; Yamamura, Anna MY; Moreira, Otacilio C; de Paula, Vanessa S; Duarte, Ana Cláudia M; Britto, Constança; Lima, Sheila Maria B

    2015-01-01

    The development and production of viral vaccines, in general, involve several steps that need the monitoring of viral load throughout the entire process. Applying a 2-step quantitative reverse transcription real time PCR assay (RT-qPCR), viral load can be measured and monitored in a few hours. In this context, the development, standardization and validation of a RT-qPCR test to quickly and efficiently quantify yellow fever virus (YFV) in all stages of vaccine production are extremely important. To serve this purpose we used a plasmid construction containing the NS5 region from 17DD YFV to generate the standard curve and to evaluate parameters such as linearity, precision and specificity against other flavivirus. Furthermore, we defined the limits of detection as 25 copies/reaction, and quantification as 100 copies/reaction for the test. To ensure the quality of the method, reference controls were established in order to avoid false negative results. The qRT-PCR technique based on the use of TaqMan probes herein standardized proved to be effective for determining yellow fever viral load both in vivo and in vitro, thus becoming a very important tool to assure the quality control for vaccine production and evaluation of viremia after vaccination or YF disease. PMID:26011746

  19. Real-time PCR based on SYBR-Green I fluorescence: An alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions

    Directory of Open Access Journals (Sweden)

    Puisieux Alain

    2003-10-01

    Full Text Available Abstract Background Real-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called 'TaqMan' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive. Results We have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC in peripheral blood mononuclear cells; the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy. Conclusion Our assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.

  20. Expression patterns of WT-1 and Bcr-Abl measured by TaqMan quantitative real-time RT-PCR during follow-up of leukemia patients with the Ph chromosome

    Institute of Scientific and Technical Information of China (English)

    CHEN Zi-xing陈子兴; Jaspal Kaeda; Sue Saunders; John M Goldman

    2004-01-01

    Background This study was designed to quantitatively measure WT-1 expression levels in patients with chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL) during follow-up and to clarify the value of WT-1 as a molecular marker in minimal residual disease monitoring.Methods The TaqMan quantitative real-time RT-PCR method was established by using cloned WT-1 cDNA or synthesized oligonucleotides resembling WT-1 cDNA fragments in limit dilution as template until a stable and reliable standard curve was obtained. In a 25-month follow-up, the transcriptional levels of WT-1, Bcr-Abl, and Abl gene, were quantitatively measured in bone marrow cells from 25 CML or acute lymphoblastic leukemia (ALL) patients with the Ph chromosome. In addition, the expression of these genes in 40 samples of normal peripheral blood was also examined using the same method. The ratios of WT-1/Abl and Bcr-Abl/Abl were both plotted, and the two expression patterns were compared as well as their clinical significance.Results The levels of WT-1 expression in normal peripheral blood were detectable. In CML and Ph positive ALL patients, WT-1 expression levels changed in parallel with the Bcr-Abl expression pattern as the disease progressed or responded to effective treatment.Conclusion WT-1 expression provides a novel molecular marker in addition to Bcr-Abl for monitoring minimal residual disease (MRD) and targeting therapy in Ph chromosome-positive leukemia patients.

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

    Science.gov (United States)

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

    2017-07-01

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

  2. Detection of Mycoplasma mycoides subsp. mycoides SC in bronchoalveolar lavage fluids of cows based on a TaqMan real-time PCR discriminating wild type strains from an lppQ− mutant vaccine strain used for DIVA-strategies

    Science.gov (United States)

    Vilei, Edy M.; Frey, Joachim

    2010-01-01

    Contagious bovine pleuropneumonia (CBPP) is the most serious cattle disease in Africa, caused by Mycoplasma mycoides subsp. mycoides small-colony type (SC). CBPP control strategies currently rely on vaccination with a vaccine based on live attenuated strains of the organism. Recently, an lppQ− mutant of the existing vaccine strain T1/44 has been developed (Janis et al., 2008). This T1lppQ− mutant strain is devoid of lipoprotein LppQ, a potential virulence attribute of M. mycoides subsp. mycoides SC. It is designated as a potential live DIVA (Differentiating Infected from Vaccinated Animals) vaccine strain allowing both serological and etiological differentiation. The present paper reports on the validation of a control strategy for CBPP in cattle, whereby a TaqMan real-time PCR based on the lppQ gene has been developed for the direct detection of M. mycoides subsp. mycoides SC in ex vivo bronchoalveolar lavage fluids of cows and for the discrimination of wild type strains from the lppQ− mutant vaccine strain. PMID:20381545

  3. Development and validation of a TaqMan real-time PCR assay for the identification and quantification of roe deer (Capreolus capreolus) in food to detect food adulteration.

    Science.gov (United States)

    Druml, Barbara; Mayer, Walter; Cichna-Markl, Margit; Hochegger, Rupert

    2015-07-01

    In order to protect the consumer from meat adulteration it is necessary to identify and quantify the meat content in foodstuffs. Game meat is particularly susceptible for fraudulent labeling since it is more valuable than meat from domestic animals. The paper presents a TaqMan real-time PCR assay for the quantitative determination of roe deer in meat products. The assay developed does not show cross-reactivity with 23 animal and 43 plant species tested and is therefore specific for roe deer. The amplification efficiency determined by analyzing serially diluted roe deer DNA extracts was found to be 93.9%. For quantifying the roe deer content in % (w/w), a reference system based on the myostatin gene was used. The quantification strategy was validated by determining the roe deer content in model meat mixtures and a model sausage. In addition, the real-time PCR assay was applied to the analysis of commercially available meat products. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. New approaches for the standardization and validation of a real-time qPCR assay using TaqMan probes for quantification of yellow fever virus on clinical samples with high quality parameters.

    Science.gov (United States)

    Fernandes-Monteiro, Alice G; Trindade, Gisela F; Yamamura, Anna M Y; Moreira, Otacilio C; de Paula, Vanessa S; Duarte, Ana Cláudia M; Britto, Constança; Lima, Sheila Maria B

    2015-01-01

    The development and production of viral vaccines, in general, involve several steps that need the monitoring of viral load throughout the entire process. Applying a 2-step quantitative reverse transcription real time PCR assay (RT-qPCR), viral load can be measured and monitored in a few hours. In this context, the development, standardization and validation of a RT-qPCR test to quickly and efficiently quantify yellow fever virus (YFV) in all stages of vaccine production are extremely important. To serve this purpose we used a plasmid construction containing the NS5 region from 17DD YFV to generate the standard curve and to evaluate parameters such as linearity, precision and specificity against other flavivirus. Furthermore, we defined the limits of detection as 25 copies/reaction, and quantification as 100 copies/reaction for the test. To ensure the quality of the method, reference controls were established in order to avoid false negative results. The qRT-PCR technique based on the use of TaqMan probes herein standardized proved to be effective for determining yellow fever viral load both in vivo and in vitro, thus becoming a very important tool to assure the quality control for vaccine production and evaluation of viremia after vaccination or YF disease.

  5. 大鲵虹彩病毒TaqMan实时荧光定量PCR检测方法的建立%Establishment of a TaqMan real-time PCR assay for detecting the giant salamander iridovirus

    Institute of Scientific and Technical Information of China (English)

    周勇; 曾令兵; 孟彦; 周群兰; 张辉; 高正勇; 肖艺; 孙建滨

    2012-01-01

    利用PCR技术扩增出大鲵虹彩病毒(giant salamander iridovirus,GSIV)主要衣壳蛋白(MCP)编码区长度为1 392 bp的片段,克隆到pMD19-T载体上,构建重组质粒pMD 19-T-MCP.经PCR鉴定确认正确后,以10倍梯度稀释pMD19-T-MCP重组质粒,作为标准模板进行TaqMan实时荧光定量PCR扩增,制作标准曲线,建立了大鲵虹彩病毒的TaqMan实时荧光定量PCR检测方法.制作的标准曲线有极好的线性关系,且线性范围宽,相关系数为0.990 19.组内重复试验的CT值标准偏差为0.52%.检测结果显示,该方法对大鲵虹彩病毒的检测有高度的特异性,与锦鲤疱疹病毒、弗氏柠檬酸杆菌、嗜水气单胞菌以及鲤上皮瘤细胞基因组DNA之间均无交叉反应,特异性好,检测总DNA灵敏度为10个病毒核酸分子拷贝数,约1.1×10-3 pg/μL病毒核酸,较之常规PCR的敏感度高出约1 000倍.研究建立的大鲵虹彩病毒TaqMan实时荧光定量PCR方法灵敏度高、特异性强,对大鲵虹彩病毒病的快速诊断与病毒病原定量检测有重要意义.%A 1 392 bp coding region of giant salamander iridovirus(GSIV)MCP protein was amplified by PCR and cloned into pMD19-T vector for the construction of recombinant plasmid pMD19-T-MCP. After being identified and confirmed by PCR reaction, 10-fold serial dilutions of plasmid pMD19-T-MCP were used as standard templates for TaqMan real-time PCR to generate standard curve for quantifying the virus genomic copy number. A good linear correlation was demonstrated in the standard curve for the real-time PCR assay. The coefficients of variance (CV) were 0.52% for intra-assay tests, which indicated good reliability. The detection results showed that the specificity of this assay was high for giant salamander iridovirus without cross-reactions with DNA templates from KHV, Aeromonas hydrophila, Citrobacter freundii and EPC cells. A minimum of 10 copies of GSIV DNA (l.l×10-3 pg/μL total DNA) could be detected, which

  6. Development of a Rapid TaqMan Real-Time PCR Assay for Detec-tion of Legionella pneumophila%嗜肺军团菌实时荧光定量PCR快速检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    杜昕颖; 黄留玉; 苏晓; 王玉飞; 龚春丽; 庄妤冰; 苑锡铜; 陈泽良; 袁静; 宋宏彬

    2013-01-01

    Objective: To develop a TaqMan-based real-time PCR method for rapid detection of Legionella pneu-mophila. Methods: The sequence of macrophage infectivity potentiator(Mip) gene was downloaded from GenBank and the specific primers and TaqMan probe were designed in the conserved region of the Mip gene for L.pneu-mophila. Then, the real-time PCR array for rapid detection of L.pneumophila was developed and its spceificity and sensitivity were evaluated. Simulated environment water samples were used to assess the assay. Results: Only L. pneumophila strains generated fluorescent signals, and no cross-reaction was observed for the differential control strains including three non-pneumophila strains and six other respiratory pathogens. The detection limits were 1.6 pg/μL with genomic DNA of L.pneumophila, and 10 CFU/mL with simulated water samples. Conclusion: The Taq-Man real-time PCR assay described here is specific, sensitive and rapid for detection of L.pneumophila, and this assay could be used for laboratory-based monitoring and emergency detection of L.pneumophila.%目的:建立针对嗜肺军团菌Mip基因的实时荧光定量TaqMan PCR检测方法,并进行自来水和空调冷却水模拟标本的检测评价。方法:根据嗜肺军团菌Mip基因的特异性序列设计引物和TaqMan探针,建立嗜肺军团菌的实时荧光定量TaqMan PCR快速检测方法,对方法进行灵敏度及特异性评价,并对自来水和空调冷却水模拟标本中的嗜肺军团菌进行检测。结果:建立的方法对嗜肺军团菌的检测具有高度特异性,与3种非嗜肺军团菌和6种其他呼吸道病原均没有交叉反应;基因组DNA的检测灵敏度为1.6 pg/μL,模拟自来水和空调冷却水标本的检测灵敏度为10 CFU/mL。结论:建立的TaqMan荧光定量PCR方法特异、灵敏、快速,适于嗜肺军团菌的日常监测和暴发疫情的应急诊断。

  7. Real-Time TaqMan PCR Assay for the Detection of Heat-Labile and Heat-Stable Enterotoxin Genes in a Geographically Diverse Collection of Enterotoxigenic Escherichia coli Strains and Stool Specimens.

    Science.gov (United States)

    Pattabiraman, Vaishnavi; Parsons, Michele B; Bopp, Cheryl A

    2016-04-01

    Enterotoxigenic Escherichia coli (ETEC) are an important cause of diarrhea in children under the age of 5 years in developing countries and are the leading bacterial agent of traveler's diarrhea in persons traveling to these countries. ETEC strains secrete heat-labile (LT) and/or heat-stable (ST) enterotoxins that induce diarrhea by causing water and electrolyte imbalance. We describe the validation of a real-time TaqMan PCR (RT-PCR) assay to detect LT, ST1a, and ST1b enterotoxin genes in E. coli strains and in stool specimens. We validated LT/ST1b duplex and ST1a single-plex RT-PCR assay using a conventional PCR assay as a gold standard with 188 ETEC strains and 42 non-ETEC strains. We validated LT/ST1b duplex and ST1a single-plex RT-PCR assay in stool specimens (n = 106) using traditional culture as the gold standard. RT- PCR assay sensitivities for LT, ST1a, and ST1b detection in strains were 100%, 100%, and 98%; specificities were 95%, 98%, and 99%, and Pearson correlation coefficient r was 0.9954 between RT-PCR assay and the gold standard. In stool specimens, RT-PCR assay sensitivities for LT, ST1a, and ST1b detection were 97%, 100%, and 97%; and specificities were 99%, 94%, and 97%. Pearson correlation coefficient r was 0.9975 between RT-PCR results in stool specimens and the gold standard. Limits of detection of LT, ST1a, and ST1b by RT-PCR assay were 0.1 to1.0 pg/μL and by conventional PCR assay were 100 to1000 pg/μL. The accuracy, rapidity and sensitivity of this RT-PCR assay is promising for ETEC detection in public health/clinical laboratories and for laboratories in need of an independent method to confirm results of other culture independent diagnostic tests.

  8. The Rapid Identification Method of Salmonella Typhi with Taqman Probe Real-Time Fluorescent PCR%食品中伤寒沙门氏菌TaqMan探针实时PCR检测方法

    Institute of Scientific and Technical Information of China (English)

    曹冬梅; 袁慕云; 史媛媛; 刘洋; 许龙岩; 曹际娟

    2014-01-01

    建立实时荧光PCR快速鉴定伤寒沙门氏菌(Salmonella Typhi)的方法。根据GenBank公布的伤寒沙门氏菌基因序列,设计引物和Taqman探针,采用实时荧光PCR进行特异性、灵敏性及模拟样品的检测实验。结果表明,特异性引物和探针可从31株伤寒沙门氏菌菌株、27株其他血清型沙门氏菌和7株非沙门氏菌菌株中鉴定出全部的31株伤寒沙门氏菌。以伤寒沙门氏菌梯度稀释菌液DNA为模板进行实时荧光PCR扩增,菌株模板浓度与Ct值呈良好线性关系,线性系数为0.994,扩增效率为94.5%,最低检测浓度为4cfu/mL的添加浓度。实时荧光PCR检测与传统方法相比较,两者结果一致。该方法特异性好、灵敏度高,可以快速鉴定伤寒沙门氏菌。%A method was developed for Rapid Identification of Salmonella Typhi with real-time fluorescent PCR. According to the gene of Genbank, a set of primers and Taqman probe was designed to perform specific, sensitive and simulation sample tests with real-time PCR. The results showedthe specificity probe correctly distinguished 31 Salmonella Typhi strains from 27 other Salmonella serotypes strains and 7 non-Salmonella strains. Gradient dilutions of Salmonella Typhi were used as template to perform real-time PCR assay which presented linear relationship between the concentration of template and Ct value. Linear coefficient (R2), efficiency and detection limit were 0.994, 94.5%and 4cfu/ml correspondingly. Simulation samples inoculated with Salmonella Typhi were detected with real-time PCR assay. The PCR method yielded a 100%correlation with results obtained by conventional culture method. The new method that showed a high specificity, sensibility and accuracy could be applied for the rapid identification of Salmonella Typhi.

  9. Detection of Exogenous Gene Copies in Transgenic Soybean by Taqman Quantitative PCR Technique%Taqman定量PCR技术检测转基因大豆中外源基因拷贝数

    Institute of Scientific and Technical Information of China (English)

    仇有文; 张明辉; 高学军; 曲波; 敖金霞; 袁育寒; 刘营; 霍楠

    2011-01-01

    [Objective] Taqman Quantitative PCR technique was adopted to detect the copies of exogenous nos terminator in transgenic hybrid soybean. [ Method] Wrth soybean Lectin as the endogenous reference gene, and gene complex DNA in non-GMO soybeans as the endogenous reference standard, the method of gradient dilution was used for separately calculate Ct value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies, and then to calculate the copies of samples through substituting thus-obtained Ct into the standard curve equation. [ Result] The standard curve equation of endogenous reference gene is y = -3.422×+35.201, R2 = 0.998;and the standard curve equation of exogenous gene is y = -3.348x +34.890, R2 =0.999. Nos terminator and its lower boundary sequences in transgenic soybean is of single copy. [ Conclusion] The study has provided a theoretical basis for determining exogenous gene copies in transgenic soybean.%[目的]采用Taqman定量PCR技术检测转基因杂交大豆中外源nos终止子基因的拷贝数.[方法]以大豆凝集素基因为内参照基因,以非转基因大豆基因组DNA为内参照基因标准品,通过梯度稀释法分别求取了内参照基因和质粒DNA的Ct值与拷贝数对数值的相关性标准曲线方程,并通过将得到的Ct值代入标准曲线方程求取了样品的拷贝数.[结果]内参照基因标准曲线方程为y=-3.422x+35.201,R(2)=0.998;外源基因标准曲线方程为y=-3.348x+34.890,R(2)=0.999.nos终止子基因及其下游边界序列在转基因杂交大豆中为单拷贝.[结论]该研究为确定转基因大豆外源基因拷贝数提供厂理论依据.

  10. Detection of Foreign Gene Copies in Transgenic Soybean by Taqman Quantitative PCR Technique%Taqman定量PCR技术检测转基因大豆中外源基因拷贝数

    Institute of Scientific and Technical Information of China (English)

    仇有文; 张明辉; 高学军; 曲波; 敖金霞; 袁肖寒; 刘营; 霍楠

    2011-01-01

    [目的[采用Taqman定量PCR技术检测转基因杂交大豆中外源nos终止子基因的拷贝数.[方法]以大豆凝集素基因为内参照基因,以非转基因大豆基因组DNA为内参照基因标准品,通过梯度稀释法分别求取了内参照基因和质粒DNA的Ct值与拷贝数对数值的相关性标准曲线方程,并通过将得到的Ct值代入标准曲线方程求取了样品的拷贝数.[结果]内参照基因标准曲线方程为y=-3.422x+35.201,R2=0.998;外源基因标准曲线方程为y=-3.348x+34.890,R2=0.999.nos终止子基因及其下游边界序列在转基因杂交大豆中为单拷贝.[结论]为确定转基因大豆外源基因拷贝数提供了理论依据.%[ Objective ] It is to adopt Taqman quantitative PCR technique to detect the copies of foreign nos terminator in transgenic hybrid soybean. [ Method ] With endogenous reference gene of soybean lectin, and endogenous reference standard of gene complex DNA in non-GMO soybeans, the method of gradient dilution was used to separately calculate Ct value of endogenous reference gene and plasmid DNA and relevance standard curve equation of logarithm of copies, and then to calculate the copies of samples through substituting thus-obtained Ct into standard curve equation. [ Result ] The standard curve equation of endogenous reference gene isy = - 3.422x + 35. 201 , R2 = 0. 998; and the standard curve equation of foreign gene is y = - 3. 348x + 34. 890, R2 = 0.999. Nos terminator and its lower boundary sequences in transgenic soybean is of single copy. [ Conclusion] The study has provided a theoretical basis for determining foreign gene copies in transgenic soybean.

  11. Taqman实时定量PCR检测产毒艰难梭菌方法的建立%Detection of toxigenic genes Clostridium difficile by TaqMan real-time quantitative PCR

    Institute of Scientific and Technical Information of China (English)

    吴琳; 王毅谦; 邵景东; 吴福平; 傅春玲

    2011-01-01

    OBJECTIVE To develop a rapid real-time quantitative PCR assay targeting on toxin gene tcdA and tcdB of clostridium difficile. METHODS The special sequence of tcdA and tcdB gene of C. difficile was amplified with a pair primers and Taqman probe. The standard curves of the reaction for the detection of each gene were generated from the standard toxgenic clostridium difficile strain. RESULTS The specificity of each gene was demonstrated by the absence of amplification with DNA purified from bacterial species other than toxigenic C. difficile. Both amplification reactions showed a linear relationship between Ct and DNA amounts which yielded the R values of 0. 9975 and 0. 9984 for tcdA and tcdB gene respectively. And the detecting limit was 2. 5× 10-3. CONCLUSION It is a rapid, special, sensitive, method for quantitative detection of C. difficile and will allow the detection of toxigenic C. difficile in clinical specimens.%目的 建立以毒素基因A/B为靶基因的产毒艰难梭菌的快速定量检测方法.方法 通过设计艰难梭菌毒素A/B基因的特异引物及探针,建立标准产毒菌株DNA(ng)含量与Ct值的标准曲线.结果 该方法仅对产毒艰难梭菌进行特异性扩增,11种其他常见的致病菌及非产毒艰难梭菌均不能扩增; tcdA和tcdB基因扩增标准曲线线性关系R值分别为0.9975、0.9984,检测低限均为2.5×10-3ng.结论 该研究建立的方法具有快速、灵敏、特异性高等优点,可用于艰难梭菌毒素基因的定量检测.

  12. Smallpox and pan-orthopox virus detection by real-time 3'-minor groove binder TaqMan assays on the roche LightCycler and the Cepheid smart Cycler platforms.

    Science.gov (United States)

    Kulesh, David A; Baker, Robert O; Loveless, Bonnie M; Norwood, David; Zwiers, Susan H; Mucker, Eric; Hartmann, Chris; Herrera, Rafael; Miller, David; Christensen, Deanna; Wasieloski, Leonard P; Huggins, John; Jahrling, Peter B

    2004-02-01

    We designed, optimized, and extensively tested several sensitive and specific real-time PCR assays for rapid detection of both smallpox and pan-orthopox virus DNAs. The assays are based on TaqMan 3'-minor groove binder chemistry and were performed on both the rapid-cycling Roche LightCycler and the Cepheid Smart Cycler platforms. The hemagglutinin (HA) J7R, B9R, and B10R genes were used as targets for the variola virus-specific assays, and the HA and DNA polymerase-E9L genes were used as targets for the pan-orthopox virus assays. The five orthopox virus assays were tested against a panel of orthopox virus DNAs (both genomic and cloned) at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID). The results indicated that each assay was capable of detecting both the appropriate cloned gene and genomic DNA. The assays showed no cross-reactivity to the 78 DNAs in the USAMRIID bacterial cross-reactivity panel. The limit of detection (LOD) of each assay was determined to be between 12 and 25 copies of target DNA. The assays were also run against a blind panel of DNAs at the Centers for Disease Control and Prevention (CDC) on both the LightCycler and the Smart Cycler. The panel consisted of eight different variola virus isolates, five non-variola virus orthopox virus isolates, two varicella-zoster virus isolates, and one herpes simplex virus isolate. Each sample was tested in triplicate at 2.5 ng, 25 pg, 250 fg, and 2.5 fg, which represent 1.24 x 10(7), 1.24 x 10(5), 1.24 x 10(3), and 1.24 x 10(1) genome equivalents, respectively. The results indicated that each of the five assays was 100% specific (no false positives) when tested against both the USAMRIID panels and the CDC blind panel. With the CDC blind panel, the LightCycler was capable of detecting 96.2% of the orthopox virus DNAs and 93.8% of the variola virus DNAs. The Smart Cycler was capable of detecting 92.3% of the orthopox virus DNAs and between 75 and 93.8% of the variola virus DNAs

  13. Smallpox and pan-Orthopox Virus Detection by Real-Time 3′-Minor Groove Binder TaqMan Assays on the Roche LightCycler and the Cepheid Smart Cycler Platforms

    Science.gov (United States)

    Kulesh, David A.; Baker, Robert O.; Loveless, Bonnie M.; Norwood, David; Zwiers, Susan H.; Mucker, Eric; Hartmann, Chris; Herrera, Rafael; Miller, David; Christensen, Deanna; Wasieloski, Leonard P.; Huggins, John; Jahrling, Peter B.

    2004-01-01

    We designed, optimized, and extensively tested several sensitive and specific real-time PCR assays for rapid detection of both smallpox and pan-orthopox virus DNAs. The assays are based on TaqMan 3′-minor groove binder chemistry and were performed on both the rapid-cycling Roche LightCycler and the Cepheid Smart Cycler platforms. The hemagglutinin (HA) J7R, B9R, and B10R genes were used as targets for the variola virus-specific assays, and the HA and DNA polymerase-E9L genes were used as targets for the pan-orthopox virus assays. The five orthopox virus assays were tested against a panel of orthopox virus DNAs (both genomic and cloned) at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID). The results indicated that each assay was capable of detecting both the appropriate cloned gene and genomic DNA. The assays showed no cross-reactivity to the 78 DNAs in the USAMRIID bacterial cross-reactivity panel. The limit of detection (LOD) of each assay was determined to be between 12 and 25 copies of target DNA. The assays were also run against a blind panel of DNAs at the Centers for Disease Control and Prevention (CDC) on both the LightCycler and the Smart Cycler. The panel consisted of eight different variola virus isolates, five non-variola virus orthopox virus isolates, two varicella-zoster virus isolates, and one herpes simplex virus isolate. Each sample was tested in triplicate at 2.5 ng, 25 pg, 250 fg, and 2.5 fg, which represent 1.24 × 107, 1.24 × 105, 1.24 × 103, and 1.24 × 101 genome equivalents, respectively. The results indicated that each of the five assays was 100% specific (no false positives) when tested against both the USAMRIID panels and the CDC blind panel. With the CDC blind panel, the LightCycler was capable of detecting 96.2% of the orthopox virus DNAs and 93.8% of the variola virus DNAs. The Smart Cycler was capable of detecting 92.3% of the orthopox virus DNAs and between 75 and 93.8% of the variola virus DNAs. However

  14. 肝螺杆菌TaqMan MGB探针实时荧光定量PCR快速检测方法的建立及应用研究%Develonment and application of TaqMan MGB probe real-time fluorescence quantitative PCR for rapid detection of Helicobacter hepaticus

    Institute of Scientific and Technical Information of China (English)

    高正琴; 邢进; 冯育芳; 岳秉飞; 贺争鸣

    2011-01-01

    目的 建立特异、敏感、快速检测肝螺杆菌的TaqMan MGB探针实时荧光定量PCR方法.方法 针对肝螺杆菌flaB 基因的保守区设计特异性引物和探针,建立肝螺杆菌TaqMan MGB探针实时荧光定最PCR方检测方法,验证方法的特异性、敏感性和稳定性.对2008-2011年期间采集的1081份临床样本中的肝螺杆菌进行检测,同时进行分离培养和常规PCR检测.结果 建立的TaqMan MGB探针实时荧光定量PCR方法对肝螺杆菌的检测具有高度的特异性,对幽门螺杆菌、空肠弯曲菌、泰泽氏菌、侵肺巴斯德氏菌、大肠埃希菌、铜绿假单胞菌均无交叉反应,检测的灵敏度达8.3拷贝.标准曲线显示各浓度范围内具有良好的线性关系,相关系数为0.999,斜率为-3.227,TaqManMGB探针实时荧光定量PCR效率为100%.对1081份临床样本进行检测,TaqMan MGB探针实时荧光定量PCR和常规PCR均能检出86份肝螺杆菌阳性样本,而细菌分离培养则仅检出4份阳性.结果显示,建立的TaqMan MGB探针实时荧光定量PCR方法比细菌分离培养方法更敏感,能够直接从临床样本中检出肝螺杆菌DNA,检测时间仅为2h.结论 研究建立的TaqMan MGB探针实时荧光定量PCR方法具有可靠、特异、敏感的特点,适用于肝螺杆菌的快速检测.%Objective To develop a TaqMan MGB probe-based,sensitive and specific real-time fluorescence quantitative PCR assay for rapid detection of Helicobacter hepaticus.Methods Primers and probes specific toflaB gene of Helicobacter hepaticus were designed.A TaqMan MGB probe-based,real-time fluorescence quantitative PCR was established.The specificity,sensitivity and stability of the assay were assassed.Then,the established TaqMan MGB probe real-time fluorescence quantitative PCR assay was applied to detect Helicobacter hepaticus in 1081 clinical specimens during 2008-2011,compared with bacterial isolation and culture method and conventional PCR assay

  15. Sequence heterogeneity in the equi merozoite antigen gene (ema-1) of Theileria equi and development of an ema-1-specific TaqMan MGB assay for the detection of T. equi.

    Science.gov (United States)

    Bhoora, Raksha; Quan, Melvyn; Matjila, Paul T; Zweygarth, Erich; Guthrie, Alan J; Collins, Nicola E

    2010-08-27

    Although a quantitative real-time PCR assay (qPCR) assay for the detection of Theileria equi has been developed and evaluated, it is possible that additional, as yet undetected 18S rRNA gene sequence variants may exist. A qPCR assay targeting a different gene, used in conjunction with the T. equi 18S rRNA qPCR assay, could assist in the detection of all T. equi genotypes in field samples. A T. equi ema-1-specific qPCR (Ueti et al., 2003) was tested on 107 South African field samples, 90 of which tested positive for T. equi antibody using the immuno-fluorescent antibody test (IFAT). The qPCR assay performed poorly, as T. equi was detected in only 67 of the 90 IFAT-positive field samples at quantification cycle (C(q)) values ranging from 27 to 39.95. Furthermore, a high C(q) value of 36.18 was obtained from DNA extracted from a South African in vitro-cultured T. equi WL isolate [1.38% parasitized erythrocytes (PE)] when a low C(q) value (indicative of a high T. equi DNA concentration) was expected. Approximately 600 bp of the ema-1 gene from 38 South African samples were sequenced and BLASTN analysis confirmed all sequences to be merozoite surface protein genes, with an identity of 87.1-100% to previously published T. equi ema-1 gene sequences. Alignment of the sequences revealed extensive sequence variations in the target regions of the primers and probes (Ueti et al., 2003), explaining the poor performance of the qPCR assay. Based on these observations, we developed a new TaqMan minor-groove binder (MGB) probe-based qPCR assay, targeting a more conserved region of the ema-1 gene. This assay was shown to be efficient and specific, and the detection limit, defined as the concentration at which 95% of T. equi-positive samples are detected, was determined to be 1.4 x 10(-4)% PE. The two ema-1 assays were compared by testing 41 South African field samples in parallel. The results suggested that the new assay was more sensitive than the original assay, as T. equi was

  16. Comparision of clinical diagnostic value between PCR and TaqMan RT-PCR for Mycoplasma pneumoniae in throat swabs%PCR及RT-PCR检测咽拭子标本肺炎支原体的比较

    Institute of Scientific and Technical Information of China (English)

    李淳; 吴移谋; 朱翠明; 钟礼立; 陈丹; 吕建华

    2012-01-01

    To compare the clinical diagnostic value between PCR and RT PCR for M. Pneumoniae in swab, we per formed both PCR and RT PCR assays analysis for M. Pneumoniae DNA on a total of 566 samples of throat swab from 106 ped iatric children with M. Pneumoniae and in whom M. Pneumoniae was suspected. Among the 566 pediatric children, there were 45(7. 95%) PCR positive specimens and 175(30. 92%) RT PCR positive specimens. In the 106 pediatric children with M. Pneumoniae, 5 were positive for PCR, and 95 were positive for RT PCR. In the 460 pediatric children with symptom of M. Pneumoniae, 40 were positive for PCR, and 80 were positive for RT PCR. The sensitivy of Rt PCR for M. Pneumoniae detec tion appeared to be better than that of PCR. (sensitivity . RT PCR 89. 62 % , PCR 4. 72 % , x2=146. 322, P = 0. 000), but there was no significant difference in the specificity between RT PCR and PCR (specifitivity: RT PCR 82. 60 %, PCR 91. 30%,x2 - 3. 331, P - 0. 068). It is concluded that the TaqMan based RT PCR assay is a rapid, sensitive and specific meth od for the detection of M. Pneumoniae in throat swabs of children in early period of diagnosis.%目的 应用聚合酶链反应(PCR)与实时taqMan荧光定量PCR(RT-PCR)检测咽拭子标本中的肺炎支原体DNA(Mp-DNA),比较2种方法检测结果的临床诊断价值.方法 随机选取I临床儿科门诊患儿566例,包括临床治诊Mp感染患儿106例和临床疑似Mp感染忠儿460例,分别采用PCR法和RT-PCR法检测,以临床治诊Mp作为参照标准,采用x2检验评定2种检测方法诊断的灵敏度和特异度,比较2种检测方法对Mp的诊断价值.结果 566份受检患儿的咽拭子标本中,PCR法检测阳性45例(7.95%)(临床治诊Mp感染患儿5例,临床疑似Mp感染患儿40例),RT-PCR法检测阳性175例(30.92%)(临床治诊Mp感染患儿95例,临床疑似Mp感染患儿80例).RT-PCR法检测咽拭子Mp-DNA诊断Mp感染的敏感度显著高于PCR法(敏感度RT-PCR 89.62%,PCR 4.72%,x2=146.322,P

  17. DEVELOPMENT OF DUPLEX TAQMAN REAL-TIME PCR TO DETECT CTX AND TDH%霍乱毒素基因(ctx)和耐热直接溶血素基因(tdh)双重TaqMan实时PCR检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    韩辉; 李海山; 胡群; 姚李四; 谭绪良; 贾琳

    2011-01-01

    [目的]建立霍乱毒素和耐热直接溶血素基因双重TaqMan实时-PCR实验室检测方法.[方法]根据霍乱毒素基因(Cholera toxin gene,ctx)和耐热直接溶血素基因(thermostable direct hemolysin,tdh)的保守序列设计引物和TaqMan探针,建立检测霍乱毒素和耐热直接溶血素两种毒力基因的双重TaqMan实时PCR方法.对所建立的霍乱毒素基因和耐热直接溶血素基因双重TaqMan实时PCR检测方法进行灵敏度和特异度评价.[结果]建立了霍乱毒素基因和酎热直接溶血素基因双重TaqMan实时PCR的实验室检测方法.优化的tdh和ct双重TaqMan实时PCR反应体系中,ct引物和探针的浓度分别为200 nmol/L和100 nmol/L;tdh引物和探针的浓度分别为200 nmoL/L和100 nmol/L.反应体系的灵敏度和特异度均为100%.优化的反应体系对两种质粒模板的检测下限均为1.0x10(2)拷贝/μl,扩增效率分别为94%和97.7%.[结论]本研究建立了基于TaqMan探针的tdh和ct双重实时PCR检测方法,具有令人满意的灵敏度和特异度.检测下限能达到1.0x10(2)拷贝/μl,高于普通PCR 100倍.并且双重实时PCR能够在一个反应体系中同时检测两种毒力基因,这为费时又繁琐的传统检测方法提供了一种可靠又快速的替代选择.%[Objective] To develop a duplex TaqMan real-time PCR for the detection of ctx and tdh. [Methods] The conserved region of TDH and CTX gene were used to design primers and probes, and the duplex TaqMan real-time PCR system of detecting ctx and tdh was established. The sensitivity and specificity of duplex TaqMan real-time PCR system was evaluated. [Results] The duplex TaqMan real-time PCR system detecting ctx and tdh-was established. In the optimized reaction system of ctx and tdh duplex TaqMan real-time PCR, the concentrations of ctx primers and probe were 200 nmol/L and 100 nmol/L, respectively; the concentrations of tdh primers and probe were 200 nmol/L and 100 nmol/L, respectively. The

  18. Diagnostic challenges of tuberculous lymphadenitis using polymerase chain reaction analysis: a case study.

    Science.gov (United States)

    Taniguchi, Hirokazu; Nakamura, Masahiko; Shimokawa, Kazuki; Kamiseki, Fumi; Ishizawa, Shin; Abo, Hitoshi; Furuse, Hideaki; Tsuda, Takeshi; Masaki, Yasuaki; Suzuki, Kensuke

    2015-01-01

    This report presents a case of tuberculous lymphadenitis that was difficult to diagnose using polymerase chain reaction analysis. An 80-year-old Japanese female was hospitalized due to swollen cervical lymph nodes. Her lymph node tests revealed paradoxical polymerase chain reaction results. Polymerase chain reaction analysis of two biopsy tissues using the Cobas TaqMan revealed a positive result for Mycobacterium avium and a negative result for Mycobacterium tuberculosis. However, polymerase chain reaction analysis of a cultured colony of acid-fast bacteria from biopsy tissue using the Cobas TaqMan and an alternative polymerase chain reaction analysis of biopsy tissue yielded discordant results. The patient was diagnosed as having tuberculous lymphadenitis. She was treated with antitubercular drugs and subsequently had a reduction in cervical lymph node swelling. Polymerase chain reaction analysis is not 100% accurate; hence, its use as a diagnostic tool for mycobacterial infection requires increased attention.

  19. Early detection of Mycobacterium tuberculosis complex in BACTEC MGIT cultures using nucleic acid amplification.

    Science.gov (United States)

    Lin, S Y; Hwang, S C; Yang, Y C; Wang, C F; Chen, Y H; Chen, T C; Lu, P L

    2016-06-01

    We evaluated the application of nucleic acid amplification (NAA) in liquid cultures for the early detection of Mycobacterium tuberculosis. The Cobas TaqMan MTB test, IS6110 real-time PCR, and hsp65 PCR-restriction fragment length polymorphism (RFLP) analysis were used to detect BACTEC MGIT 960 (MGIT) cultures on days 3, 5, 7, and 14. The procedure was initially tested with a reference strain, H37Rv (ATCC 27294). Subsequently, 200 clinical specimens, including 150 Acid Fast bacillus (AFB) smear-positive and 50 AFB smear-negative samples, were examined. The Cobas TaqMan MTB test and IS6110-based PCR analysis were able to detect M. tuberculosis after 1 day when the inoculum of H37Rv was >3 x 10(-2) CFU/ml. After a 5-day incubation in the MGIT system, all three NAA assays had a positive detection regardless of the inoculum size. After a 1-day incubation of the clinical specimens in the MGIT system, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for the Cobas TaqMan MTB assay were 70.2%, 100%, 100%, and 82.3% respectively. For IS6110-based PCR analysis, these values were 63.1%, 100%, 100%, and 78.9%, and were 88.1%, 100%, 100%, and 92.1% respectively for hsp65 PCR-RFLP analysis. After a 3-day incubation, the specificity and PPV were 100% for all three NAA tests; the Cobas TaqMan MTB assay had the best sensitivity (97.6%) and NPV (98.3%). The sensitivity, specificity, PPV, and NPV for conventional culture analysis were 98.8%, 100%, 100%, and 99.1%. Thus, NAA may be useful for the early detection of M. tuberculosis after 3 days in MGIT.

  20. 弗氏枸橼酸杆菌TaqMan实时荧光定量-聚合酶链反应检测方法的建立%Establishment of novel real-time TaqMan PCR assay for detection of Citrobacter freundii

    Institute of Scientific and Technical Information of China (English)

    金东; 王艺婷; 白雪梅; 叶长芸; 刘丽云

    2013-01-01

    目的 建立针对弗氏枸橼酸杆菌的TaqMan实时荧光定量-聚合酶链反应(real time-PCR)检测方法.方法 针对弗氏枸橼酸杆菌的特有序列设计引物和TaqMan探针,扩增目的基因建立标准曲线,确定检测方法的灵敏度;对20种其他肠道致病菌及院内感染中常见的致病菌进行检测,评价该检测方法的特异性;使用牛奶模拟标本评价方法在实际检测工作中应用性.结果 TaqMan real time-PCR检测方法对弗氏枸橼酸杆菌重组质粒的检测灵敏度为1.0×101拷贝/反应体系;该检测方法在检测30种其他肠道致病菌及院内感染中常见的致病菌时未出现特异性扩增.该检测方法对牛奶模拟样本中弗氏枸橼酸杆菌检测下限为1.0×102cfu/ml的菌量;通过对1.0×107、1.0×105和1.0×103三个浓度质粒标准品的重复检测,确定本方法的组内变异系数为1.90%~3.91%;组间变异系数为1.52% ~ 1.69%.结论 本研究建立的TaqMan real time-PCR检测方法可作为检测弗氏枸橼酸杆菌灵敏、特异、快速的方法.%Objective To establish a real-time TaqMan polymerase chain reaction (PCR) assay for the detection of Citrobacter freundii.Methods Primers and probe were designed based on the sequences of tricarboxylic transport (tct)gene.The target gene was cloned to pMD20-T vector to build the standard curve of this assay and evaluate the sensitivity of the assay.The specificity was evaluated by using 20 other enteropathogenic bacteria and isolates causing nosocomial infection.Results Sensitivity test of recombinant plasmids showed that the sensitivity could reach 1 × 101copies /reaction.Specificity test showed that no specific amplifications were presented for the 20 other enteropathogenic bacteria and the isolates causing nosocomial infection.The detection limit of this assay for artificially contaminated milk was 1.0 × 102cfu/ml.Conclusion This real-time TaqMan PCR assay is sensitive and specific for

  1. Use of the duplex TaqMan MGB probe for simultaneous detection of Perkinsus and Bonamia in marine shellfish%同时检测海洋贝类包纳米虫和派琴虫的双重TaqMan MGB探针实时荧光 PCR方法

    Institute of Scientific and Technical Information of China (English)

    郭书林; 陈信忠; 肖懿哲; 朱苏琴; 龚艳清; 杨俊萍

    2014-01-01

    A duplex TaqMan MGB real-time PCR method was optimized to simultaneously detect Perkinsus sp.and Bonamia sp..The primers and TaqMan MGB probes were designed and chosen to amplify the conserved SSU seg-ment of genus Bonamia sp.ribosomal DNA and ITS segment of genus Perkinsus sp.ribosomal DNA.The duplex real-time PCR identified and differentiated the two protozoan parasite groups.The sensitivity of the duplex real-time PCR assay was 446 and 171 template copies and it had higher sensitivity.Tenfold serial dilutions of the plasmid DNAs of Bonamia sp.and Perkinsus sp.were quantified the actual copy numbers using the duplex real-time PCR.The corre-lation coefficient of calibration curves were 0.999 and 1 .000,respectively.Meanwhile,this method had no cross reaction with other species of protozoa in mollusks and the common pathogenic bacteria in mariculture.The method showed advantages of rapid and high efficiency when applied to detect 296 clinical specimens from Meizhou bay, Pinghai bay and Xinghua bay of Fujian.This assay is proved to be sensitive and specific and can be widely used for the protozoan infection survey,disease surveillance and the quarantine of shell fish.%根据包纳米虫(Bonamia sp.)SSU rDNA和派琴虫(Perkinsus sp.)ITS rDNA的保守区序列,设计特异性引物和Taqman MGB探针,建立了同时检测上述两种贝类原虫的双重实时荧光PCR方法.该方法可检测到包纳米虫基因的446拷贝质粒,以及派琴虫基因的171拷贝质粒,具有较高的灵敏度.以10倍系列稀释的包纳米虫阳性标准品质粒和派琴虫阳性标准品质粒为模板,测得该方法的定量标准曲线相关系数分别为0.999和1.000,显示出很好的扩增效率.同时该方法与尼氏单孢子虫(Haplosporidium nelsoni)、沿岸单孢子虫(H.costale)等其他贝类原虫,以及副溶血性弧菌(Vibrio parahaemolyticus)、迟缓爱德华氏菌(Edwardsiella tarda)和#爱德华氏菌

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

    Institute of Scientific and Technical Information of China (English)

    毛立; 李文良; 李彬; 江杰元

    2012-01-01

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

  3. Comparative evaluation of the performance of the Abbott RealTime HIV-1 assay for measurement of HIV-1 plasma viral load on genetically diverse samples from Greece

    Directory of Open Access Journals (Sweden)

    Paraskevis Dimitrios

    2011-01-01

    Full Text Available Abstract Background HIV-1 is characterized by increased genetic heterogeneity which tends to hinder the reliability of detection and accuracy of HIV-1 RNA quantitation assays. Methods In this study, the Abbott RealTime HIV-1 (Abbott RealTime assay was compared to the Roche Cobas TaqMan HIV-1 (Cobas TaqMan and the Siemens Versant HIV-1 RNA 3.0 (bDNA 3.0 assays, using clinical samples of various viral load levels and subtypes from Greece, where the recent epidemiology of HIV-1 infection has been characterized by increasing genetic diversity and a marked increase in subtype A genetic strains among newly diagnosed infections. Results A high correlation was observed between the quantitative results obtained by the Abbott RealTime and the Cobas TaqMan assays. Viral load values quantified by the Abbott RealTime were on average lower than those obtained by the Cobas TaqMan, with a mean (SD difference of -0.206 (0.298 log10 copies/ml. The mean differences according to HIV-1 subtypes between the two techniques for samples of subtype A, B, and non-A/non-B were 0.089, -0.262, and -0.298 log10 copies/ml, respectively. Overall, differences were less than 0.5 log10 for 85% of the samples, and >1 log10 in only one subtype B sample. Similarly, Abbott RealTime and bDNA 3.0 assays yielded a very good correlation of quantitative results, whereas viral load values assessed by the Abbott RealTime were on average higher (mean (SD difference: 0.160 (0.287 log10 copies/ml. The mean differences according to HIV-1 subtypes between the two techniques for subtype A, B and non-A/non-B samples were 0.438, 0.105 and 0.191 log10 copies/ml, respectively. Overall, the majority of samples (86% differed by less than 0.5 log10, while none of the samples showed a deviation of more than 1.0 log10. Conclusions In an area of changing HIV-1 subtype pattern, the Abbott RealTime assay showed a high correlation and good agreement of results when compared both to the Cobas TaqMan and bDNA 3

  4. 戊型肝炎病毒TaqMan Real-time RT-PCR法的建立及应用%Establishment and application of TaqMan real-time RT-PCR for the detection of hepatitis E virus

    Institute of Scientific and Technical Information of China (English)

    孟庆玲; 邱丰; 沈立萍; 毕胜利

    2012-01-01

    目的 建立灵敏、特异、稳定的戊型肝炎病毒(HEV) TaqMan Real-time PCR检测方法.方法 根据GenBank中的HEV相关序列,选取HEV基因组ORF2的保守区域设计合成特异性引物和探针,建立TaqMan HEV Real-time RT-PCR检测体系,评价体系的特异性、敏感度和稳定性,并应用于临床样本的检测.结果 本研究建立的HEV Real-time RT-PCR检测体系最低检测极限达到10个拷贝/反应,重复性实验Ct值的变异系数(CV)最大为1.53%,并且该体系能特异检测出戊肝临床样本中的HEV,其拷贝数从1.87×104拷贝/ml到8.12×106拷贝/ml不等.结论 成功建立特异性强、灵敏度高的HEV Real-time RT-PCR检测方法,应用于临床样本检测时取得了良好效果,为HEV分子病原学诊断打下基础.%Objective To establish a specific TaqMan-based Real-time PCR assay for the detection of hepatitis E virus (HEV).Methods According to the references,primers-probe sets which were located in ORF2,the conservative part of HEV genome were designed and therefore we established a HEV TaqMan real-time RT-PCR assay with great performance of specificity,sensitivity and reproducibility.And then it was used in the detection of HEV RNA in clinical samples.Results The HEV Real-time RT-PCR assay established in this study were able to detect HEV RNA with a detection limit of 10 copies/reaction.When the detection of a same sample was repeated for several times,coefficients of variation (CV) was all less than 1.53%.Our data also suggested that there were 1.87 × 106-8.12 × 109 RNA copies in 1ml of the clinical samples.Conclusion The TaqMan-based Real-time PCR assay established in this study was specific and precise for the rapid detection of HEV RNA.It was applied successfully in the pathogen detection of clinical samples.

  5. Establishment and application of a multiplex TaqMan real-time RT-POR assay for detecting porcine proinflammatory cytokines%猪促炎细胞因子多重TaqMan荧光定量RT-PCR检测方法的建立及应用

    Institute of Scientific and Technical Information of China (English)

    施开创; 梁媛; 陈芳芳; 屈素洁; 莫胜兰; 李军

    2012-01-01

    This study is to establish and apply quantitative methods for detecting the mRNA expression of porcine proinflammatory cytokine.In order to study the pathogenesis of encephalomyocarditis virus(EMCV) in molecular level,a recombinant plasmid containing the fragment of target gene,i.e.porcine IL-1β,IL-6 and TNF-α genes and housekeeping gene β-actin,were constructed as standard control.Thereafter,one multiplex real-time RT-PCR assay based on TaqMan probe for detection of IL-1β/β-actin,IL-6/β-actin,TNF-α/β-actin genes was established.The correlation coefficient of the standard curves was over 0.998;The detection limit reached 10 copies/μL of initial templates;The fluorescent signals could only be detected by the reaction with cDNA,specific primer and probe for each cytokine;The coefficient of variation was less than 2 percent for both intra-and inter-assay.The established assays were successfully used to detect IL-1β,IL-6 and TNF-α mRNA expression levels in heart tissue from piglets experimentally infected with porcine EMCV GXLC strain.The multiplex TaqMan real-time RT-PCR could be used as an effective tool for detection and quantification of these proinflammatory cytokines with high sensitivity,specificity and reproducibility.%为建立及应用定量检测猪促炎细胞因子mRNA表达水平的方法,从分子水平研究脑心肌炎病毒(EMCV)的致病机制,分别构建含有猪促炎细胞因子IL-1β、IL-6、TNF-α以及管家基因β-actin基因片段的重组质粒标准品,建立了检测IL-1β/β-actin、IL-6/β-actin、TNF-α/β-actin的多重TaqMan real-time PCR检测方法。标准曲线的相关系数均达到0.998以上;初始模板的检出下限均达到10拷贝/μL;只有以目标cDNA为模板,并加入特异性引物和探针的反应才能检测到荧光信号;组内与组间的变异系数均小于2%。应用所建立的检测方法,对猪源EMCV GXLC株感染仔猪心肌中IL-1β、TNF-α、IL-6mRNA的表达水平进行检测

  6. Comparative analysis of real-time quantitative PCR-Sanger sequencing method and TaqMan probe method for detection of KRAS/BRAF mutation in colorectal carcinomas%即时定量PCR-Sanger测序与TaqMan探针法检测结直肠癌KRAS、BRAF基因突变的对比分析

    Institute of Scientific and Technical Information of China (English)

    张汛; 王跃华; 高宁; 王晋芬

    2014-01-01

    Objective To compare the application values of real-time quantitative PCR-Sanger sequencing and TaqMan probe method in the detection of KRAS and BRAF mutations,and to correlate KRAS/BRAF mutations with the clinicopathological characteristics in colorectal carcinomas.Methods Genomic DNA of the tumor cells was extracted from formalin fixed paraffin embedded (FFPE) tissue samples of 344 colorectal carcinomas by microdissection.Real-time quantitative PCR-Sanger sequencing and TaqMan probe method were performed to detect the KRAS/BRAF mutations.The frequency and types of KRAS/BRAF mutations,clinicopathological characteristics and survival time were analyzed.Results KRAS mutations were detected in 39.8% (137/344) and 38.7% (133/344) of 344 colorectal carcinomas by using real-time quantitative PCR-Sanger sequencing and TaqMan probe method,respectively.BRAF mutation was detected in 4.7% (16/344) and 4.1% (14/344),respectively.There was no significant correlation between the two methods.The frequency of the KRAS mutation in female was higher than that in male (P <0.05).The frequency of the BRAF mutation in colon was higher than that in rectum.The frequency of the BRAF mutation in stage Ⅲ-Ⅳ cases was higher than that in stage Ⅰ-Ⅱ cases.The frequency of the BRAF mutation in signet ring cell carcinoma was higher than that in mucinous carcinoma and nonspecific adenocarcinoma had the lowest mutation rate.The frequency of the BRAF mutation in grade Ⅲ cases was higher than that in grade Ⅱ cases (P < 0.05).The overall concordance for the two methods of KRAS/BRAF mutation detection was 98.8% (kappa =0.976).There was statistic significance between BRAF and KRAS mutations for the survival time of colorectal carcinomas (P =0.039).There were no statistic significance between BRAF mutation type and BRAF/KRAS wild type (P =0.058).Conclusions (1) Compared with real-time quantitative PCR-Sanger sequencing,TaqMan probe method is better with regard to handling time

  7. USE OF TAQMAN TO ENUMERATE ENTEROCOCCUS FAECALIS IN WATER

    Science.gov (United States)

    The Polymerase Chain Reaction (PCR) has become a useful tool in the detection of microorganisms. However, conventional PCR is somewhat time-consuming considering that additional steps (e.g., gel electrophoresis and gene sequencing) are required to confirm the presence of the tar...

  8. TaqMan Real-Time Polymerase Chain Reaction and ...

    African Journals Online (AJOL)

    against the development of alcoholism. .... heating block. .... Native Americans. Turkish. Spanish. Uzbekistan. Korean. Indian. Polish. 22.30 ... This work was supported by grants from Natural ... 59th World Medical Association Assembly, 2008.

  9. Establishment and applying of TaqMan real-time PCR for detection and identification of Haemophilus influenzae and Streptococcus pneumoniae%TaqMan荧光定量PCR检测流感嗜血杆菌和肺炎链球菌方法的建立及应用

    Institute of Scientific and Technical Information of China (English)

    朱兵清; 李马超; 徐丽; 任红宇; 田国忠; 高源; 邵祝军

    2009-01-01

    Objective To establish TaqMan real-time PCR method for detection and identification of Haemophilus influenzae and Streptococcus pneumonia. Methods Two sets of primers and FAM-labeled probes targeting different genes of Haemophilus influenzae and Streptococcus pneumoniae were designed and synthesized. The bexA gene was used for identification of Haemophilus influenzae and lytA for Streptococcus pneumoniae. The sensitivity and specificity of real-time PCR were assessed for different primers and probes. Cut-off values of cycle threshold (Ct) were determined. Two hundred and seventy-eight cerebrospinal fluid (CSF) specimens from suspected bacterial meningitis cases were detected by real-time PCR assay, latex agglutination test and bacteria culture simultaneously. Results Haemophilus influenzae isolates of serotype a to d could be detected and identified by bexA primers and probe. All Streptococcus pneumoniae isolates of different serotypes could be detected and identified by lytA primers and probe. The respective sensitivities for Haemophilus influenzae and Streptococcus pneumoniae were 10 and 90 genome DNA copies in each PCR reaction. Of the 278 CSF specimens, four were positive by Haemophilus influenzae and seven positive by Streptococcus pneumoniae when detected by real-time PCR. Of the four Haemophilus influenzae positive specimens, two were positive by culture and one positive hy latex. Of the seven Streptococcus pneumonia positive specimens, two were positive by culture and two positive by latex. Conclusions Real-time PCR could rapidly detect and identify Haemophilus influenzae of serotype a to d and Streptococcus pneumoniae of different serotypes with high sensitivity. TaqMan real-time PCR could be widely used for the diagnosis of invasive meningitis caused by Haemophilus influenzae and Streptococcus pneumoniae. It can improve the rate of positivity for diagnosis of suspicious bacterial meningitis cases.%目的 建立TaqMan荧光定量PCR检测方法 ,用于流

  10. 小鼠IL-1β、TNF-αTaqMan荧光定量RT-PCR检测方法的建立及脑心肌炎病毒感染小鼠的检测%Development of a TaqMan real-time PCR assay for detection of IL-1β and TNF-α mRNA in mice experimentally infected with encephalomyocarditis virus

    Institute of Scientific and Technical Information of China (English)

    陈宏备; 施开创; 李向涛; 郑敏; 郑喜邦; 李军

    2011-01-01

    In this study, a real-time RT-PCR assay based on TaqMan probe for detection of mouse proinflammatory cytokine gene IL-1 p and TNF-α was established, respectively. The assays were highly specific, sensitive and reproducible, of which the correlation coefficient of the standard curve was over 0.998, the sensitivity was 10 copies/μL of standard recombinant plasmid and the coefficient of variation was less than 2 percent for both intra-assay and inter-assay. The established assays were used to detect IL-1 P and TNF-a mRNA levels in brain, heart and spleen tissues of mice experimentally infected with porcine encephalomyocarditis virus (EMCV) GXLC strain. The results showed that IL-1β and TNF-α mRNA expression levels reached peak value at 4 day post EMCV infection, with a time correlation between the expression levels and the mortality of infected mice. The results indicated that the TaqMan real-time PCR assay could be used as an effective tool for detection and quantification of these proinflammatory cytokines.%为探讨脑心肌炎病毒(EMCV)感染后促炎细胞因子的表达水平、从分子水平深入研究EMCV的致病机制,本研究分别建立了检测小鼠IL-1β、TNF-α和管家基因β-actin的TaqMan real-time PCR检测方法.该方法标准曲线的相关系数均达到0.998以上,检出下限均达到10 copies/μL质粒标准品,组内与组间的变异系数均小于2%.应用该方法对猪源EMCV GXLC株人工感染小鼠的脑、心、脾中IL-1 β、TNF-α mRNA的转录水平进行检测,发现感染后第4d IL-1β、TNF-α mRNA的转录水平达到峰值,并且与小鼠发病死亡高峰存在明显的时间相关性.本研究所建立的TaqMan real-time PCR检测方法为小鼠促炎细胞因子的检测及定量分析提供了技术手段.

  11. Detection of foodborne Salmonella typhimurium and Salmonella enteritidis by diplex real-time PCR using TaqMan probe%基于TaqMan探针双重荧光PCR检测食品中鼠伤寒沙门菌和肠炎沙门菌

    Institute of Scientific and Technical Information of China (English)

    袁慕云; 许龙岩; 曹际娟; 阳静; 张旺; 陈碧玲; 相大鹏

    2013-01-01

    Objective To establish a method to detect Salmonella typhimurium (ST) and Salmonella enteritidis (SE) simultaneously with a dual real-time PCR assay using double-color fluorescent TaqMan probes.Methods The primers and probes were designed based on the conservative domain of STM4599 sequence of ST (GenBank:AERV01000023.1) and the specific sequence of SE (GenBank:AF370707.1)respectively.The probes were labeled with reporter dye FAM for ST or VIC for SE at the 5' end.The dual real-time fluorescence PCR assay was set up and conditions were modified.Results The dual real-time fluorescence PCR method for ST and SE was developed successfully.ST and SE specific primers and probes amplified 16 SE and 15 ST strains,while other 28 different Sa serotypes and 17 negative control Proteus strains showed negative results.The amplification efficiency of ST and SE with the dual fluorescent PCR were all 94.2% and R2 were 0.998 and 0.995 respectively,while the minimum detectable concentration reached 300 CFU/ml for ST and 260 CFU/ml for SE.The entire test can be completed within 31 hours.Conclusion The method is highly specific,sensitive,and fast.The present study thus provides a rapid and effective method to detect ST and SE simultaneously from food samples.%目的 建立基于TaqMan探针双重荧光PCR检测鼠伤寒沙门菌和(Salmonella typhimurium,ST)和肠炎沙门菌(Salmonella enteritidis,SE)的方法.方法 根据ST的STM4599序列(GenBank:AERV01000023.1)和SE特异序列(GenBank:AF370707.1),分别设计引物和探针,ST探针的5′端标记FAM、SE探针的5′端标记VIC,建立基于TaqMan探针双重荧光PCR检测方法.结果 ST和SE的引物和探针分别特异性地扩增出16株ST和15株SE,而28种不同血清型沙门菌和17株变形杆菌等扩增结果均为阴性.ST和SE的双重荧光PCR扩增效率均为94.2%,R2分别为0.998和0.995,最低检测浓度分别达到300 CFU/ml、260 CFU/ml.结论 建立的方法特异性好、

  12. 牛病毒性腹泻病毒和牛轮状病毒TaqMan二重实时荧光RT-PCR检测方法的建立%Detection of bovine viral diarrhea virus and bovine rotavirus by TaqMan based real-time RT-PCR

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

    根据牛病毒性腹泻病毒(BVDV)5′端非编码区和牛轮状病毒(BRV)VP6基因序列,设计特异性引物和探针。通过对引物和探针浓度、Mg2+浓度、dNTP浓度和Taq酶用量以及反应条件等因素的优化筛选,建立了能同时鉴别BVDV和BRV的二重荧光RT-PCR方法。该方法特异性好,与其他病原如CSFV、MB和IBRV不发生交叉反应;敏感性高,能够检测100个BVDV RNA和100个BRV RNA;稳定性好,批内重复和批间重复变异系数小;干扰性试验表明该方法能同时检测2个模板的不同浓度组合。本研究建立的二重荧光RT-PCR方法可用于BVDV和BRV检测,具有特异、敏感、快速、稳定等优点,是BVDV和BRV基础研究、流行病学调查和临床检测的良好工具。%Two pairs of primers and two TaqMan probes were designed and synthesized according to the conserved gene sequence of BVDV 5′ untrascription region and BRV VP6.The reaction parameters such as the concentration of two pair of primers,two probes and other conditions were optimized to develop a duplex real-time RT-PCR assay for rapid detection of BVDV and BRV.It was found that the specificity of this assay was high,and be able to detected BVDV and BRV without other any cross-reactions to CSFV,MB and IBRV.The detection limit of the real-time RT-PCR assay was 100 copies of BVDV viral RNA and BRV viral RNA,indicating a good sensitivity of the assay.The coefficients of variation were both low for the intra-assay and inter-assay tests respectively,indicating a good reliability.When different concentration of BVDV and BRV was mixed together,the result was without any interference.All the resuls indicate that this duplex real-time PCR assay is a specific,sensitive,rapid and reproducible method for detection of BVDV and BRV,and is could applied in fundamental research,clinical detection and epidemiological investigation of BVDV and BRV.

  13. COBAS® TaqMan® MTB, smear positivity grade and MGIT culture; correlation analyses of three methods for bacillary quantification.

    Science.gov (United States)

    Chikamatsu, Kinuyo; Aono, Akio; Kato, Tomoko; Takaki, Akiko; Yamada, Hiroyuki; Sasaki, Yuka; Izumi, Kiyohiko; Yi, Lina; Mitarai, Satoshi

    2016-01-01

    We investigated the correlation between the cycle threshold (Ct) value of the COBAS(®) TaqMan(®) MTB (TaqMan MTB), the mycobacterial smear positivity grade, and the time to detection (TTD) in the Mycobacteria Growth Indicator Tube (MGIT) for quantification of Mycobacterium tuberculosis (MTB). For 57 sputum samples, significant correlations were observed between the Ct value, the smear positivity grade, and the MGIT TTD (Spearman's rank correlation coefficient: r(s) = -0.940, P real-time PCR system, for diagnostic samples.

  14. Routine screening of blood donations at Qingdao central blood bank, China, for hepatitis B virus (HBV) DNA with a real-time, multiplex nucleic acid test for HBV, hepatitis C virus, and human immunodeficiency virus Types 1 and 2.

    Science.gov (United States)

    Yang, Zhongsi; Xu, Lei; Liu, Li; Feng, Qiuxia; Zhang, Longmu; Ma, Weijuan; Saldanha, John; Wang, Mingmin; Zhao, Lin

    2013-10-01

    The Roche cobas TaqScreen MPX test was used to evaluate the rate of hepatitis B surface antigen (HBsAg)-negative donations that were hepatitis B virus (HBV) DNA reactive from June 2010 to January 2011 in Qingdao, China. HBsAg-negative samples from 65,800 voluntary blood donors were tested with the cobas TaqScreen MPX test in pools of 6 on the Roche cobas s 201 blood screening platform. Samples positive for HBV DNA and negative for HBsAg were quantitated with the Roche COBAS AmpliPrep/COBAS TaqMan HBV test. In addition, serologic tests for HBsAg, hepatitis B surface antibody, anti-hepatitis B core antigen (anti-HBc), anti-hepatitis B e antigen (anti-HBe), and hepatitis B e antigen (HBe) were done using the Roche electrochemiluminescence immunoassay. A total of 80 nucleic acid amplification technology (NAT) test-reactive pools were identified and 59 pools (74%) resolved to a reactive sample. All samples were HBV DNA reactive and the viral load in each sample was quantitated. The viral loads of the samples ranged from less than 20 to 34,600 IU/mL; 13 samples (22%) had viral loads of more than 20 IU/mL, 27 samples (45.8%) had viral loads of less than 20 IU/mL, and 19 samples (32.2%) had undetectable viral loads. Of the 59 NAT-reactive samples, 40 (67.8%) were anti-HBc positive. Fifteen of the 59 samples could not be confirmed as NAT reactive either by an alternative NAT test or by serology. The HBV NAT yield in blood donors in Qingdao is 0.06% (38/65,800). This study confirmed the value of NAT for interdicting HBV-positive donations and preventing transfusion-transmitted HBV infections. © 2013 American Association of Blood Banks.

  15. Uji Coba Penggunaan Limbah Air Kelapa Tua sebagai Bahan Dasar Media Isolasi

    Directory of Open Access Journals (Sweden)

    Hanna Yolanda

    2011-09-01

    Full Text Available he culture media commonly used for isolation are imported and expensive. Many organic materials are naturally decomposed from complex organic compounds to simple ones by microbes. Based on this principles, this study wants to make isolation media with ripe coconut waste-water as based substance, so it can be considered as economical culture media. The method was laboratoric experimental by isolating tested bacteria with ripe coconut waste-water as based substance. The composition of the media were adjusted with MacConkey agar and blood agar base. Standard media were MacConkey agar and blood agar base. Control media were agar 15 g/L media and ripe coconut waste-water agar media. Tested bacteria were a number species of Enterobacteriaceae and positive gram cocci. The evaluated variables were macroscopic and microscopic images. Data was analized by Wilcoxon matched pairs test and sign test methods. This study did not find a significant differences (p>0.05 between standard media and ripe coconut waste-water media. Specific characteristics of tested bacteria, such as red colonies, mucoid, and hemolitic zone, were similar between standard media and ripe coconut waste-water media. The conclusion is ripe coconut waste-water can be used as base for isolation media substance of Enterobacteriaceae and gram positive cocci.

  16. On Coba and Cocok: youth-led drug-experimentation in Eastern Indonesia

    NARCIS (Netherlands)

    Hardon, A.; Idrus, N.I.

    2014-01-01

    The everyday lives of contemporary youths are awash with drugs to boost pleasure, moods, sexual performance, vitality, appearance and health. This paper examines pervasive practices of chemical ‘self-maximization’ from the perspectives of youths themselves. The research for this paper was conducted

  17. UJI COBA PEMULIHAN GIZI BURUK CARA KLINIK GIZI PUSLITBANG GIZI DI POSYANDU

    Directory of Open Access Journals (Sweden)

    Djoko Kartono

    2012-11-01

    Full Text Available Trial The Management of Severe Malnourished Children of Nutrition Clinic Method, The Nutrition Research and Development Centre at Village Level.Background: Managerrent of severe malnutrition recommended by WHO should be in hospital. For family with severe malnourished child, generally poor, hospitalization means spend a lot of money. The alternative method is the out patient management developed by Nutrition Clinic of the Nutrition Research ard Development Centre.Objectives: To study the effectiveneess of management for severe malnourished of Nutrition Clinic method in village level (posyandu by village cadre.Methods: The study was carried out at 4 sub-districts in Bogor and Sukabumi, West Java. Sixty under-five children for group 1 and 60 for group 2 were selected. Three to five posyandu's cadres were selected in each village. Visit schedule to posyandu for group 1 was similar to that Nutrition Clinic while group 2 was every 1 week. Nutrition package for group 1 and 2 was same. Data collection included body measurements, morbiddity and food consumption. Observation to the cadres performance include ownership and he use of guidance book.Results: Seven percent of children aged 6-11 months, 20% aged 12-17 months, 60% aged 18-35 months and 13% aged > 36 months. Around 30% of children had been grven fruit and porridge on the age 1-4 months old. Nutritional status improved variously depended on the nutritional indices. Energy consumption was low but protein consumption had reached the recommended allowance. Compliance to come to posyandu and nutrition package was high.Conclusions: Around 10% of cchildren had changed from under-weight to well-nourished, but most of severe malnourished children remained severe in 3 months. Stunted was over 75% and remained stunted in 3 months. Wasted was 50% and began to decline in 3 months. The average of weight increment in 3 months was 0,6 kg. Cadre could give simple education to mothers using the available guidance book.Recommendations: To use wasted as an indicator in the evaluation of management of severe malnutrition. Active role of health Centre is needed to have maximum effect of the implementation of Nutrition Clinic method at village level.Key words: management, severe malnutrition, nutrition clinic, out-patient, village cadre.

  18. STUDI DAN UJI COBA TEKNOLOGI BLUETOOTH SEBAGAI ALTERNATIF KOMUNIKASI DATA NIRKABEL

    Directory of Open Access Journals (Sweden)

    Yulia Yulia

    2004-01-01

    Full Text Available Bluetooth is a new emerging technology. This technology gives significant changes for electronic devices that we are using. If we look around, a keyboard is connected to a computer. So does a printer, a mouse, a monitor and so on. This condition creates a problem of so many scattered wires installed in the offices, houses and other places. Another problem is how to inspect the damaging or boken wires. In this paper, we will have a discussion on specific applications of bluetooth such as services provided by the bluetooth technology; bluetooth method - how bluetooth devices make connections in a piconet; as well as investigation on bluetooth protocol stack. Bluetooth has succesfully built easy connection among devices from many vendor without using cables, with less power dan money. By using bluetooth, we can build small network or Piconet, consisting of several devices without cables. Abstract in Bahasa Indonesia : Bluetooth adalah suatu teknologi baru yang mulai dikenal dan digunakan. Teknologi ini memberikan perubahan yang signifikan terhadap peralatan elektronik yang kita gunakan. Jika kita melihat sekeliling kita dimana keyboard dihubungkan pada komputer. Demikian juga halnya dengan printer, mouse, monitor dan lain sebagainya. Semua peralatan itu dihubungkan dengan menggunakan kabel. Akibatnya terjadi masalah banyak kabel yang dibutuhkan di kantor, rumah atau tempat-tempat lainnya. Masalah lain yang ditemui adalah bagaimana menelusuri kabel-kabel yang terpasang jika ada suatu kesalahan atau kerusakan. Bluetooth memperbaiki penggunaan teknologi kabel yang cenderung menyulitkan ini dengan cara menghubungkan beberapa peralatan tanpa menggunakan kabel. Pada karya tulis ini, dibahas aplikasi spesifik bluetooth, antara lain servis-servis apa saja yang disediakan oleh teknologi bluetooth; cara kerja bluetooth yaitu bagaimana bluetooth device melakukan koneksi di dalam sebuah piconet serta bluetooth protocol stack. Bluetooth telah berhasil memudahkan koneksi antar beberapa alat dari berbagai vendor tanpa kabel dengan tenaga yang kecil serta biaya yang ringan. Dengan bluetooth dapat dibentuk sebuah jaringan kecil atau Piconet yang terdiri dari beberapa peralatan dan sekali lagi, tanpa memerlukan kabel. Kata kunci: Bluetooth, Bluetooth Protocol Stack, Piconet, Komunikasi data, Nirkabel.

  19. EFEK DIURETIK Desmodium Triquetrum (L DC (DAUN DUDUK PADA HEWAN COBA

    Directory of Open Access Journals (Sweden)

    Sa'roni Sa'roni

    2012-10-01

    Full Text Available Secara empiris Desmodium triquetrum (L DC (Daun duduk digunakan sebagai pelancar air seni (diuretik. Untuk mendukung pemakaian empiris maka dilakukan penelitian efek diuretik pada tikus putih. Penelitian dilakukan dalam bentuk ekstrak dengan dosis 3,1 mg; 9,3 mg dan 31 mg/100 gram bobot badan. Sebagai blangko digunakan akuades 1ml/100 g bobot badan dan sebagai pembanding efek diuretik HCT dosis 0,16 mg/100 gram bobot badan. Perlakuan diberikan secara oral. Penelitian efek diuretik dilakukan menurut cara Lipschitz. Diuretik selain meningkatkan volume air, juga meningkatkan ekskresi Na dan K. Untuk mengukur kadar Na dan K dalam urine digunakan AAS. Hasil penelitian menunjukkan bahwa ekstrak daun duduk dosis 31 mg/100 g bobot badan mempunyai efek diuretik paling kuat dibandingkan dengan kontrol akuades, sedang kadar Na dan K dalam urine tidak berbeda dengan kadar Na dan K dalam urine tikus pembanding HCT dosis 0,16 mg/100 gram bobot badan.   Kata Kunci :  Diuretik; Desmodium triquetrum (L DC; Daun duduk

  20. TINJAUAN HASIL UJI COBA PENGOBATAN DAN PENCEGAHAN MALARIA DI BEBERAPA TEMPAT INDONESIA, 1986- 1995

    Directory of Open Access Journals (Sweden)

    Emiliana Tjitra

    2012-09-01

    Full Text Available In Indonesia, only antimalarials chloroquine, sulfadoxine/ sulfalene-pyrimethamine, quinine, and primaquine are available. The development of chloroquine and multidrug resistance poses a therapeutic challenge. In order to obtain alternative antimalarial drugs, trials were conducted of malaria treatment and prophylaxis in several chloroquine or multidrug resistance areas. The objective of these trials was to assess the efficacy and safety of the alternative antimalarial drugs. All the trials were mostly open studies in the fields and hospitals. These were collaboration studies between Communicable Disease Research Center, Communicable Disease Control and Environmental Health, Faculty of Medicine of the University of Indonesia, NAMRU-2, and local health staff. The patients were selected according to the WHO criteria for in-vivo antimalarial drug sensitivity testing. They should sign the informed consent form and they were followed up during the study, for 2 weeks - 4 months. In chloroquine and multidrug resistance areas, mefloquine, halofantrine, and artemether are effective and safe for treatment of uncomplicated falciparum malaria. While artesunate was noted effective and safe only in the first 14 days. Halofantrine was also documented effective and safe for vivax malaria treatment. Intramuscular artemether was effective and safe for treatment of severe and complicated falciparum malaria, particularly in remote areas lacking hospitals and the capability for intravenous infusion. Primaquine, doxycycline and mefloquine are effective and safe for malaria prophylaxis. Since the new antimalarials are not yet available in Indonesia, the improvement of efficacy of antimalarial drugs currently available should be studied. Prophylactic drugs which are effective and safe for children, pregnant and lactating women should also be studied.

  1. MENGETAHUI KEAMANAN JAMU MADURA "SARI RAPAT" MELALUI UJI MIKROSKOPIS DAN TOKSISITAS SUB AKUT PADA BINATANG COBA

    Directory of Open Access Journals (Sweden)

    Lestari Handayani

    2012-11-01

    Full Text Available ''Jamu Sari Rapat" made in Madura is a famous traditional medicine especially after promoted at television cable. A cut utilition of the "jamu" was safe, it was provided by no complain from consumers. The problem is how the effect of "jamu" in the longterm consumption. A chosen famous "jamu Sari Rapat'' was taken to be examined. Microscopic examinations were done to know the herbs composition and contamination to patogen bacteries. A sub a cut toxicity exami­nation was done  to find out the safety of "jamu'' to the consumer because of the longterm consumption. The result showed that the ''jamu" was consisted of 9 herbs those were Caryophylli Flos, Kaemferiae rotundae Rhizoma, Curcuma domesticae Rhizoma, Paramcriae Cort ex, Theae Folium, Guazumae Folium, Arecae Semen, Glycyrrhi­ zae Radix and Psidii Folium. A herb (Gallae was written in the label but actually it did not find in microcopic test. It was found that Most Probable Number colliform were contaminated the "jamu" and the Total Plate Count Bacterial number was bigger than standard. Toxicity test showed that there was no toxic effect after 3 month intervention to 54 mice. The conclusion of this study: the "jamu" was safe to be consume in the longterm (more or less 3 months but it might cause illness to the consumers because of patogen bacteries. The production process should be improved to solve those problem.

  2. 运用COBAS AMPLICOR HCV系统快速检测HCV RNA

    Institute of Scientific and Technical Information of China (English)

    江志奎; Albad,J

    1999-01-01

    近年来实验室诊断丙型肝炎病毒(HCV)PCR法已向标准化和“即可用”的方向发展。本试验用来自5个检测中心的2000多份标本对首台自动化扩增和检测HCV RNA的仪器COBAS在日常实验室工作中的性能进行了评估。结果表明,自动的COBAS法与AMPLICOR手工法同样准确,符合率达99.8%。PCR法与血清学方法的结果并不完全一致,因为抗HCV抗体的存在有可能表示隐性感染或既往感染。如果以抗HCV抗

  3. UJI COBA PEDOMAN EVALUASI PENGELOLAAN DAN PEMBIAYAAN OBAT DI 20 PUSKESMAS JAWA TIMUR

    Directory of Open Access Journals (Sweden)

    Sriana Azis

    2012-10-01

    Full Text Available Drug financing is the biggest cost component of health care which is relatively easy to be intervened, especially the government sector. If key outcome indicators have been determined at the beginning of an intervention measuring whether the objectives of the intervention have been met through changes in these indicators makes it possible to assess the impact of an intervention. The objective of the study is essential regarding the recent decentralization policy. Guidelines on Drug Management and Financiing at Puskesmas with indicators adopted from WHO-PAHO "Manual of Rapid Assessment Pharmaceutical Management, 1995" as well as indicators recently developed from a study in Pekalongan District in 1999/2000, i.e. cost recovery, real cost and ability to pay, is intended to increase efficiency, to assess the system and broaden knowledge of drug managing staff at District Health office, District (Dinkes kabupaten, Pharmaceutical Warehouse (GFK and Health Centre (puskesmas. In this cross-sectional retrospective study training of data collector, 5 health provider (H.P from Puskesmas, 1 H.P from GFK and 1 H.P from Dinkes Kabupaten, should prepare them for different situations. The design for this study to characterize drug use practices in a region would call for a sample of at least 20 health facilities, with at least 30 encounters being recorded in each facility. Studying 20 Puskesmas from 4 districts: Jombang, Lumajang, Malang and Pasuruan through their medical records (600 from each Puskesmas will increase  the reliability and generalizability of indicators. The results of this study shows that In all districts drug budget per capita increased during 1998-2001 for different reasons and patients' ability to pay was higher than the real treatment cost for certain diseases. On the other hand, the average drug budget per encounter was higher than the real cost and cost recovery was declining for all districts except for Jombang, More intensive socialization of rational prescribing for Puskesmas physician to achieve efficiency in drug budgeting and of this guidelines implementation is necessary, regional authority should better return all Puskesmas retribution entirely.   Key words : pharmacoeconomtc, health center, assessment guidelines

  4. Cross-reactivity profiles of hybrid capture II, cobas, and APTIMA human papillomavirus assays

    DEFF Research Database (Denmark)

    Preisler, Sarah Nørgaard; Rebolj, Matejka; Ejegod, Ditte Møller

    2016-01-01

    Background High-risk Human Papillomavirus (HPV) testing is replacing cytology in cervical cancer screening as it is more sensitive for preinvasive cervical lesions. However, the bottleneck of HPV testing is the many false positive test results (positive tests without cervical lesions). Here, we...... assays. None of the 35 genotypes was detected in 49 (1.0 %), 162 (3.2 %), and 56 (1.1 %) samples, respectively. In primary screening at age 30 to 65 years (n = 2859), samples of 72 (25 %) out of 289 with high-risk infections on HC2 and 

  5. UJI COBA PEDOMAN APLIKASI PERUMUSAN PESAN UMUM GIZI SEIMBANG (PUGS SESUAI KONDISI DAERAH

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    Tjetjep Syarif Hidayat

    2012-11-01

    Full Text Available Test The Guidance For The Impelentation of "Dietary Guidelines" For Different Area Conditions.Background: Dietary guidelines for nutrition education tool developed by the health department until now still difficult in the field. Dietary guidelines should be arranged by local officials. Guidelines that needs to be made how to craft a balanced nutrition messages in accordance with local conditions.Objectives: To develop a guidance on how to implement the PUGS which is suitable for the local conditions.Methods: Exploratory research methods as applied research for local officials. Implematation manual has been composed PUGS who first disseminated to local officials at the district level. And than local officials to practice through the following stages; how do identify nutritional problems and then how to make balanced nutrition messages in accordance with local conditions. This guidance was tested in the district of Tasikmalaya, West Java and Magelang of Central Java.Results: Showed that there was a significant difference after and before test the guidance for the implementation of dietary guidelines in local officers were able to identify nutrition problems and to develop messages on PUGS.Keywords: PUGS, implementation of dietary guidelines and local condition.

  6. EFEK ANALGETIK DAN TOKSISITAS AKUT EKSTRAK RIMPANG DRINGO (Acorus calamus L. PADA HEWAN COBA

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    Sa'roni Sa'roni

    2012-10-01

    Full Text Available Secara empiris/tradisional rimpang dringo (Acorus calamus L. digunakan sebagai obat encok (reumatik, bengkak, demam, dll. Rimpang dringo, antara lain mengandung minyak atsiri, tanin, protein dan kalsium oksalat. Berdasarkan pemakaian empiris dan kandungan kimianya terutama minyak atsiri diduga rimpang dringo mempunyai efek terhadap susunan saraf pusat antara lain efek analgetik. Untuk membuktikan adanya efek analgetik, maka dilakukan penelitian efek analgetik ekstrak rimpang dringo pada mencit menurut cara Witkin dengan dosos 0,15 mg, 1,5 mg dan 4,5 mg /10g.bb. serta penelitian toksisitas akut menurut Weil C.S untuk mengetahui besarnya LD50 ekstrak rimpang dringo.Hasil penelitian menunjukkan bahwa ekstrak rimpang dringo dosis 0,15 mg/10 g.bb belum mempunyai efek analgetik. Ekstrak rimpang dringo dosis 1,5 mg dan 4,5 mg /10 g.bb mempunyai efek analgetik yang tidak berbeda dengan asetosal dosis 0,52 mg/10 g.bb. Ekstrak rimpang dringo mempunyai LD50 = 15,2 (13,4 - 17,3 mg/10 g.bb. secara i.p. pada mencit. Kata kunci: Acorus calamus L; Analgetik; Toksisitas Akut

  7. On Coba and Cocok: youth-led drug-experimentation in Eastern Indonesia

    NARCIS (Netherlands)

    A. Hardon; N.I. Idrus

    2014-01-01

    The everyday lives of contemporary youths are awash with drugs to boost pleasure, moods, sexual performance, vitality, appearance and health. This paper examines pervasive practices of chemical ‘self-maximization’ from the perspectives of youths themselves. The research for this paper was conducted

  8. Impact of occult HBV infection in HIV/HCV co-infected patients: HBV-DNA detection in liver specimens and in serum samples.

    Science.gov (United States)

    Fabris, Paolo; Biasin, Maria R; Giordani, Maria T; Berardo, Laura; Menini, Vania; Carlotto, Antonio; Miotti, Maria G; Manfrin, Vinicio; Baldo, Vincenzo; Nebbia, Gaia; Infantolino, Domenico

    2008-03-01

    Prevalence and impact of occult HBV infection in HIV positive patients is controversial. The aims of this study were to determine the prevalence of occult HBV infection and its impact on histological and virological parameters. 52 HIV/HCV (but HBsAg-negative) co-infected patients, 29 HBsAg and anti-HCV negative chronic hepatitis, and 20 HBsAg positive chronic hepatitis controls were studied. DNA was extracted from frozen biopsies and amplified with primers for S, C and X regions, and for (ccc) HBV-DNA. Sera were tested for HBV-DNA with two quantitative assays (Cobas Amplicor HBV Monitor, and the real-time COBAS (r) Taqman HBV Test, Roche Diagnostics, UK). Occult HBV infection was detected in 7 (13.4%) liver biopsies of the study group, and in none case of the non viral chronic hepatitis group (p=0.04). All serum samples were HBV-DNA negative with Cobas Amplicor HBV monitor assay, while 3 cases were found positive with real time PCR. Statistical analysis didn't show any impact of occult HBV infection on liver histology, CD4+ cells count, HIV and HCV load, and ALT levels. Occult B infection is relatively frequent in HIV/HCV co-infected patients, and is underestimated by common HBV-DNA serological assays. However, it doesn't seem to exert a relevant impact.

  9. [Comparison of commercial HIV-1 viral load tests by using proficiency test results in China, 2013- 2015].

    Science.gov (United States)

    Zhang, L; Jin, C; Jiang, Z; Tang, T; Jiang, Y; Pan, P L

    2017-09-10

    Objective: To compare the bio-equivalence among commercial HIV-1 viral load tests, including EasyQ HIV-1 v2.0 (EasyQ) from bioMerieux NucliSens of France; VERSANT HIV-1 RNA 3.0 assay (bDNA) from Siemens Healthcare Diagnostics of USA; COBAS AmpliPrep/COBAS TaqMan HIV-1 test (Taqman) from Roche Molecular Diagnosis of USA; Abbott Real Time HIV-1 Kit (M2000) from Abbott Molecular of USA and two domestic HIV-1 viral load test kits (domestic kit) from DaAn Gene Company of Sun Yat-Sen University and Liaoning Bio-Pharmaceutical company of Northeast pharmaceutical group, by using proficiency test results in China from 2013 to 2015. Methods: A total of 2 954 proficiency test results, obtained from 22 positive samples of 6 proficiency tests in 155 laboratories conducted by China CDC were analyzed during 2013-2015. The results from each sample were first logarithmic transformed and then grouped according to the method used, the mean value of logarithmic results was calculated. Subsequently, 22 clusters of mean values were analyzed by Bland-Altman analysis for the consistency, and linear regression analysis for the interdependency. Results: The results indicated that, by taking Taqman as the reference, EasyQ, M2000, bDNA and domestic kit had good consistency (90%-100%) and interdependency. Conclusion: All the viral load tests were bio-equivalent. Moreover, according to the conversion formula derived from domestic proficiency test results, all the viral load results could be converted, which is critical for epidemiological analysis.

  10. Real-time PCR assays for hepatitis B virus DNA quantification may require two different targets.

    Science.gov (United States)

    Liu, Chao; Chang, Le; Jia, Tingting; Guo, Fei; Zhang, Lu; Ji, Huimin; Zhao, Junpeng; Wang, Lunan

    2017-05-12

    Quantification Hepatitis B virus (HBV) DNA plays a critical role in the management of chronic HBV infections. However, HBV is a DNA virus with high levels of genetic variation, and drug-resistant mutations have emerged with the use of antiviral drugs. If a mutation caused a sequence mismatched in the primer or probe of a commercial DNA quantification kit, this would lead to an underestimation of the viral load of the sample. The aim of this study was to determine whether commercial kits, which use only one pair of primers and a single probe, accurately quantify the HBV DNA levels and to develop an improved duplex real-time PCR assay. We developed a new duplex real-time PCR assay that used two pairs of primers and two probes based on the conserved S and C regions of the HBV genome. We performed HBV DNA quantitative detection of HBV samples and compared the results of our duplex real-time PCR assays with the COBAS TaqMan HBV Test version 2 and Daan real-time PCR assays. The target region of the discordant sample was amplified, sequenced, and validated using plasmid. The results of the duplex real-time PCR were in good accordance with the commercial COBAS TaqMan HBV Test version 2 and Daan real-time PCR assays. We showed that two samples from Chinese HBV infections underestimated viral loads when quantified by the Roche kit because of a mismatch between the viral sequence and the reverse primer of the Roche kit. The HBV DNA levels of six samples were undervalued by duplex real-time PCR assays of the C region because of mutations in the primer of C region. We developed a new duplex real-time PCR assay, and the results of this assay were similar to the results of commercial kits. The HBV DNA level could be undervalued when using the COBAS TaqMan HBV Test version 2 for Chinese HBV infections owing to a mismatch with the primer/probe. A duplex real-time PCR assay based on the S and C regions could solve this problem to some extent.

  11. Diagnostic efficacy of a real time-PCR assay for Chlamydia trachomatis infection in infertile women in north India

    Directory of Open Access Journals (Sweden)

    Benu Dhawan

    2014-01-01

    Full Text Available Background & objectives: Little is known about the prevalence of Chlamydia trachomatis infection in Indian women with infertility. To improve the diagnosis of C. trachomatis infection in developing countries, there is an urgent need to establish cost-effective molecular test with high sensitivity and specificity. This study was conducted to determine the diagnostic utility of a real time-PCR assay for detention of C. trachomatis infection in infertile women attending an infertility clinic in north India. The in house real time-PCR assay was also compared with a commercial real-time PCR based detection system. Methods: Endocervical swabs, collected from 200 infertile women were tested for C. trachomatis by three different PCR assays viz. in-house real time-PCR targeting the cryptic plasmid using published primers, along with omp1 gene and cryptic plasmid based conventional PCR assays. Specimens were also subjected to direct fluorescence assay (DFA and enzyme immunoassay (EIA Performance of in-house real time-PCR was compared with that of COBAS Taqman C. trachomatis Test, version 2.0 on all in-house real time-PCR positive sample and 30 consecutive negative samples. Results: C. trachomatis infection was found in 13.5 per cent (27/200 infertile women by in-house real time-PCR, 11.5 per cent (23/200 by cryptic plasmid and/or omp1 gene based conventional PCR, 9 per cent (18/200 by DFA and 6.5 per cent (7/200 by EIA. The in-house real time-PCR exhibited a sensitivity and specificity of 100 per cent, considering COBAS Taqman CT Test as the gold standard. The negative and positive predictive values of the in-house real time-PCR were 100 per cent. The in-house real time-PCR could detect as low as 10 copies of C. trachomatis DNA per reaction. Interpretation & conclusions: In-house real time-PCR targeting the cryptic plasmid of C. trachomatis exhibited an excellent sensitivity and specificity similar to that of COBAS Taqman CT Test, v2.0 for detection of C

  12. UJI COBA LARVISIDA SPHERIFIX (Bacillus sphaericus VCRC B 42 TERHADAP LARVA Anopheles sundaicus Di GERUMBUL KLACES, UJUNG ALANG - KABUPATEN CILACAP

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    Umi Widyastuti

    2012-09-01

    Full Text Available A biological larvicide spherifix containing Bacillus sphaericus VCRC B 42 was investigated against Anopheles sundaicus in Klaces hamlet, Cilacap regency. This study was conducted to determine the effectivity of spherifix on An. sundaicus larvae at a dosage of 2.5 kg/Ha. Observations were conducted one day before application of the larvicide, 24, 36, 48 hours, day 4, 7, and 14 after application. The larval reduction rates were calculated using the formula of Mulia et al, 1971, and a reduction of the results were 16.69 % after 24 hours, 20.95 % after 36 hours, 34.07 % after 48 hours, 65.08 % after 4 days, 85.98 % after 7 days, and 90.81 % after 14 days. B. sphaericus has capabilities to function as a biological larvicide.

  13. PERBANDINGAN PENGARUH BIOSIDA SANDOZ DENGAN BACTIMOS TERHADAP PENCEMAR BIOLOGIS, CULEX QUINQUEFASCIATUS DALAM SATU UJI COBA LAPANGAN DI JAKARTA, INDONESIA

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    I. G. Seregeg

    2012-09-01

    Full Text Available A field trial of Bacillus thuringiensis serotype H-14 has been done in Rawasari, Jakarta. Bacillus thuringiensis serotype H-14 under the trade-name of Sandoz 402 I in wde formulation and under the trade-name of Bactimos in wdp formulation is a specific agent against mosquito larvae (WHO mimeo. series, 1980. The result of the trial showed that both formulations have a short killing ef­fect, not more than one day against the larvae of Culex quinquefasciatus in Jakarta, Indonesia. Bactimos wdp has a better killing effect against early instar larvae as compared to Sandoz wdc.

  14. Igualación equipercentil del Examen de Habilidades y Conocimientos Básicos (EXH-COBA)

    OpenAIRE

    Luz Elena Antillón; Norma Larrazolo; Eduardo Backhoff

    2006-01-01

    La igualación equipercentil es un método estadístico en el cual los puntajes crudos de dos versiones de una prueba se conside-ran igualados si ellos corresponden al mismo rango percentilar en un grupo de examinados. En la igualación equipercentil se presenta una curva para describir las diferencias de dificultad de versión a versión. Este trabajo tuvo como objetivo estimar la igualación equipercentil sin suavizado de las versiones 3 y 4, con la versión 2, por área temática del Examen de Habil...

  15. DESAIN PERANGKAT-KERAS KOMPONEN PENDUKUNG PENGEMBANGAN RADAR SEKUNDER UNTUK TRACKING TIGA DIMENSI TRAYEKTORI UJI COBA ROKET

    Directory of Open Access Journals (Sweden)

    Darjat Darjat

    2012-02-01

    Full Text Available Topic executed by is " Development of Secondary Radar For The Tracking Of Three Dimension Trajectory Test-Drive The National Rocket".In first year of research focussed at desain and making of component of hardware of producer of radio frequency 900 MHZ. Technological Development of rocket is not quit of other technology, one of them is secondary radar. Communications of radio data overcome through the secondary radar need a component hardware capable to awaken the radio frequency as according to wanted job frequency.This article study about method of hardware desain transceiver which usually implementation by PLL ( Phase Locked Loop or underlayer modulation locked by the phase to arrange the VCO ( Voltage Controlled Oscillator what is used. Component used by is IC transceiver RFM12BP integrating all function of radio frequency in single chipPrototype which is being developed by to build the transceiver FSK use the unit microcontroller , crystal, capacitor and antenna; separated. Obtained by result that system of radio frequency developed by earn the implementation for the system of secondary radar ably stable frequency at specified frequency arrangement

  16. UJI COBA PENYEMPROTAN ULV (ULV SPRAYING INSEKTISIDA BENDIOCARB 20 % (FICAM ULV TERHADAP VEKTOR DEMAM BERDARAH DENGUE Aedes aegypti

    Directory of Open Access Journals (Sweden)

    Hadi Suwasono

    2012-09-01

    Full Text Available A trial was conducted using ULV spraying of Bendiocarb 20% (Ficam ULV in Salatiga municipality at a dosage of 45 ml/ha and 75 ml/ha. The trial was conducted in the morning. Two cycles were implemented at an interval of 7 days, using a vehicle-mounted ULV generator. Results revealed no significant changes in mosquito population densitities, in any of the parameters observed.

  17. Uji Coba Alat Penghasil Asap Cair Skala Laboratorium dengan Bahan Pengasap Serbuk Gergaji Kayu Jati Sabrang atau Sungkai (Peronema canescens

    Directory of Open Access Journals (Sweden)

    Rodiah Nurbaya Sari

    2014-05-01

    energi yang dilepas dari pembentukan asap menjadi asap cair (- 476,45 kJ/kg asap, energi yang diserap air kondensor sebesar 2,1 kJ/kg air sehingga jumlah air bersuhu 30,4oC yang dibutuhkan untuk mengembunkan 1 kg asap menjadi asap cair dengan suhu pirolisis 316,7oC adalah sebanyak 226,88 liter. Kinerja alat adalah 6,98 g/(jam. m. Komponen dominan dalam asap cair yang dihasilkan adalah senyawa 1,2-benzenedicarboxylic acid, diethyl ester (C12H14O4 sebanyak 23,61%.

  18. UJI COBA PENYEMPROTAN ULV (ULV SPRAYING INSEKTISIDA BENDIOCARB 20 % (FICAM ULV TERHADAP VEKTOR DEMAM BERDARAH DENGUE Aedes aegypti

    Directory of Open Access Journals (Sweden)

    Hadi Suwasono

    2012-09-01

    Full Text Available A trial was conducted using ULV spraying of Bendiocarb 20% (Ficam ULV in Salatiga municipality at a dosage of 45 ml/ha and 75 ml/ha. The trial was conducted in the morning. Two cycles were implemented at an interval of 7 days, using a vehicle-mounted ULV generator. Results revealed no significant changes in mosquito population densitities, in any of the parameters observed.

  19. Rekayasa Alat Penghasil Asap Cair untuk Produksi Ikan Asap 1. Uji Coba Alat Penghasil Asap Cair Skala Laboratorium

    Directory of Open Access Journals (Sweden)

    Rodiah Nurbaya Sari

    2006-04-01

    penelitian menunjukkan bahwa pada suhu pembakaran 200-250o C, tempurung kelapa dengan kadar air 11,40% menghasilkan asap cair sebanyak 48,10%, sisa pembakaran berupa arang sebanyak 31,33%, jumlah komponen yang hilang sebanyak 20,56% dengan kinerja alat sebesar 250,52 g/jam.m kondensor. Komponen terbanyak asap cair yang dihasilkan adalah senyawa 9-octadecenoic acid (Z-, tetradecyl ester (C32H62O2 sebanyak 71,68%. Pada suhu pembakaran 300–450oC asap cair yang dihasilkan sebanyak 48,66%, sisa pembakaran berupa arangsebanyak 26,30%, komponen asap yang hilang sebanyak 25,04% dengan kinerja alat 253,44 g/jam.m kondensor. Pada suhu tersebut komponen terbanyak asap cair adalah senyawa 2-lauro-1,3-didecoinyaitu 37,53%.

  20. UJI COBA PENEBARAN JASAD HAYATI NEMATODA ROMANOMERMIS IYENGARI DI PERINDUKAN VEKTOR MALARIA DAN FILARIASIS DI FLORES TIMUR

    Directory of Open Access Journals (Sweden)

    Widiarti Widiarti

    2012-09-01

    Full Text Available A study to evaluate the effect of R. iyengari, a parasitic nematod as a biological agent of vector control toward mosquito larvae in the sandpool, an ideal breeding places for malaria and fdariasis vectors, was conducted in East Flores. The aim of the present study was to determine : (1. The adaptability and recycling ability of post parasites stage of the nematode in the sandpool. (2. The ability of pre parasites (infective stage to parasitize Cx. quinquefasciatus (laboratory larvae in centinel cage and An. barbirostris (natural larvae/sandpool. The distribution of R. iyengari post parasites at a dosage of500 nematodes/m2 revealed that the nematodes mature to adults, mated and layed eggs (adapted, however recycling could not be observed yet. The infection rate of Cx. quinquefasciatus sentinel cages after release through dissections was observed in 70.55%, 48.68% and 3.64% respectively after 1 month, 2 months and 3 months. The mean monthly infection level for An. barbirostris was 28.24%, 16.66% and 31.25% for 1 month, 2 months and 3 months post release dissections respectively.

  1. Cell culture-Taqman PCR assay for evaluation of Cryptosporidium parvum disinfection.

    Science.gov (United States)

    Keegan, Alexandra R; Fanok, Stella; Monis, Paul T; Saint, Christopher P

    2003-05-01

    Cryptosporidium parvum represents a challenge to the water industry and a threat to public health. In this study, we developed a cell culture-quantitative PCR assay to evaluate the inactivation of C. parvum with disinfectants. The assay was validated by using a range of disinfectants in common use in the water industry, including low-pressure UV light (LP-UV), ozone, mixed oxidants (MIOX), and chlorine. The assay was demonstrated to be reliable and sensitive, with a lower detection limit of a single infectious oocyst. Effective oocyst inactivation was achieved (>2 log(10) units) with LP-UV (20 mJ/cm(2)) or 2 mg of ozone/liter (for 10 min). MIOX and chlorine treatments of oocysts resulted in minimal effective disinfection, with disinfection systems for drinking water and recycled water.

  2. Optimized Multiplex Detection of 7 KRAS Mutations by Taqman Allele-Specific qPCR

    Science.gov (United States)

    Orue, Andrea; Rieber, Manuel

    2016-01-01

    Establishing the KRAS mutational status of tumor samples is essential to manage patients with colorectal or lung cancer, since these mutations preclude treatment with monoclonal anti-epidermal growth factor receptor (EGFR) antibodies. We report an inexpensive, rapid multiplex allele-specific qPCR method detecting the 7 most clinically relevant KRAS somatic mutations with concomitant amplification of non-mutated KRAS in tumor cells and tissues from CRC patients. Positive samples evidenced in the multiplex assay were further subjected to individual allele-specific analysis, to define the specific mutation. Reference human cancer DNA harbouring either G12A, G12C, G12D, G12R, G12S, G12V and G13D confirmed assay specificity with ≤1% sensitivity of mutant alleles. KRAS multiplex mutation analysis usefulness was also demonstrated with formalin-fixed paraffin embedded (FFPE) from CRC biopsies. Conclusion. Co-amplification of non-mutated DNA avoided false negatives from degraded samples. Moreover, this cost effective assay is compatible with mutation detection by DNA sequencing in FFPE tissues, but with a greater sensitivity when mutant DNA concentrations are limiting. PMID:27632281

  3. Single rapid TaqMan fluorogenic probe based PCR assay that detects all four dengue serotypes.

    Science.gov (United States)

    Warrilow, David; Northill, Judith A; Pyke, Alyssa; Smith, Greg A

    2002-04-01

    Public health laboratories require rapid diagnosis of dengue outbreaks for application of measures such as vector control. We have developed a rapid single fluorogenic probe-based polymerase chain reaction assay for the detection of all four dengue serotypes (FUDRT-PCR). The method employs primers and probe that are complementary to the evolutionarily conserved 3' untranslated region of the dengue genome. The assay detected viral RNA of strains of all four dengue serotypes but not of the flaviviruses Japanese encephalitis virus, Murray Valley encephalitis virus, Kunjin, Stratford, West Nile, Alfuy or Yellow fever. When compared to an existing nested-PCR assay for the detection of dengue on clinical samples, FUDRT-PCR detected dengue 1 (100%, n=14), dengue 2 (85%, n=13), dengue 3 (64%, n=14) and dengue 4 (100%, n=3) with the indicated sensitivities. FUDRT-PCR enables diagnosis of acute dengue infection in four hours from sample receipt. In addition, a single-test procedure should result in a reduction in the number of tests performed with considerable cost savings for diagnostic laboratories.

  4. DETECTION AND IDENTIFICATION OF PATHOGENIC CANDIDA SPECIES IN WATER USING FLOW CYTOMETRY COUPLED WITH TAQMAN PCR

    Science.gov (United States)

    As the incidence of human fungal infection increases, the ability to detect and identify pathogenic fungi in potential environmental reservoirs becomes increasingly important for disease control. PCR based assays are widely used for diagnostic purposes, but may be inadequate for...

  5. Specificity of a novel TaqMan PCR method for detection of poultry DNA

    NARCIS (Netherlands)

    Scholtens-Toma, Ingrid; Prins, Theo W.; Raamsdonk, Van Leo W.D.

    2017-01-01

    After the Bovine Spongiform Encephalopathy (BSE) crisis emerged in 1985/1986, all processed animal proteins (PAPs) were finally banned for use in animal feed in the European Union. To partially lift this feed ban, paths for re-introduction of PAPs from species other than ruminants e.g. pig and poult

  6. QUANTITATIVE MEASUREMENT OF HELICOBACTER PYLORI BY THE TAQMAN FLUOROGENIC PROBE SYSTEM

    Science.gov (United States)

    Culturing of H. pylori from environmental sources continues to be an obstacle in detecting and enumerating this organism. Successful methods of isolation and growth from water samples have not yet been developed. In this study a method involving real tme PCR product detection wit...

  7. QUANTITATIVE MEASUREMENT OF HELICOBACTER PYLORI BY THE TAQMAN FLUOROGENIC PROBE SYSTEM

    Science.gov (United States)

    Culturing of H. pylori from environmental sources continues to be an obstacle in detecting and enumerating this organism. Successful methods of isolation and growth from water samples have not yet been developed. In this study a method involving real tme PCR product detection wit...

  8. Semiautomated TaqMan PCR screening of GMO labelled samples for (unauthorised) GMOs

    NARCIS (Netherlands)

    Scholtens-Toma, Ingrid; Molenaar, Bonnie; Hoof, van Richard A.; Zaaijer, Stephanie; Prins, Theo W.; Kok, Esther J.

    2017-01-01

    In most countries, systems are in place to analyse food products for the potential presence of genetically modified organisms (GMOs), to enforce labelling requirements and to screen for the potential presence of unauthorised GMOs. With the growing number of GMOs on the world market, a larger

  9. Semiautomated TaqMan PCR screening of GMO labelled samples for (unauthorised) GMOs.

    Science.gov (United States)

    Scholtens, Ingrid M J; Molenaar, Bonnie; van Hoof, Richard A; Zaaijer, Stephanie; Prins, Theo W; Kok, Esther J

    2017-06-01

    In most countries, systems are in place to analyse food products for the potential presence of genetically modified organisms (GMOs), to enforce labelling requirements and to screen for the potential presence of unauthorised GMOs. With the growing number of GMOs on the world market, a larger diversity of methods is required for informative analyses. In this paper, the specificity of an extended screening set consisting of 32 screening methods to identify different crop species (endogenous genes) and GMO elements was verified against 59 different GMO reference materials. In addition, a cost- and time-efficient strategy for DNA isolation, screening and identification is presented. A module for semiautomated analysis of the screening results and planning of subsequent event-specific tests for identification has been developed. The Excel-based module contains information on the experimentally verified specificity of the element methods and of the EU authorisation status of the GMO events. If a detected GMO element cannot be explained by any of the events as identified in the same sample, this may indicate the presence of an unknown unauthorised GMO that may not yet have been assessed for its safety for humans, animals or the environment.

  10. Performance of the new automated Abbott RealTime MTB assay for rapid detection of Mycobacterium tuberculosis complex in respiratory specimens.

    Science.gov (United States)

    Chen, J H K; She, K K K; Kwong, T-C; Wong, O-Y; Siu, G K H; Leung, C-C; Chang, K-C; Tam, C-M; Ho, P-L; Cheng, V C C; Yuen, K-Y; Yam, W-C

    2015-09-01

    The automated high-throughput Abbott RealTime MTB real-time PCR assay has been recently launched for Mycobacterium tuberculosis complex (MTBC) clinical diagnosis. This study would like to evaluate its performance. We first compared its diagnostic performance with the Roche Cobas TaqMan MTB assay on 214 clinical respiratory specimens. Prospective analysis of a total 520 specimens was then performed to further evaluate the Abbott assay. The Abbott assay showed a lower limit of detection at 22.5 AFB/ml, which was more sensitive than the Cobas assay (167.5 AFB/ml). The two assays demonstrated a significant difference in diagnostic performance (McNemar's test; P = 0.0034), in which the Abbott assay presented significantly higher area under curve (AUC) than the Cobas assay (1.000 vs 0.880; P = 0.0002). The Abbott assay demonstrated extremely low PCR inhibition on clinical respiratory specimens. The automated Abbott assay required only very short manual handling time (0.5 h), which could help to improve the laboratory management. In the prospective analysis, the overall estimates for sensitivity and specificity of the Abbott assay were both 100 % among smear-positive specimens, whereas the smear-negative specimens were 96.7 and 96.1 %, respectively. No cross-reactivity with non-tuberculosis mycobacterial species was observed. The superiority in sensitivity of the Abbott assay for detecting MTBC in smear-negative specimens could further minimize the risk in MTBC false-negative detection. The new Abbott RealTime MTB assay has good diagnostic performance which can be a useful diagnostic tool for rapid MTBC detection in clinical laboratories.

  11. One window-period donation in two years of individual donor-nucleic acid test screening for hepatitis B, hepatitis C and human immunodeficiency virus

    Directory of Open Access Journals (Sweden)

    Jose Eduardo Levi

    2013-06-01

    Full Text Available Objective: To describe general data on nucleic acid/serology testing and report the first hepatitis B-nucleic acid testing yield case of an immunized donor in Brazil. Methods: A total of 24,441 donations collected in 2010 and 2011 were submitted to individual nucleic acid testing for hepatitis B, hepatitis C and human immunodeficiency virus using the TaqMan® MPX kit (Roche on the Cobas s201 platform, in addition to routine screening for serological markers. Nucleic acid testing-reactive donations were further evaluated by real-time polymerase chain reaction using Cobas AmpliPrep/Cobas TaqMan hepatitis B virus, hepatitis C virus and human immunodeficiency virus tests. Results: Thirty-two donations were reactive by nucleic acid testing, 31 were also serologically reactive and one first-time donor was identified as having hepatitis B in the window period. Follow-up samples showed increasing titers of anti-HBs rising from 19 UI/mL in the index donation to 109 IU/mL seven months later attributable to his vaccination history. Curiously, this donor was never reactive for HbsAg nor for anti-HBc. In the yield donation, he was concomitantly reactive for syphilis (enzyme immunoassay and fluorescent treponemal antibody-absorption; venereal disease research laboratory non-reactive. Overall, six donors (0.02% were characterized as occult hepatitis B. A total of 35% of the confirmed (recombinant immunoblot assay positive hepatitis C donations were nucleic acid testing non-reactive and no human immunodeficiency virus "elite controller" was identified. Conclusion: The yield rate (1:24,441; 95% confidence interval: 1:9,537 - 1:89,717 contrasts to the North American rate (1:410,540 donations and strongly advocates the adoption of nucleic acid testing for hepatitis B in Brazil despite the increasing rate of anti-HBs reactive subjects due to the successful immunization program.

  12. Evaluation of a Novel PCR-Based Assay for Detection and Identification of Chlamydia trachomatis Serovars in Cervical Specimens▿

    Science.gov (United States)

    Quint, Koen; Porras, Carolina; Safaeian, Mahboobeh; González, Paula; Hildesheim, Allan; Quint, Wim; van Doorn, Leen-Jan; Silva, Sandra; Melchers, Willem; Schiffman, Mark; Rodríguez, Ana Cecilia; Wacholder, Sholom; Freer, Enrique; Cortes, Bernal; Herrero, Rolando

    2007-01-01

    The aims of this study were to compare a novel PCR-based Chlamydia trachomatis detection and genotyping (Ct-DT) assay with the FDA-approved, commercially available C. trachomatis detection Hybrid Capture 2 (HC2) assay and to investigate the C. trachomatis serovar distribution among young women in a rural Costa Rican study population. A total of 5,828 sexually active women participating in a community-based trial in Costa Rica were tested for C. trachomatis by HC2. A sample of 1,229 specimens consisting of 100% HC2 C. trachomatis-positive specimens (n = 827) and a random sample of 8% HC2 C. trachomatis-negative specimens (n = 402) were tested with the Ct-DT assay. Agreement between the two assays was determined by the unweighted kappa statistic. Discrepant specimens were tested with a second commercially available test (COBAS TaqMan). The Ct-DT-positive specimens were further analyzed with the Ct-DT genotyping step to investigate the distribution of 14 different C. trachomatis serovars (A, B/Ba, C, D/Da, E, F, G/Ga, H, I/Ia, J, K, L1, L2/L2a, and L3). After accounting for the sampling fraction selected for Ct-DT testing, crude agreement with the HC2 assay was 98% and the kappa was 0.92 (95% confidence interval [CI], 0.89 to 0.97). The 33 discordant samples that were further analyzed with the COBAS TaqMan test showed better agreement with the Ct-DT assay (31/33, P < 0.001). Among the 806 Ct-DT-positive samples, serovar E was the most common serovar (31%), followed by serovars F and D (both 21%) and serovar I (15%). In conclusion, the novel Ct-DT assay permits reliable detection and identification of C. trachomatis serovars. PMID:17959760

  13. Performance and Logistical Challenges of Alternative HIV-1 Virological Monitoring Options in a Clinical Setting of Harare, Zimbabwe

    Directory of Open Access Journals (Sweden)

    Pascale Ondoa

    2014-01-01

    Full Text Available We evaluated a low-cost virological failure assay (VFA on plasma and dried blood spot (DBS specimens from HIV-1 infected patients attending an HIV clinic in Harare. The results were compared to the performance of the ultrasensitive heat-denatured p24 assay (p24. The COBAS AmpliPrep/COBAS TaqMan HIV-1 test, version 2.0, served as the gold standard. Using a cutoff of 5,000 copies/mL, the plasma VFA had a sensitivity of 94.5% and specificity of 92.7% and was largely superior to the VFA on DBS (sensitivity = 61.9%; specificity = 99.0% or to the p24 (sensitivity = 54.3%; specificity = 82.3% when tested on 302 HIV treated and untreated patients. However, among the 202 long-term ART-exposed patients, the sensitivity of the VFA decreased to 72.7% and to 35.7% using a threshold of 5,000 and 1,000 RNA copies/mL, respectively. We show that the VFA (either on plasma or on DBS and the p24 are not reliable to monitor long-term treated, HIV-1 infected patients. Moreover, achieving acceptable assay sensitivity using DBS proved technically difficult in a less-experienced laboratory. Importantly, the high level of virological suppression (93% indicated that quality care focused on treatment adherence limits virological failure even when PCR-based viral load monitoring is not available.

  14. Comparison of two molecular assays for detection of cytomegalovirus DNA in whole blood and plasma samples from transplant recipients.

    Science.gov (United States)

    Costa, Cristina; Sidoti, Francesca; Mantovani, Samantha; Gregori, Gabriella; Proietti, Alex; Ghisetti, Valeria; Cavallo, Rossana

    2016-07-01

    In immunosuppressed patients, pre-emptive therapy and a strict follow-up of CMV infection are the standard of care for the prevention of CMV disease. Several real-time PCR assays for CMV DNA quantification on whole blood (WB) and plasma (PL) are commercially available. This study compared and correlated CMV viral loads obtained by the Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) platform on plasma specimens with those obtained on corresponding whole blood specimens by the real-time PCR assay (ELITe MGB-CMV) in 185 sequential samples from 41 immunosuppressed patients. Correlation between the two assays was good. Kinetics of CMV DNA within the same patient was similar, but PL viral load was constantly 1 log lower than WB. In patients under antiviral therapy, low level of CMV DNA persisted in WB, while it was absent in PL. The good correlation between CMV DNA detected on both PL and WB supports the reliability of the two matrices for viral monitoring and the therapeutic management of CMV infection. Nevertheless, due to significant quantification differences between PL and WB CMV DNA, the same biological specimen should be used for a sequential and reliable follow-up of patients at high risk of CMV infection.

  15. Performance evaluation of new automated hepatitis B viral markers in the clinical laboratory: two quantitative hepatitis B surface antigen assays and an HBV core-related antigen assay.

    Science.gov (United States)

    Park, Yongjung; Hong, Duck Jin; Shin, Saeam; Cho, Yonggeun; Kim, Hyon-Suk

    2012-05-01

    We evaluated quantitative hepatitis B surface antigen (qHBsAg) assays and a hepatitis B virus (HBV) core-related antigen (HBcrAg) assay. A total of 529 serum samples from patients with hepatitis B were tested. HBsAg levels were determined by using the Elecsys (Roche Diagnostics, Indianapolis, IN) and Architect (Abbott Laboratories, Abbott Park, IL) qHBsAg assays. HBcrAg was measured by using Lumipulse HBcrAg assay (Fujirebio, Tokyo, Japan). Serum aminotransferases and HBV DNA were respectively quantified by using the Hitachi 7600 analyzer (Hitachi High-Technologies, Tokyo, Japan) and the Cobas AmpliPrep/Cobas TaqMan test (Roche). Precision of the qHBsAg and HBcrAg assays was assessed, and linearity of the qHBsAg assays was verified. All assays showed good precision performance with coefficients of variation between 4.5% and 5.3% except for some levels. Both qHBsAg assays showed linearity from 0.1 to 12,000.0 IU/mL and correlated well (r = 0.9934). HBsAg levels correlated with HBV DNA (r = 0.3373) and with HBcrAg (r = 0.5164), and HBcrAg also correlated with HBV DNA (r = 0.5198; P HBcrAg assays.

  16. A novel duplex real-time reverse transcriptase-polymerase chain reaction assay for the detection of hepatitis C viral RNA with armored RNA as internal control

    Directory of Open Access Journals (Sweden)

    Meng Shuang

    2010-06-01

    Full Text Available Abstract Background The hepatitis C virus (HCV genome is extremely heterogeneous. Several HCV infections can not be detected using currently available commercial assays, probably because of mismatches between the template and primers/probes. By aligning the HCV sequences, we developed a duplex real-time reverse transcriptase-polymerase chain reaction (RT-PCR assay using 2 sets of primers/probes and a specific armored RNA as internal control. The 2 detection probes were labelled with the same fluorophore, namely, 6-carboxyfluorescein (FAM, at the 5' end; these probes could mutually combine, improving the power of the test. Results The limit of detection of the duplex primer/probe assay was 38.99 IU/ml. The sensitivity of the assay improved significantly, while the specificity was not affected. All HCV genotypes in the HCV RNA Genotype Panel for Nucleic Acid Amplification Techniques could be detected. In the testing of 109 serum samples, the performance of the duplex real-time RT-PCR assay was identical to that of the COBAS AmpliPrep (CAP/COBAS TaqMan (CTM assay and superior to 2 commercial HCV assay kits. Conclusions The duplex real-time RT-PCR assay is an efficient and effective viral assay. It is comparable with the CAP/CTM assay with regard to the power of the test and is appropriate for blood-donor screening and laboratory diagnosis of HCV infection.

  17. Development of a new ultra sensitive real-time PCR assay (ultra sensitive RTQ-PCR for the quantification of HBV-DNA

    Directory of Open Access Journals (Sweden)

    Varaklioti Agoritsa

    2010-03-01

    Full Text Available Abstract Background Improved sensitivity of HBV-DNA tests is of critical importance for the management of HBV infection. Our aim was to develop and assess a new ultra sensitive in-house real-time PCR assay for HBV-DNA quantification (ultra sensitive RTQ-PCR. Results Previously used HBV-DNA standards were calibrated against the WHO 1st International Standard for HBV-DNA (OptiQuant® HBV-DNA Quantification Panel, Accrometrix Europe B.V.. The 95% and 50% HBV-DNA detection end-point of the assay were 22.2 and 8.4 IU/mL. According to the calibration results, 1 IU/mL equals 2.8 copies/mL. Importantly the clinical performance of the ultra sensitive real-time PCR was tested similar (67% to the Procleix Ultrio discriminatory HBV test (dHBV (70% in low-titer samples from patients with occult Hepatitis B. Finally, in the comparison of ultra sensitive RTQ-PCR with the commercially available COBAS TaqMan HBV Test, the in-house assay identified 94.7% of the 94 specimens as positive versus 90.4% identified by TaqMan, while the quantitative results that were positive by both assay were strongly correlated (r = 0.979. Conclusions We report a new ultra sensitive real time PCR molecular beacon based assay with remarkable analytical and clinical sensitivity, calibrated against the WHO 1st International standard.

  18. Diagnostic value of nine nucleic acid amplification test systems for Mycobacterium tuberculosis complex

    Directory of Open Access Journals (Sweden)

    Gülnur Tarhan

    2015-09-01

    Full Text Available Objective: In this study, nine commercial Nucleic Acid Amplification Test Systems (NAATs were evaluated for diagnostic performance of Mycobacterium tuberculosis complex (MTBC from smear positive sputum species (SPss and smear negative sputum specimens (SNss. Methods: Sixty SPss and 55 SNss were examined icroscopically by Ehrlich Ziehl Neelsen (EZN staining method, and also inoculated on Löwenstein Jensen (LJ medium for culture. The sensitivity and specificity of nine NAATs were calculated according to LJ culture method accepted as gold standard. Results: When LJ culture results were taken as gold standard; the sensitivity rates of method COBAS Amplicor MTB (Method A, GenProbe MTD (Method B, Cobas TaqMan MTB PCR Method C, iCycler iQ RT PCR (Method D, TaqMan PCR AB 5700 (Method E, TaqMan PCR AB7700 (Method F, ightCycler® 480 RT PCR (Method G, Rotor Gene RT PCR (Method H and the AdvanSure TB/NTM RT PCR (Method I for SPss were 98.3 %, 93.3 %, 96.7 %, 100 %, 93.3 %, 100 %, 100 %, 100 % and 100 %, respectively. The sensitivity was 53.84% for the methods A, B, D, E, G and I; 38.46% for the method C and H; 61.5% for the method F for the method I in SNss. There were no statistical significant differences between the nine NAATs (p≥0.05. The specificity was 100% for all nine NAATs in SNss. The positivity rates of methods were 53.8% for methods A, B, D, E, G, I; 38.5% for methods C and H, and 61.5% for method F in SNss. These rates were 100% for D, F, G, H and I; 98.3% for method A; 96.7% for method C; 93,3% for methods B and E in SPss. Statistical analysis showed that there was no statistically significant differences among the nine NAATs (p≥0.05. Conclusion: It is concluded that the nine NAATs might be useful for detecting MTBC from SPss, but not effective for SNss. J Microbiol Infect Dis 2015;5(3: 103-109

  19. TaqMan 实时荧光定量RT-PCR检测变异剪接体%Quantification of Splicing Variants by TaqMan Real-time RT-PCR

    Institute of Scientific and Technical Information of China (English)

    蒋胜; 李力

    2009-01-01

    目的:建立应用TaqMan实时荧光定量RT-PCR技术对基因mRNA的各个变异剪接体分别进行定量检测的技术.方法:以对ERCCI的变异剪接体进行检测为例.使用TRIzol裂解新鲜手术切除的卵巢癌组织后提取总RNA,逆转录为eDNA后,PCR扩增ERCCI基因以及beta-actin基因,琼脂糖电泳分离PCR产物,纯化后连接到pMD18-T载体,转化人感受态大肠杆菌,提取并纯化质粒,稀释成不同浓度作为标准品以便制作标准曲线.结果:成功构建了缺失第Ⅷ外显子的ERCCI质粒以及beta-actin质粒,设计了两套针针对ERCCI的TaqMan探针和引物,建立了稳定的定量检测EBCCI mRNA变异剪接体表达水平的平台.结论:TaqMan实时荧光定量RT-PCR方法可以成功地分别对基因的变异剪接定体进行定量检测.

  20. Optimization of a 12-hour TaqMan PCR-based method for detection of Salmonella bacteria in meat

    DEFF Research Database (Denmark)

    Josefsen, Mathilde Hartmann; Krause, Michael; Hansen, F.

    2007-01-01

    no positive effects and resulted in decreased reproducibility. Increasing the amount of PCR template DNA from 5 to 20 mu l improved the threshold cycle value by approximately 2. The improved 12-h PCR method was successfully compared to a reference culture method with 100 minced meat and poultry samples...... the highest number of salmonellae. When analyzing minced meat samples, positive effects of increasing the initial sampling volume from 1 to 5 ml and increasing the amount of paramagnetic particles to 90 mu l were observed. However, washing the pellet and eluting the DNA in reduced volumes (25 and 50 mu l) had...

  1. Development and evaluation of a TaqMan Real-Time PCR assay for Fusarium oxysporum f. sp. spinaciae

    Science.gov (United States)

    Fusarium oxysporum f. sp. spinaciae, causal agent of spinach Fusarium wilt, is an important soilborne pathogen in many areas of the world where spinach is grown. The pathogen is persistent in acid soils of maritime western Oregon and Washington, the only region of the USA suitable for commercial spi...

  2. A Taqman real-time PCR assay for detection of Meloidogyne hapla in root galls and in soil

    Science.gov (United States)

    Meloidogyne hapla is one of the most widespread and serious soil-borne nematodes causing root knot diseases in various crops. Early and accurate detection and quantification of M. hapla in soil is essential for effective disease management. The purpose of this study was to develop an assay for detec...

  3. A TaqMan PCR method for routine diagnosis of the quarantine fungus guignardia citricarpa on citrus fruit

    NARCIS (Netherlands)

    Gent-Pelzer, van M.P.E.; Brouwershaven, van I.R.; Kox, L.F.F.; Bonants, P.J.M.

    2007-01-01

    With respect to disease risk for the quarantine fungus Guignardia citricarpa on citrus fruit an accurate diagnosis for routine analysis is required. Also, when inspections have to be performed on imported citrus fruits, a fast detection method is urgently needed. A fast automated DNA extraction

  4. Development of a TaqMan assay for the six major genotypes of hepatitis C virus: Comparison with commercial assays

    DEFF Research Database (Denmark)

    Engle, Ronald E; Russell, Rodney S; Purcell, Robert H

    2008-01-01

    A quantitative real-time PCR assay was developed that detects genomic RNA from reference strains representing the six major genotypes of hepatitis C virus (HCV) with equal sensitivity and accurately measured HCV RNA in JFH1 HCV-infected Huh7.5 cells. The method is indirectly calibrated to the first...

  5. A TaqMan PCR method for routine diagnosis of the quarantine fungus guignardia citricarpa on citrus fruit

    NARCIS (Netherlands)

    Gent-Pelzer, van M.P.E.; Brouwershaven, van I.R.; Kox, L.F.F.; Bonants, P.J.M.

    2007-01-01

    With respect to disease risk for the quarantine fungus Guignardia citricarpa on citrus fruit an accurate diagnosis for routine analysis is required. Also, when inspections have to be performed on imported citrus fruits, a fast detection method is urgently needed. A fast automated DNA extraction meth

  6. Optimization of a 12-hour TaqMan PCR-based method for detection of Salmonella bacteria in meat.

    Science.gov (United States)

    Josefsen, M H; Krause, M; Hansen, F; Hoorfar, J

    2007-05-01

    We developed a 12-h Salmonella detection method, based on 8 h of preenrichment, followed by automated DNA extraction and a sensitive real-time PCR. The method was optimized to obtain the highest possible yield of cells and DNA. The growth of different Salmonella strains in various preenrichment media and the effects of adding growth-promoting and selective reagents were explored, taking into account their PCR compatibility. The effects of (i) analyzing larger volumes (1 to 5 ml) from preenriched samples and introducing wash steps prior to DNA extraction, (ii) regulating the amount of paramagnetic particles (increasing it from 60 to 90 microl) in the DNA extraction, (iii) eluting the DNA in reduced volumes (25 or 50 microl rather than 100 microl), and (iv) increasing the PCR template volume (from 5 to 20 microl) were investigated. After 8 h of preenrichment, buffered peptone water yielded the highest number of salmonellae. When analyzing minced meat samples, positive effects of increasing the initial sampling volume from 1 to 5 ml and increasing the amount of paramagnetic particles to 90 microl were observed. However, washing the pellet and eluting the DNA in reduced volumes (25 and 50 microl) had no positive effects and resulted in decreased reproducibility. Increasing the amount of PCR template DNA from 5 to 20 mul improved the threshold cycle value by approximately 2. The improved 12-h PCR method was successfully compared to a reference culture method with 100 minced meat and poultry samples, with a relative accuracy of 99%, a relative sensitivity of 98%, and a relative specificity of 100%.

  7. Postnatal and non-invasive prenatal detection of β-thalassemia mutations based on Taqman genotyping assays

    Science.gov (United States)

    Breveglieri, Giulia; Travan, Anna; D’Aversa, Elisabetta; Cosenza, Lucia Carmela; Pellegatti, Patrizia; Guerra, Giovanni; Gambari, Roberto

    2017-01-01

    The β-thalassemias are genetic disorder caused by more than 200 mutations in the β-globin gene, resulting in a total (β0) or partial (β+) deficit of the globin chain synthesis. The most frequent Mediterranean mutations for β-thalassemia are: β039, β+IVSI-110, β+IVSI-6 and β0IVSI-1. Several molecular techniques for the detection of point mutations have been developed based on the amplification of the DNA target by polymerase chain reaction (PCR), but they could be labor-intensive and technically demanding. On the contrary, TaqMan® genotyping assays are a simple, sensitive and versatile method suitable for the single nucleotide polymorphism (SNP) genotyping affecting the human β-globin gene. Four TaqMan® genotyping assays for the most common β-thalassemia mutations present in the Mediterranean area were designed and validated for the genotype characterization of genomic DNA extracted from 94 subjects comprising 25 healthy donors, 33 healthy carriers and 36 β-thalassemia patients. In addition, 15 specimens at late gestation (21–39 gestational weeks) and 11 at early gestation (5–18 gestational weeks) were collected from pregnant women, and circulating cell-free fetal DNAs were extracted and analyzed with these four genotyping assays. We developed four simple, inexpensive and versatile genotyping assays for the postnatal and prenatal identification of the thalassemia mutations β039, β+IVSI-110, β+IVSI-6, β0IVSI-1. These genotyping assays are able to detect paternally inherited point mutations in the fetus and could be efficiently employed for non-invasive prenatal diagnosis of β-globin gene mutations, starting from the 9th gestational week. PMID:28235086

  8. Intratumor genetic heterogeneity of breast carcinomas as determined by fine needle aspiration and TaqMan low density array

    DEFF Research Database (Denmark)

    Lyng, Maria B.; Laenkholm, Anne-Vibeke; Pallisgaard, Niels

    2007-01-01

    BACKGROUND: Gene expression profiling is thought to be an important tool in determining treatment strategies for breast cancer patients. Tissues for such analysis may at a preoperative stage be obtained, by fine needle aspiration (FNA) allowing initiation of neoadjuvant treatment. To evaluate...... the extent of the genetic heterogeneity within primary breast carcinomas, we examined whether a gene expression profile obtained by FNA was representative of the tumor. METHODS: Tumors from 12 consecutive cases of early predominantly estrogen receptor positive (ER+) breast cancer patients undergoing primary...... by statistical analysis. High correlations between the gene profiles of tumor FNAs and tissue biopsies from the same patient were observed for all patients. A cluster analysis identified clustering of both the two FNAs and the tissue biopsy of the same 9 patients. CONCLUSION: The overall genetic heterogeneity...

  9. Simultaneous detection of Hepatitis B virus and Hepatitis C virus in human plasma using Taq-man chemistry

    Directory of Open Access Journals (Sweden)

    Khaja M N

    2011-07-01

    Full Text Available Designing a rapid, reliable and sensitive assay, for detection of hepatitis B virus and Hepatitis C virus variants by real-time PCR, is challenging at best. A recent approach for quantifying the viral load using the sensitive fluorescence principle, was used in this study. A total of 350 samples were collected from outpatient unit, Center for Liver Research and Diagnostics (CLRD. Complete Human HBV DNA and HCV sequences were obtained from the National Centre for Biotechnology Information (NCBI; primers and probes were designed and synthesized from core, surface and x region of Hepatitis B and UTR region of HCV. Real-time based detection was done, using standard kit and in-house generated standards and RT-PCR protocols. A standard curve was generated by using the Smart Cycler II software and serial dilution 102 to 108 of cloned viral regions, the calibration curve was linear in a range from 102 to108 cp/ml for both HBV and HCV, with R2 value of 0.999 and 0.995. Out of 100 predetermined HCV negative samples, 02 samples were found positive with in-house developed RT-PCR assay, the positivity of this sample was confirmed by sequencing the amplified product. Low cost of this assay procedure and précised sample volume will permit the assay to be implemented for routine screening of Hepatitis B and C virus mono-infection and co-infection using Real Time PCR , Nucleic acid Chip technology and Fluorescent End Point detection systems. This assay is reproducible showing limited inter and intra assay variability. Our results correlated well with the standard kit for HBV and HCV virus monitor.

  10. Detection and quantitation of the new world Squash leaf curl virus by TaqMan real-time PCR.

    Science.gov (United States)

    Abrahamian, Peter E; Abou-Jawdah, Yusuf

    2013-07-01

    Squash leaf curl diseases are caused by distinct virus species that are separated into two major phylogenetic groups, western and eastern hemisphere groups. The western group includes the new world Squash leaf curl virus (SLCV) which causes major losses to cucurbit production and induces severe stunting and leaf curl in squash plants. A TaqMan-based real time polymerase chain reaction (qPCR) assay has been developed for detection and quantitation of SLCV. Designed primers and probe targeted the AV1 (coat protein) gene and in silico analysis showed that they detect a large number of SLCV isolates. The developed assay could detect the virus in 18fg of total nucleic acid and 30 genomic units. The qPCR assay was about 1000 times more sensitive than PCR and amplified successfully SLCV from a wide range of cucurbit hosts and from viruliferous whiteflies. The developed qPCR assay should be suitable for detection and quantitation purposes for all reported SLCV isolates of the western hemisphere.

  11. SPESIFIKASI SIMPLISIA DAN EKSTRAK ETANOL BIJI PINANG (ARECA CATECHU L ASAL TAWANGMANGU SERTA TOKSISITAS AKUT DAN KHASIAT HEMOSTATIKNYA PADA HEWAN COBA

    Directory of Open Access Journals (Sweden)

    Sa'roni Sa'roni

    2012-10-01

    Full Text Available Biji pinang (Areca catechu L secara tradisional diantaranya digunakan untuk obat menghentikan cucur darah dan haid banyak mengeluarkan darah. Untuk membuktikan penggunaan tersebut perlu dilakukan penelitian apakah ekstrak biji pinang mempunyai khasiat hemostatik, yaitu dapat mempercepat waktu beku darah serta untuk mendapatkan gambaran toksisitasnya ditentukan harga LD50 nya. Sebelum penelitian dilakukan spesifikasi simplisia dan ekstrak total dari biji pinang. Penelitian LD50 menurut cara Weil ,C.S dengan menggunakan hewan mencit dan penelitian khasiat hemostatik menurut cara Lee-White dengan menggunakan hewan tikus putih. Penelitian khasiat hemostatik dilakukan pada 3 macam  dosis ekstrak biji pinang yaitu 1,63mg, 4,9mg dan 16,3mg/100g.bobot badan tikus. Spesifikasi simplisia biji pinang asal Tawangmangu berwarna coklat, rasa pahit, kadar abu 4,2%± 0,1 kadar air  6,9%± 0,27. Spesifikasi ekstrak etanol biji pinang berwarna coklat kemerahan rasa pahit, kental, mengandung kaloid, saponin, flavonoid, tanin, polifenol dan antrakinon. Besar LD50 ekstrak etanol biji pinang 4,14 (3,31-5,18mg/10g. bobot badan secara ip pada mencit. Ekstrak dosis 16,3mg/100g.bobot badan tikus mempunyai khasiat hemostatik yang tidak berbeda dengan transamin dosis 4,5mg/100g.bobot badan tikus.   Kata kunci :  Areca catecu L; Toksisitas akut; Hemostatik

  12. Method for The Removal of Piumbum(II Ion from Aqueous Solutions by Corn Cobas A Natural Adsorbent and from The View Point Thermodynamics

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    Gholamali Haghdoost

    2016-06-01

    Full Text Available Corn cob as a low-cost adsorbent were used in the present work for the removal of toxic heavy metal Pb2+ from aqueous solutions. Bath experiments were used to determine the best adsorption conditions. The equilibrium adsorption level was determined as a function of solution pH, temperature(T, contact time(tc, initial adsorbate concentration, and adsorbent dosage. Effective removal of metal ions was demonstrated at pH values of 6. Metal adsorption onto Corn cobwas evaluated by Langmuir and Freundlich isotherms. Results indicate that the Langmuir isotherm model is the most suitable one for the adsorption process using Corn cob(R2=0.9742, thus indicating the applicability of monolayer coverage of Pb(II ion on Corn cob surface.The relationship between thermodynamic parameters was used to predict the absorption process. According to Thermodynamic analysis, the process endothermic and natural (ΔHo=-16.2 Jmol-1 and ΔSo =- 59.71 Jmol-1 K-1.

  13. The New Aptima HBV Quant Real-Time TMA Assay Accurately Quantifies Hepatitis B Virus DNA from Genotypes A to F.

    Science.gov (United States)

    Chevaliez, Stéphane; Dauvillier, Claude; Dubernet, Fabienne; Poveda, Jean-Dominique; Laperche, Syria; Hézode, Christophe; Pawlotsky, Jean-Michel

    2017-04-01

    Sensitive and accurate hepatitis B virus (HBV) DNA detection and quantification are essential to diagnose HBV infection, establish the prognosis of HBV-related liver disease, and guide the decision to treat and monitor the virological response to antiviral treatment and the emergence of resistance. Currently available HBV DNA platforms and assays are generally designed for batching multiple specimens within an individual run and require at least one full day of work to complete the analyses. The aim of this study was to evaluate the ability of the newly developed, fully automated, one-step Aptima HBV Quant assay to accurately detect and quantify HBV DNA in a large series of patients infected with different HBV genotypes. The limit of detection of the assay was estimated to be 4.5 IU/ml. The specificity of the assay was 100%. Intra-assay and interassay coefficients of variation ranged from 0.29% to 5.07% and 4.90% to 6.85%, respectively. HBV DNA levels from patients infected with HBV genotypes A to F measured with the Aptima HBV Quant assay strongly correlated with those measured by two commercial real-time PCR comparators (Cobas AmpliPrep/Cobas TaqMan HBV test, version 2.0, and Abbott RealTime HBV test). In conclusion, the Aptima HBV Quant assay is sensitive, specific, and reproducible and accurately quantifies HBV DNA in plasma samples from patients with chronic HBV infections of all genotypes, including patients on antiviral treatment with nucleoside or nucleotide analogues. The Aptima HBV Quant assay can thus confidently be used to detect and quantify HBV DNA in both clinical trials with new anti-HBV drugs and clinical practice. Copyright © 2017 American Society for Microbiology.

  14. Hepatitis B viral load in dried blood spots: a validation study in Zambia

    Science.gov (United States)

    Vinikoor, Michael J.; Zürcher, Samuel; Musukuma, Kalo; Kachuwaire, Obert; Rauch, Andri; Chi, Benjamin H.; Gorgievski, Meri; Zwahlen, Marcel; Wandeler, Gilles

    2016-01-01

    Background Access to hepatitis B viral load (VL) testing is poor in sub-Saharan Africa (SSA) due to economic and logistical reasons. Objectives To demonstrate the feasibility of testing dried blood spots (DBS) for hepatitis B virus (HBV) VL in a laboratory in Lusaka, Zambia, and to compare HBV VLs between DBS and plasma samples. Study design Paired plasma and DBS samples from HIV-HBV co-infected Zambian adults were analyzed for HBV VL using the COBAS AmpliPrep/COBAS TaqMan HBV test (Version 2.0) and for genotype by direct sequencing. We used Bland-Altman analysis to compare VLs between sample types and by HBV genotype. Logistic regression analysis was conducted to assess the probability of an undetectable DBS result by plasma VL. Results Among 68 participants, median age was 34 years, 61.8% were men, and median plasma HBV VL was 3.98 log IU/ml (interquartile range, 2.04–5.95). Among sequenced viruses, 28 were genotype A1 and 27 were genotype E. Bland-Altman plots suggested strong agreement between DBS and plasma VLs. DBS VLs were on average 1.59 log IU/ml lower compared to plasma with 95% limits of agreement of −2.40 to −0.83 log IU/ml. At a plasma VL ≥2,000 IU/ml, the probability of an undetectable DBS result was 1.8% (95% CI: 0.5–6.6). At plasma VL ≥20,000 IU/ml this probability reduced to 0.2% (95% CI: 0.03–1.7). Conclusions In a Zambian laboratory, we observed strong agreement between DBS and plasma VLs and high sensitivity in DBS at plasma VL ≥2,000 IU/ml. As HBV treatment expands, DBS could increase access to HBV VL testing in SSA settings. PMID:26356987

  15. Early diagnosis of in utero and intrapartum HIV infection in infants prior to 6 weeks of age.

    Science.gov (United States)

    Lilian, Rivka R; Kalk, Emma; Bhowan, Kapila; Berrie, Leigh; Carmona, Sergio; Technau, Karl; Sherman, Gayle G

    2012-07-01

    Early initiation of antiretroviral therapy reduces HIV-related infant mortality. The early peak of pediatric HIV-related deaths in South Africa occurs at 3 months of age, coinciding with the earliest age at which treatment is initiated following PCR testing at 6 weeks of age. Earlier diagnosis is necessary to reduce infant mortality. The performances of the Amplicor DNA PCR, COBAS AmpliPrep/COBAS TaqMan (CAP/CTM), and Aptima assays for detecting early HIV infection (acquired in utero and intrapartum) up to 6 weeks of age were compared. Dried blood spots (DBS) were collected at birth and at 2, 4, and 6 weeks from HIV-exposed infants enrolled in an observational cohort study in Johannesburg, South Africa. HIV status was determined at 6 weeks by DNA PCR on whole blood. Serial DBS samples from all HIV-infected infants and two HIV-uninfected, age-matched controls were tested with the 3 assays. Of 710 infants of known HIV status, 38 (5.4%) had in utero (n = 29) or intrapartum (n = 9) infections. By 14 weeks, when treatment should have been initiated, 13 (45%) in utero-infected and 2 (22%) intrapartum-infected infants had died or were lost to follow-up. The CAP/CTM and Aptima assays identified 76.3% of all infants with early HIV infections at birth and by 4 weeks were 96% sensitive. DNA PCR demonstrated lower sensitivities at birth and 4 weeks of 68.4% and 87.5%, respectively. All assays had the lowest sensitivity at 2 weeks of age. CAP/CTM was the only assay with 100% specificity at all ages. Testing at birth versus 6 weeks of age identifies a higher total number of HIV-infected infants, irrespective of the assay.

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

    Science.gov (United States)

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

    2016-01-01

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

  17. Higher specificity of nucleic acid sequence-based amplification isothermal technology than of real-time PCR for quantification of HIV-1 RNA on dried blood spots.

    Science.gov (United States)

    Mercier-Delarue, Severine; Vray, Muriel; Plantier, Jean Christophe; Maillard, Theodora; Adjout, Zidan; de Olivera, Fabienne; Schnepf, Nathalie; Maylin, Sarah; Simon, Francois; Delaugerre, Constance

    2014-01-01

    Dried blood spots (DBS) are widely proposed as a plasma surrogate for monitoring antiretroviral treatment efficacy based on the HIV-1 RNA level (viral load [VL]) in resource-limited settings. Interfering coamplification of cell-associated HIV-1 DNA during reverse transcription (RT)-PCR can be avoided by using nucleic acid sequence-based amplification (NASBA) technology, which is based on an RNA template and isothermic conditions. We analyzed VL values obtained with DBS and plasma samples by comparing isothermic NASBA (NucliSENS EasyQ HIV-1 V2.0; bioMérieux) with real-time RT-PCR (Cobas TaqMan HIV-1 V2.0; Roche). Samples from 197 HIV-1-infected patients were tested (non-B subtypes in 51% of the cases). Nucleic acid extractions were performed by use of NucliSENS EasyMAG (bioMérieux) and Cobas AmpliPrep (Roche) before the NASBA and RT-PCR quantifications, respectively. Both quantification assays have lower limits of detection of 20 (1.3) and 800 (2.9) log10 copies/ml (log) in plasma and DBS, respectively. The mean (DBS minus plasma) differences were -0.39 and -0.46 log, respectively, for RT-PCR and NASBA. RT-PCR on DBS identified virological failure in 122 of 126 patients (sensitivity, 97%) and viral suppression in 58 of 70 patients (specificity, 83%), yielding 12 false-positive results (median, 3.2 log). NASBA on DBS identified virological failure in 85 of 96 patients (sensitivity, 89%) and viral suppression in 95 of 97 patients (specificity, 98%) and yielded 2 false-positive results (3.0 log for both). Both technologies detected HIV-1 RNA in DBS at a threshold of 800 copies/ml. This higher specificity of NASBA technology could avoid overestimation of poor compliance or the emergence of resistance when monitoring antiretroviral efficacy with the DBS method.

  18. Performance of an Early Infant Diagnostic Test, AmpliSens DNA-HIV-FRT, Using Dried Blood Spots Collected from Children Born to Human Immunodeficiency Virus-Infected Mothers in Ukraine

    Science.gov (United States)

    Shanmugam, Vedapuri; Azarskova, Marianna; Nguyen, Shon; Hurlston, Mackenzie; Sabatier, Jennifer; Zhang, Guoqing; Osmanov, Saladin; Ellenberger, Dennis; Yang, Chunfu; Vitek, Charles; Liulchuk, Maria; Nizova, Natalya

    2015-01-01

    An accurate accessible test for early infant diagnosis (EID) is crucial for identifying HIV-infected infants and linking them to treatment. To improve EID services in Ukraine, dried blood spot (DBS) samples obtained from 237 HIV-exposed children (≤18 months of age) in six regions in Ukraine in 2012 to 2013 were tested with the AmpliSens DNA-HIV-FRT assay, the Roche COBAS AmpliPrep/COBAS TaqMan (CAP/CTM) HIV-1 Qual test, and the Abbott RealTime HIV-1 Qualitative assay. In comparison with the paired whole-blood results generated from AmpliSens testing at the oblast HIV reference laboratories in Ukraine, the sensitivity was 0.99 (95% confidence interval [CI], 0.95 to 1.00) for the AmpliSens and Roche CAP/CTM Qual assays and 0.96 (95% CI, 0.90 to 0.98) for the Abbott Qualitative assay. The specificity was 1.00 (95% CI, 0.97 to 1.00) for the AmpliSens and Abbott Qualitative assays and 0.99 (95% CI, 0.96 to 1.00) for the Roche CAP/CTM Qual assay. McNemar analysis indicated that the proportions of positive results for the tests were not significantly different (P > 0.05). Cohen's kappa (0.97 to 0.99) indicated almost perfect agreement among the three tests. These results indicated that the AmpliSens DBS and whole-blood tests performed equally well and were comparable to the two commercially available EID tests. More importantly, the performance characteristics of the AmpliSens DBS test meets the World Health Organization EID test requirements; implementing AmpliSens DBS testing might improve EID services in resource-limited settings. PMID:26447114

  19. Evaluation of Hologic Aptima HIV-1 Quant Dx Assay on the Panther System on HIV Subtypes.

    Science.gov (United States)

    Manak, Mark M; Hack, Holly R; Nair, Sangeetha V; Worlock, Andrew; Malia, Jennifer A; Peel, Sheila A; Jagodzinski, Linda L

    2016-10-01

    Quantitation of the HIV-1 viral load in plasma is the current standard of care for clinical monitoring of HIV-infected individuals undergoing antiretroviral therapy. This study evaluated the analytical and clinical performances of the Aptima HIV-1 Quant Dx assay (Hologic, San Diego, CA) for monitoring viral load by using 277 well-characterized subtype samples, including 171 cultured virus isolates and 106 plasma samples from 35 countries, representing all major HIV subtypes, recombinants, and circulating recombinant forms (CRFs) currently in circulation worldwide. Linearity of the Aptima assay was tested on each of 6 major HIV-1 subtypes (A, B, C, D, CRF01_AE, and CRF02_AG) and demonstrated an R(2) value of ≥0.996. The performance of the Aptima assay was also compared to those of the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 v.2 (CAP/CTM) and Abbott m2000 RealTime HIV-1 (RealTime) assays on all subtype samples. The Aptima assay values averaged 0.21 log higher than the CAP/CTM values and 0.30 log higher than the RealTime values, and the values were >0.4 log higher than CAP/CTM values for subtypes F and G and than RealTime values for subtypes C, F, and G and CRF02_AG. Two samples demonstrated results with >1-log differences from RealTime results. When the data were adjusted by the average difference, 94.9% and 87.0% of Aptima results fell within 0.5 log of the CAP/CTM and RealTime results, respectively. The linearity and accuracy of the Aptima assay in correctly quantitating all major HIV-1 subtypes, coupled with the completely automated format and high throughput of the Panther system, make this system well suited for reliable measurement of viral load in the clinical laboratory. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  20. The New Aptima HCV Quant Dx Real-time TMA Assay Accurately Quantifies Hepatitis C Virus Genotype 1-6 RNA.

    Science.gov (United States)

    Chevaliez, Stéphane; Dubernet, Fabienne; Dauvillier, Claude; Hézode, Christophe; Pawlotsky, Jean-Michel

    2017-06-01

    Sensitive and accurate hepatitis C virus (HCV) RNA detection and quantification is essential for the management of chronic hepatitis C therapy. Currently available platforms and assays are usually batched and require at least 5hours of work to complete the analyses. The aim of this study was to evaluate the ability of the newly developed Aptima HCV Quant Dx assay that eliminates the need for batch processing and automates all aspects of nucleic acid testing in a single step, to accurately detect and quantify HCV RNA in a large series of patients infected with different HCV genotypes. The limit of detection was estimated to be 2.3 IU/mL. The specificity of the assay was 98.6% (95% confidence interval: 96.1%-99.5%). Intra-assay and inter-assay coefficients of variation ranged from 0.09% to 5.61%, and 1.05% to 3.65%, respectively. The study of serum specimens from patients infected with HCV genotypes 1 to 6 showed a satisfactory relationship between HCV RNA levels measured by the Aptima HCV Quant Dx assay, and both real-time PCR comparators (Abbott RealTime HCV and Cobas AmpliPrep/Cobas TaqMan HCV Test, version 2.0, assays). the new Aptima HCV Quant Dx assay is rapid, sensitive, reasonably specific and reproducible and accurately quantifies HCV RNA in serum samples from patients with chronic HCV infection, including patients on antiviral treatment. The Aptima HCV Quant Dx assay can thus be confidently used to detect and quantify HCV RNA in both clinical trials with new anti-HCV drugs and clinical practice in Europe and the US. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Evaluation of the NucliSens EasyQ v2.0 assay in comparison with the Roche Amplicor v1.5 and the Roche CAP/CTM HIV-1 Test v2.0 in quantification of C-clade HIV-1 in plasma.

    Science.gov (United States)

    Muenchhoff, Maximilian; Madurai, Savathee; Hempenstall, Allison Jo; Adland, Emily; Carlqvist, Anna; Moonsamy, Angeline; Jaggernath, Manjeetha; Mlotshwa, Busisiwe; Siboto, Emma; Ndung'u, Thumbi; Goulder, Philip Jeremy Renshaw

    2014-01-01

    Human immunodeficiency virus type 1 (HIV-1) genetic diversity poses a challenge to reliable viral load monitoring. Discrepancies between different testing platforms have been observed, especially for non-clade-B virus. Therefore we compare, in antiretroviral therapy (ART)-naïve South African subjects predominantly infected with HIV-1 clade-C, three commercially available assays: the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test version 2.0 by Roche (CAP/CTM v2.0), the BioMérieux NucliSens Version 2.0 Easy Q/Easy Mag (NucliSens v2.0) and the Roche COBAS Amplicor HIV-1 Monitor Test Version 1.5 (Amplicor v1.5). Strong linear correlation was observed and Bland-Altman analyses showed overall good agreement between the assays with mean viral load differences of 0.078 log cp/ml (NucliSens v2.0 - Amplicor v1.5), 0.260 log cp/ml (CAP/CTM v2.0 - Amplicor v1.5) and 0.164 log cp/ml (CAP/CTM v2.0 - NucliSens v2.0), indicating lower mean viral load results for the Amplicor v1.5 and higher mean readings for the CAP/CTM v2.0. Consistent with observations following previous comparisons of CAP/CTM v2.0 versus Amplicor v1.5, the CAP/CTM v2.0 assay detected low-level viremia (median 65 cp/ml) in more than one-third of those in whom viremia had been undetectable (<20 cp/ml) in assays using the NucliSens platform. These levels of viremia are of uncertain clinical significance but may be of importance in early detection of ART resistance in those on treatment. Overall the three assays showed good comparability of results but with consistent, albeit relatively small, discrepancies for HIV-1 clade-C samples, especially in the low-viremic range that should be taken into account when interpreting viral load data.

  2. Evaluation of the NucliSens EasyQ v2.0 assay in comparison with the Roche Amplicor v1.5 and the Roche CAP/CTM HIV-1 Test v2.0 in quantification of C-clade HIV-1 in plasma.

    Directory of Open Access Journals (Sweden)

    Maximilian Muenchhoff

    Full Text Available Human immunodeficiency virus type 1 (HIV-1 genetic diversity poses a challenge to reliable viral load monitoring. Discrepancies between different testing platforms have been observed, especially for non-clade-B virus. Therefore we compare, in antiretroviral therapy (ART-naïve South African subjects predominantly infected with HIV-1 clade-C, three commercially available assays: the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test version 2.0 by Roche (CAP/CTM v2.0, the BioMérieux NucliSens Version 2.0 Easy Q/Easy Mag (NucliSens v2.0 and the Roche COBAS Amplicor HIV-1 Monitor Test Version 1.5 (Amplicor v1.5. Strong linear correlation was observed and Bland-Altman analyses showed overall good agreement between the assays with mean viral load differences of 0.078 log cp/ml (NucliSens v2.0 - Amplicor v1.5, 0.260 log cp/ml (CAP/CTM v2.0 - Amplicor v1.5 and 0.164 log cp/ml (CAP/CTM v2.0 - NucliSens v2.0, indicating lower mean viral load results for the Amplicor v1.5 and higher mean readings for the CAP/CTM v2.0. Consistent with observations following previous comparisons of CAP/CTM v2.0 versus Amplicor v1.5, the CAP/CTM v2.0 assay detected low-level viremia (median 65 cp/ml in more than one-third of those in whom viremia had been undetectable (<20 cp/ml in assays using the NucliSens platform. These levels of viremia are of uncertain clinical significance but may be of importance in early detection of ART resistance in those on treatment. Overall the three assays showed good comparability of results but with consistent, albeit relatively small, discrepancies for HIV-1 clade-C samples, especially in the low-viremic range that should be taken into account when interpreting viral load data.

  3. Comparison of the Abbott RealTime CT new formulation assay with two other commercial assays for detection of wild-type and new variant strains of Chlamydia trachomatis

    DEFF Research Database (Denmark)

    Møller, Jens Kjølseth; Pedersen, Lisbeth Nørum; Persson, Kenneth

    2010-01-01

    In an analytical methods comparison study on clinical samples, the Abbott RealTime CT new formulation assay (m2000 real-time PCR) consisting of a duplex PCR targeting different parts of the cryptic plasmid in Chlamydia trachomatis was compared with version 2 of the Roche COBAS(R) TaqMan(R) CT assay...... comprising a duplex PCR for a target in the cryptic plasmid and the omp1 gene, and compared with the Gen-Probe APTIMA COMBO 2(R) assay (AC2) targeting the C. trachomatis 23S rRNA molecule. First-catch urine samples from Sweden were tested in Malmoe for C. trachomatis with the m2000 real-time PCR assay......, and with an in-house PCR for the new variant C. trachomatis strain with a deletion in the cryptic plasmid. Aliquots of the urine samples were sent to Aarhus, Denmark and further examined with the TaqMan CT and the AC2 assay. A positive prevalence of 9.1% (148/1,632 urine samples examined) was detected according...

  4. Multicentric performance analysis of HCV quantification assays and its potential relevance for HCV treatment.

    Science.gov (United States)

    Wiesmann, F; Naeth, G; Berger, A; Hirsch, H H; Regenass, S; Ross, R S; Sarrazin, C; Wedemeyer, H; Knechten, H; Braun, P

    2016-06-01

    An accurate quantification of low viremic HCV RNA plasma samples has gained importance since the approval of direct acting antivirals and since only one single measurement predicts the necessity of a prolonged or shortened therapy. As reported previously, HCV quantification assays such as Abbott RealTime HCV and Roche COBAS AmpliPrep/COBAS TaqMan HCV version 2 (CTM v2) may vary in sensitivity and precision particularly in low-level viremia. Importantly, substantial variations were previously demonstrated between some of these assays compared to the Roche High Pure System/COBAS TaqMan assay (HPS) reference assay, which was used to establish the clinical decision points in clinical studies. In this study, the reproducibility of assay performances across several laboratories was assessed by analysing quantification results generated by six independent laboratories (3× RealTime, 3× CTM v2) in comparison with one HPS reference laboratory. The 4th WHO Standard was diluted to 100, 25 and 10 IU/ml, and aliquots were tested in triplicates in 5 independent runs by each assay in the different laboratories to assess assay precision and detection rates. In a second approach, 2 clinical samples (GT 1a & GT 1b) were diluted to 100 and 25 IU/ml and tested as described above. While the result range for WHO 100 IU/ml replicates across all laboratories was similar in this analysis, the CVs of each laboratory ranged from 19.3 to 25.6 % for RealTime laboratories and were lower than CVs of CTM v2 laboratories with a range of 26.1-47.3 %, respectively, and also in comparison with the CV of the HPS reference laboratory (34.9 %). At WHO standard dilution of 25 IU/ml, 24 replicates were quantified by RealTime compared to 8 replicates with CTM v2. Results of clinical samples again revealed a higher variation of CTM v2 results as compared to RealTime values. (CVs at 100 IU/ml: RealTime: 13.1-21.0 % and CTM v2: 15.0-32.3 %; CVs at 25 IU/ml: RealTime 17.6-34.9 % and CTM v2 28

  5. Systematic Review of the Performance of HIV Viral Load Technologies on Plasma Samples

    Science.gov (United States)

    Sollis, Kimberly A.; Smit, Pieter W.; Fiscus, Susan; Ford, Nathan; Vitoria, Marco; Essajee, Shaffiq; Barnett, David; Cheng, Ben; Crowe, Suzanne M.; Denny, Thomas; Landay, Alan; Stevens, Wendy; Habiyambere, Vincent; Perrins, Jos; Peeling, Rosanna W.

    2014-01-01

    Background Viral load (VL) monitoring is the standard of care in developing country settings for detecting HIV treatment failure. Since 2010 the World Health Organization has recommended a phase-in approach to VL monitoring in resource-limited settings. We conducted a systematic review of the accuracy and precision of HIV VL technologies for treatment monitoring. Methods and Findings A search of Medline and Embase was conducted for studies evaluating the accuracy or reproducibility of commercially available HIV VL assays. 37 studies were included for review including evaluations of the Amplicor Monitor HIV-1 v1.5 (n = 25), Cobas TaqMan v2.0 (n = 11), Abbott RealTime HIV-1 (n = 23), Versant HIV-1 RNA bDNA 3.0 (n = 15), Versant HIV-1 RNA kPCR 1.0 (n = 2), ExaVir Load v3 (n = 2), and NucliSens EasyQ v2.0 (n = 1). All currently available HIV VL assays are of sufficient sensitivity to detect plasma virus levels at a lower detection limit of 1,000 copies/mL. Bias data comparing the Abbott RealTime HIV-1, TaqMan v2.0 to the Amplicor Monitor v1.5 showed a tendency of the Abbott RealTime HIV-1 to under-estimate results while the TaqMan v2.0 overestimated VL counts. Compared to the Amplicor Monitor v1.5, 2–26% and 9–70% of results from the Versant bDNA 3.0 and Abbott RealTime HIV-1 differed by greater than 0.5log10. The average intra and inter-assay variation of the Abbott RealTime HIV-1 were 2.95% (range 2.0–5.1%) and 5.44% (range 1.17–30.00%) across the range of VL counts (2log10–7log10). Conclusions This review found that all currently available HIV VL assays are of sufficient sensitivity to detect plasma VL of 1,000 copies/mL as a threshold to initiate investigations of treatment adherence or possible treatment failure. Sources of variability between VL assays include differences in technology platform, plasma input volume, and ability to detect HIV-1 subtypes. Monitoring of individual patients should be performed on the same

  6. Systematic review of the performance of HIV viral load technologies on plasma samples.

    Directory of Open Access Journals (Sweden)

    Kimberly A Sollis

    Full Text Available BACKGROUND: Viral load (VL monitoring is the standard of care in developing country settings for detecting HIV treatment failure. Since 2010 the World Health Organization has recommended a phase-in approach to VL monitoring in resource-limited settings. We conducted a systematic review of the accuracy and precision of HIV VL technologies for treatment monitoring. METHODS AND FINDINGS: A search of Medline and Embase was conducted for studies evaluating the accuracy or reproducibility of commercially available HIV VL assays. 37 studies were included for review including evaluations of the Amplicor Monitor HIV-1 v1.5 (n = 25, Cobas TaqMan v2.0 (n = 11, Abbott RealTime HIV-1 (n = 23, Versant HIV-1 RNA bDNA 3.0 (n = 15, Versant HIV-1 RNA kPCR 1.0 (n = 2, ExaVir Load v3 (n = 2, and NucliSens EasyQ v2.0 (n = 1. All currently available HIV VL assays are of sufficient sensitivity to detect plasma virus levels at a lower detection limit of 1,000 copies/mL. Bias data comparing the Abbott RealTime HIV-1, TaqMan v2.0 to the Amplicor Monitor v1.5 showed a tendency of the Abbott RealTime HIV-1 to under-estimate results while the TaqMan v2.0 overestimated VL counts. Compared to the Amplicor Monitor v1.5, 2-26% and 9-70% of results from the Versant bDNA 3.0 and Abbott RealTime HIV-1 differed by greater than 0.5log10. The average intra and inter-assay variation of the Abbott RealTime HIV-1 were 2.95% (range 2.0-5.1% and 5.44% (range 1.17-30.00% across the range of VL counts (2log10-7log10. CONCLUSIONS: This review found that all currently available HIV VL assays are of sufficient sensitivity to detect plasma VL of 1,000 copies/mL as a threshold to initiate investigations of treatment adherence or possible treatment failure. Sources of variability between VL assays include differences in technology platform, plasma input volume, and ability to detect HIV-1 subtypes. Monitoring of individual patients should be performed on the same

  7. Comparative, Collaborative, and On-Site Validation of a TaqMan PCR Method as a Tool for Certified Production of Fresh, Campylobacter-Free Chickens

    DEFF Research Database (Denmark)

    Krause, Michael; Josefsen, Mathilde Hartmann; Lund, Marianne

    2006-01-01

    Certified Campylobacter-free poultry products have been produced in Denmark since 2002, the first example of fresh (unprocessed and nonfrozen) chickens labeled "Campylobacter free." This success occurred partly through use of a 4-hour gel-based PCR testing scheme on fecal swabs. In this study......, a faster, real-time PCR approach was validated in comparative and collaborative trials, based on recommendations from the Nordic system for validation of alternative microbiological methods (NordVal). The comparative real-time PCR trial was performed in comparison to two reference culture protocols...... as positive. The comparative trial resulted in relative accuracy, sensitivity, and specificity of 98%, 95%, and 97%, respectively. The collaborative trial included nine laboratories testing neck skin, cloacal swab, and shoe cover samples, spiked with low, medium, and high concentrations of Campylobacter...

  8. Comparative analytical evaluation of the respiratory TaqMan Array Card with real-time PCR and commercial multi-pathogen assays.

    Science.gov (United States)

    Harvey, John J; Chester, Stephanie; Burke, Stephen A; Ansbro, Marisela; Aden, Tricia; Gose, Remedios; Sciulli, Rebecca; Bai, Jing; DesJardin, Lucy; Benfer, Jeffrey L; Hall, Joshua; Smole, Sandra; Doan, Kimberly; Popowich, Michael D; St George, Kirsten; Quinlan, Tammy; Halse, Tanya A; Li, Zhen; Pérez-Osorio, Ailyn C; Glover, William A; Russell, Denny; Reisdorf, Erik; Whyte, Thomas; Whitaker, Brett; Hatcher, Cynthia; Srinivasan, Velusamy; Tatti, Kathleen; Tondella, Maria Lucia; Wang, Xin; Winchell, Jonas M; Mayer, Leonard W; Jernigan, Daniel; Mawle, Alison C

    2016-02-01

    In this study, a multicenter evaluation of the Life Technologies TaqMan(®) Array Card (TAC) with 21 custom viral and bacterial respiratory assays was performed on the Applied Biosystems ViiA™ 7 Real-Time PCR System. The goal of the study was to demonstrate the analytical performance of this platform when compared to identical individual pathogen specific laboratory developed tests (LDTs) designed at the Centers for Disease Control and Prevention (CDC), equivalent LDTs provided by state public health laboratories, or to three different commercial multi-respiratory panels. CDC and Association of Public Health Laboratories (APHL) LDTs had similar analytical sensitivities for viral pathogens, while several of the bacterial pathogen APHL LDTs demonstrated sensitivities one log higher than the corresponding CDC LDT. When compared to CDC LDTs, TAC assays were generally one to two logs less sensitive depending on the site performing the analysis. Finally, TAC assays were generally more sensitive than their counterparts in three different commercial multi-respiratory panels. TAC technology allows users to spot customized assays and design TAC layout, simplify assay setup, conserve specimen, dramatically reduce contamination potential, and as demonstrated in this study, analyze multiple samples in parallel with good reproducibility between instruments and operators.

  9. A Multi-Species TaqMan PCR Assay for the Identification of Asian Gypsy Moths (Lymantria spp.) and Other Invasive Lymantriines of Biosecurity Concern to North America

    Science.gov (United States)

    Stewart, Donald; Zahiri, Reza; Djoumad, Abdelmadjid; Freschi, Luca; Lamarche, Josyanne; Holden, Dave; Cervantes, Sandra; Ojeda, Dario I.; Potvin, Amélie; Nisole, Audrey; Béliveau, Catherine; Capron, Arnaud; Kimoto, Troy; Day, Brittany; Yueh, Hesther; Duff, Cameron; Levesque, Roger C.; Hamelin, Richard C.; Cusson, Michel

    2016-01-01

    Preventing the introduction and establishment of forest invasive alien species (FIAS) such as the Asian gypsy moth (AGM) is a high-priority goal for countries with extensive forest resources such as Canada. The name AGM designates a group of closely related Lymantria species (Lepidoptera: Erebidae: Lymantriinae) comprising two L. dispar subspecies (L. dispar asiatica, L. dispar japonica) and three closely related Lymantria species (L. umbrosa, L. albescens, L. postalba), all considered potential FIAS in North America. Ships entering Canadian ports are inspected for the presence of suspicious gypsy moth eggs, but those of AGM are impossible to distinguish from eggs of innocuous Lymantria species. To assist regulatory agencies in their identification of these insects, we designed a suite of TaqMan® assays that provide significant improvements over existing molecular assays targeting AGM. The assays presented here can identify all three L. dispar subspecies (including the European gypsy moth, L. dispar dispar), the three other Lymantria species comprising the AGM complex, plus five additional Lymantria species that pose a threat to forests in North America. The suite of assays is built as a “molecular key” (analogous to a taxonomic key) and involves several parallel singleplex and multiplex qPCR reactions. Each reaction uses a combination of primers and probes designed to separate taxa through discriminatory annealing. The success of these assays is based on the presence of single nucleotide polymorphisms (SNPs) in the 5’ region of mitochondrial cytochrome c oxidase I (COI) or in its longer, 3’ region, as well as on the presence of an indel in the “FS1” nuclear marker, generating North American and Asian alleles, used here to assess Asian introgression into L. dispar dispar. These assays have the advantage of providing rapid and accurate identification of ten Lymantria species and subspecies considered potential FIAS. PMID:27513667

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

    Directory of Open Access Journals (Sweden)

    Zheng Pang

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

  11. Quantification of ochratoxin A-producing molds in food products by SYBR Green and TaqMan real-time PCR methods

    DEFF Research Database (Denmark)

    Rodríguez, Alicia; Rodríguez, Mar; Luque, M. Isabel

    2011-01-01

    Ochratoxin A (OTA) is a mycotoxin synthesized by a variety of different fungi, most of them from the genera Penicillium and Aspergillus. Early detection and quantification of OTA producing species is crucial to improve food safety. In the present work, two protocols of real-time qPCR based on SYBR...

  12. Interlaboratory validation of an improved U.S. Food and Drug Administration method for detection of Cyclospora cayetanensis in produce using TaqMan real-time PCR

    Science.gov (United States)

    A collaborative validation study was performed to evaluate the performance of a new U.S. Food and Drug Administration method developed for detection of the protozoan parasite, Cyclospora cayetanensis, on cilantro and raspberries. The method includes a sample preparation step in which oocysts are re...

  13. Development of a multiplex taqMan real-time PCR assay for typing of Mycoplasma pneumoniae based on type-specific indels identified through whole genome sequencing.

    Science.gov (United States)

    Wolff, Bernard J; Benitez, Alvaro J; Desai, Heta P; Morrison, Shatavia S; Diaz, Maureen H; Winchell, Jonas M

    2017-03-01

    We developed a multiplex real-time PCR assay for simultaneously detecting M. pneumoniae and typing into historically-defined P1 types. Typing was achieved based on the presence of short type-specific indels identified through whole genome sequencing. This assay was 100% specific compared to existing methods and may be useful during epidemiologic investigations.

  14. Genome Analysis and Development of a Multiplex TaqMan Real-Time PCR for Specific Identification and Detection of Clavibacter michiganensis subsp. nebraskensis.

    Science.gov (United States)

    Tambong, James T; Xu, Renlin; Daayf, Fouad; Brière, Stephan; Bilodeau, Guillaume J; Tropiano, Raymond; Hartke, Allison; Reid, Lana M; Cott, Morgan; Cote, Tammy; Agarkova, Irina

    2016-12-01

    The reemergence of the Goss's bacterial wilt and blight disease in corn in the United States and Canada has prompted investigative research to better understand the genome organization. In this study, we generated a draft genome sequence of Clavibacter michiganensis subsp. nebraskensis strain DOAB 395 and performed genome and proteome analysis of C. michiganensis subsp. nebraskensis strains isolated in 2014 (DOAB 397 and DOAB 395) compared with the type strain, NCPPB 2581 (isolated over 40 years ago). The proteomes of strains DOAB 395 and DOAB 397 exhibited a 99.2% homology but had 92.1 and 91.8% homology, respectively, with strain NCPPB 2581. The majority (99.9%) of the protein sequences had a 99.6 to 100% homology between C. michiganensis subsp. nebraskensis strains DOAB 395 and DOAB 397, with only four protein sequences (0.1%) exhibiting a similarity michiganensis subsp. nebraskensis. The specificity of the assay was validated using 122 strains of Clavibacter and non-Clavibacter spp. A blind test and naturally infected leaf samples were used to confirm specificity. The sensitivity (0.1 to 1.0 pg) compared favorably with previously reported real-time PCR assays. This tool should fill the current gap for a reliable diagnostic technique.

  15. A Multi-Species TaqMan PCR Assay for the Identification of Asian Gypsy Moths (Lymantria spp.) and Other Invasive Lymantriines of Biosecurity Concern to North America.

    Science.gov (United States)

    Stewart, Donald; Zahiri, Reza; Djoumad, Abdelmadjid; Freschi, Luca; Lamarche, Josyanne; Holden, Dave; Cervantes, Sandra; Ojeda, Dario I; Potvin, Amélie; Nisole, Audrey; Béliveau, Catherine; Capron, Arnaud; Kimoto, Troy; Day, Brittany; Yueh, Hesther; Duff, Cameron; Levesque, Roger C; Hamelin, Richard C; Cusson, Michel

    2016-01-01

    Preventing the introduction and establishment of forest invasive alien species (FIAS) such as the Asian gypsy moth (AGM) is a high-priority goal for countries with extensive forest resources such as Canada. The name AGM designates a group of closely related Lymantria species (Lepidoptera: Erebidae: Lymantriinae) comprising two L. dispar subspecies (L. dispar asiatica, L. dispar japonica) and three closely related Lymantria species (L. umbrosa, L. albescens, L. postalba), all considered potential FIAS in North America. Ships entering Canadian ports are inspected for the presence of suspicious gypsy moth eggs, but those of AGM are impossible to distinguish from eggs of innocuous Lymantria species. To assist regulatory agencies in their identification of these insects, we designed a suite of TaqMan® assays that provide significant improvements over existing molecular assays targeting AGM. The assays presented here can identify all three L. dispar subspecies (including the European gypsy moth, L. dispar dispar), the three other Lymantria species comprising the AGM complex, plus five additional Lymantria species that pose a threat to forests in North America. The suite of assays is built as a "molecular key" (analogous to a taxonomic key) and involves several parallel singleplex and multiplex qPCR reactions. Each reaction uses a combination of primers and probes designed to separate taxa through discriminatory annealing. The success of these assays is based on the presence of single nucleotide polymorphisms (SNPs) in the 5' region of mitochondrial cytochrome c oxidase I (COI) or in its longer, 3' region, as well as on the presence of an indel in the "FS1" nuclear marker, generating North American and Asian alleles, used here to assess Asian introgression into L. dispar dispar. These assays have the advantage of providing rapid and accurate identification of ten Lymantria species and subspecies considered potential FIAS.

  16. A Multi-Species TaqMan PCR Assay for the Identification of Asian Gypsy Moths (Lymantria spp.) and Other Invasive Lymantriines of Biosecurity Concern to North America

    National Research Council Canada - National Science Library

    Stewart, Donald; Zahiri, Reza; Djoumad, Abdelmadjid; Freschi, Luca; Lamarche, Josyanne; Holden, Dave; Cervantes, Sandra; Ojeda, Dario I; Potvin, Amélie; Nisole, Audrey; Béliveau, Catherine; Capron, Arnaud; Kimoto, Troy; Day, Brittany; Yueh, Hesther; Duff, Cameron; Levesque, Roger C; Hamelin, Richard C; Cusson, Michel

    2016-01-01

    .... The name AGM designates a group of closely related Lymantria species (Lepidoptera: Erebidae: Lymantriinae) comprising two L. dispar subspecies (L. dispar asiatica, L. dispar japonica) and three closely related Lymantria species...

  17. A multi-species TaqMan assay for the identification of Asian gypsy moths and other invasive lymantriines of biosecurity concern to North America

    National Research Council Canada - National Science Library

    Stewart, D; Zahiri, R; Djoumad, A; Freschi, L; Lamarche, J; Holden, D; Cervantes, S; Ojeda, D.I; Potvin, A; Nisole, A; Béliveau, C; Capron, A; Kimoto, T; Day, B; Yueh, H; Duff, C; Lévesque, R.C; Hamelin, R.C; Cusson, M

    2016-01-01

    .... The name AGM designates a group of closely related Lymantria species (Lepidoptera: Erebidae: Lymantriinae) comprising two L. dispar subspecies (L. dispar asiatica, L. dispar japonica) and three closely related Lymantria species...

  18. Comparison of efficiency of various DNA extraction methods from cysts of Giardia intestinalis measured by PCR and TaqMan real time PCR.

    Science.gov (United States)

    Adamska, M; Leońska-Duniec, A; Maciejewska, A; Sawczuk, M; Skotarczak, B

    2010-12-01

    The aim of the presented study was to work out an effective method of extraction of DNA from Giardia intestinalis cysts as well as a sensitive and specific method for detection of DNA of this protozoan using a polymerase chain reaction (PCR). Twelve protocols for DNA extraction have been compared. Purification and extraction of DNA were preceded by additional actions in order to destroy the cysts' wall. The highest effectiveness of DNA extraction was obtained in case of alternating application of freezing the samples in liquid nitrogen and their incubation in water bath in the temperature of 100 degrees C, and then the extraction with the QIAamp DNA Tissue Mini Kit (QIAGEN)--T kit--with an all night long incubation with proteinase K in 56 degrees C. Effectiveness of DNA extraction with the use of each kit after extraction with each treatment was measured by nested PCR product of beta-giardin gene fragment and C(T) values of real time PCR of the SSU rRNA gene of G. intestinalis. The detection limit, defined as the lowest number detected in 100% cases, was 100 cysts per 200 microl when effectiveness was evaluated with nested PCR and 50 oocysts with real time PCR after extraction DNA with T kit. Results of our comparative studies have shown that all stages preceding the molecular detection of G. intestinalis DNA are equally important, and materially influence on the final effect and this version of method seems to be very useful for the sensitive detection of DNA of G. intestinalis.

  19. A novel combination of TaqMan RT-PCR and a suspension microarray assay for the detection and species identification of pestiviruses

    OpenAIRE

    Leblanc, Neil; Leijon, Mikael; Jobs, Magnus; Blomberg, Jonas; Belák, Sándor

    2010-01-01

    Abstract The genus Pestivirus contains four recognized species: classical swine fever virus, Border disease virus, bovine viral diarrhoea virus types 1 and 2. All are economically important and globally distributed but classical swine fever is the most serious, concerning losses and control measures. It affects both domestic pigs and wild boars. Outbreaks of this disease in domestic pigs call for the most serious measures of disease control, including a stamping out policy in Europ...

  20. Prevalence of Fasciola hepatica in the intermediate host Lymnaea truncatula detected by real time TaqMan PCR in populations from 70 Swiss farms with cattle husbandry.

    Science.gov (United States)

    Schweizer, G; Meli, M L; Torgerson, P R; Lutz, H; Deplazes, P; Braun, U

    2007-11-30

    Bovine fasciolosis is an economically important parasitic disease. Quantitative real time PCR was utilized to determine the prevalence of Fasciola hepatica in the snail intermediate host Lymnaea truncatula from 70 selected, infected Swiss cattle farms, and to gain information on the infection risk to the definitive host. Snails from 130 habitats (36 streams, 21 wells, 24 drainage ditches, 33 spring swamps, 14 reeds, 1 drainage shaft and 1 pond) originating from 71 dairy cow pastures, 39 pastures for young stock, 14 hay fields and 6 dry cow pastures were collected. Of these, 51 populations were found to be infected with F. hepatica. A total of 4733 snails were examined of which 331 were infected (7.0%). The numbers of snails collected from different sites ranged from 1 to 159 snails. Clustering of infection in snails was found on the farm of origin with a mixed logistic model with random effects. The risk of infection of L. truncatula with F. hepatica was significantly higher in populations originating from spring swamps, wells and reeds compared to populations from streams. In addition the risk of snail infection was significantly lower in populations collected in young stock and dry cow pastures compared to dairy cow pastures. The greater the population size collected from a habitat also increased the risk of an individual snail being infected.

  1. 基于TaqMan探针三重荧光PCR检测MRSA%Detection of MRSA by TaqMan probe-based triple fluorescence PCR

    Institute of Scientific and Technical Information of China (English)

    袁慕云; 张旺; 卓锦雪; 谢力; 陈碧玲; 许龙岩

    2014-01-01

    目的 建立基于TaqMan探针三重荧光PCR检测耐甲氧西林金黄色葡萄球菌(MRSA)方法.方法 根据nuc(GenBank:EF529607.1)、mecA(GenBank:JN108029.1)和femA gene(GenBank:DQ352463.1)基因序列,分别设计特异性引物和Taqman探针,nuc探针5、端标记TET、mecA探针5’端标记HEX、femA探针5、端标记FAM,建立基于Taqman探针三重荧光PCR检测MRSA的方法,并对69株金黄色葡萄球菌分离株进行基因检测与耐药表型比较.结果MR-SA出现femA、mecA、nuc基因扩增曲线,而表皮葡萄球菌等14株对照菌株扩增结果呈阴性;femA、mecA、nuc基因的扩增效率分别为104%、90.3%、98%,线性系数R2分别为0.999、0.994、0.998;69株分离株中femA、nuc基因阳性率为100%,mecA基因阳性率为13.04%,mecA与耐甲氧西林表型符合率为100%.结论 建立的方法特异性强、准确,为金黄色葡萄球菌鉴定和耐药基因分析提供参考依据.

  2. The development of a validated real-time (TaqMan) PCR for detection of Stagonosporopsis andigena and S. crystalliniformis in infected leaves of potato and tomato

    NARCIS (Netherlands)

    Gruyter, de J.; Gent-Pelzer, van M.P.E.; Woudenberg, J.H.C.; Rijswick, van P.C.J.; Meekes, E.T.M.; Crous, P.W.; Bonants, P.J.M.

    2012-01-01

    Stagonosporopsis andigena and S. crystalliniformis are serious foliage pathogens on potato (Solanum tuberosum) and tomato (Solanum lycopersicum). As both species have been recorded only in the Andes area, S. andigena is listed as an A1 quarantine organism in Europe. The actin region of isolates of

  3. Monkeypox detection in rodents using real-time 3’minor groove binder Taqman assays on the Roche LightCycler, Laboratory Investigation 84:1200 - 1208

    Science.gov (United States)

    2007-11-02

    DNA 1000 MPV Zaire 79-I-05 10 + + MPXR Zaire 79-I-05 ( Cidofovir - resistant) 10 + + MPV Utrecht 10 + + MPV Sierra Leone 10 + + MPV 12003k 10 + + MPV...100 CMLR— Cidofovir -resistant Camelpox Somalia 100 CPX—cowpox Brighton Red (BR) 100 CPXR— Cidofovir -resistant cowpox Brighton Red 100 VAC...Vaccinia Copenhagen 100 VACR— Cidofovir -resistant Vaccinia Copenhagen 100 HSV-1—Herpes Simplex virus type 1 100 HSV-2—Herpes Simplex virus

  4. Novel TaqMan PCR screening methods for element cry3A and construct gat/T-pinII to support detection of both known and unknown GMOs

    NARCIS (Netherlands)

    Prins, Theo W.; Hoof, van Richard A.; Scholtens, Ingrid M.J.; Kok, Esther J.

    2017-01-01

    The import and use of genetically modified organisms (GMOs) is strictly regulated in the European Union. In order to maintain the legislation on GMOs, a genetic element screening is generally applied as a first step to detect authorised as well as unauthorised GMOs. Subsequent identification of G

  5. A TaqMan real-time PCR assay for detection of Meloidogyne hapla in root galls and in soil

    DEFF Research Database (Denmark)

    Sapkota, Rumakanta; Skantar, Andrea M.; Nicolaisen, Mogens

    2016-01-01

    . haplaand showed no significant amplification of DNA from non-target nematodes. The assay was able to detect M. haplain a background of plant and soil DNA. A dilution series of M. haplaeggs in soil showed a high correlation ( R 2 = 0 . 95 , P

  6. Real-time quantitative PCR assay with Taqman® probe for rapid detection of MCR-1 plasmid-mediated colistin resistance

    Directory of Open Access Journals (Sweden)

    S. Chabou

    2016-09-01

    Full Text Available Here we report the development of two rapid real-time quantitative PCR assays with TaqMan® probes to detect the MCR-1 plasmid-mediated colistin resistance gene from bacterial isolates and faecal samples from chickens. Specificity and sensitivity of the assay were 100% on bacterial isolates including 18 colistin-resistant isolates carrying the mcr-1 gene (six Klebsiella pneumoniae and 12 Escherichia coli with a calibration curve that was linear from 101 to 108 DNA copies. Five out of 833 faecal samples from chickens from Algeria were positive, from which three E. coli strains were isolated and confirmed to harbour the mcr-1 gene by standard PCR and sequencing.

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

    Science.gov (United States)

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

    2015-11-01

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

  8. Improved Molecular Detection of Angiostrongylus cantonensis in Mollusks and Other Environmental Samples with a Species-Specific Internal Transcribed Spacer 1-Based TaqMan Assay ▿

    Science.gov (United States)

    Qvarnstrom, Yvonne; da Silva, Ana Cristina Aramburu; Teem, John L.; Hollingsworth, Robert; Bishop, Henry; Graeff-Teixeira, Carlos; da Silva, Alexandre J.

    2010-01-01

    Angiostrongylus cantonensis is the most common cause of human eosinophilic meningitis. Humans become infected by ingesting food items contaminated with third-stage larvae that develop in mollusks. We report the development of a real-time PCR assay for the species-specific identification of A. cantonensis in mollusk tissue. PMID:20543049

  9. Rapid and Quantitative Detection of Leifsonia xyli subsp. xyli in Sugarcane Stalk Juice Using a Real-Time Fluorescent (TaqMan) PCR Assay

    National Research Council Canada - National Science Library

    Hua-Ying Fu; Sheng-Ren Sun; Jin-Da Wang; Kashif Ahmad; Heng-Bo Wang; Ru-Kai Chen; San-Ji Gao

    2016-01-01

      Ratoon stunting disease (RSD) of sugarcane, one of the most important diseases seriously affecting the productivity of sugarcane crops, was caused by the bacterial agent Leifsonia xyli subsp. xyli (Lxx...

  10. Novel TaqMan PCR screening methods for element cry3A and construct gat/T-pinII to support detection of both known and unknown GMOs

    NARCIS (Netherlands)

    Prins, Theo W.; Hoof, van Richard A.; Scholtens, Ingrid M.J.; Kok, Esther J.

    2016-01-01

    The import and use of genetically modified organisms (GMOs) is strictly regulated in the European Union. In order to maintain the legislation on GMOs, a genetic element screening is generally applied as a first step to detect authorised as well as unauthorised GMOs. Subsequent identification of G

  11. Novel TaqMan PCR screening methods for element cry3A and construct gat/T-pinII to support detection of both known and unknown GMOs

    NARCIS (Netherlands)

    Prins, Theo W.; Hoof, van Richard A.; Scholtens, Ingrid M.J.; Kok, Esther J.

    2017-01-01

    The import and use of genetically modified organisms (GMOs) is strictly regulated in the European Union. In order to maintain the legislation on GMOs, a genetic element screening is generally applied as a first step to detect authorised as well as unauthorised GMOs. Subsequent identification of

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

    Directory of Open Access Journals (Sweden)

    Ashutosh Wadhwa

    2017-01-01

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

  13. Efficacy of caspofungin against central nervous system Aspergillus fumigatus infection in mice determined by TaqMan PCR and CFU methods.

    Science.gov (United States)

    Singh, Gaurav; Imai, Jackie; Clemons, Karl V; Stevens, David A

    2005-04-01

    We have reported previously that prolonged caspofungin (CAS) dosing enhances survival in a murine model of central nervous system aspergillosis. In this study we determined by quantitative PCR (qPCR) and CFU enumeration whether CAS could reduce fungal burdens, prior to the deaths of untreated animals, and also assessed progressive infection in untreated mice. Mice were infected intracranially and treated for 4 days with CAS (1, 5, or 10 mg/kg of body weight/day) or amphotericin B (AMB) (3 mg/kg/day) starting 1 day postinfection. Fungal burdens in brains and kidneys of untreated controls were determined on days 1, 3, and 5 to assess progressive infection; burdens in treated animals were determined on day 5. qPCR showed higher burdens than CFU enumeration in all comparisons. In untreated animals, qPCR showed transiently increased burdens in brains, while CFU enumeration showed a decrease. qPCR showed increased burdens in kidneys, but CFU enumeration did not. Neither method indicated drug efficacy in the brain. Both methods showed AMB efficacy in the kidneys, and qPCR demonstrated CAS efficacy at all doses. Spearman correlations of qPCR and CFU determination results showed a significant correlation for most untreated groups; results correlated well for kidneys (P CFU groups indicated different slopes for progressive infection in untreated animals but the same slopes for CAS dose-response efficacy. qPCR appeared to better reflect the progression of untreated infection. The lack of demonstration of efficacy in the brain suggests that longer dosing is necessary to cause burden reduction. These results also suggest that, when there is drug efficacy in a therapeutic study, either method appears to be useful for determining Aspergillus fumigatus burdens.

  14. Comparison of efficiency of various DNA extraction methods from cysts of Giardia intestinalis measured by PCR and TaqMan real time PCR

    Directory of Open Access Journals (Sweden)

    Adamska M.

    2010-12-01

    Full Text Available The aim of the presented study was to work out an effective method of extraction of DNA from Giardia intestinalis cysts as well as a sensitive and specific method for detection of DNA of this protozoan using a polymerase chain reaction (PCR. Twelve protocols for DNA extraction have been compared. Purification and extraction of DNA were preceded by additional actions in order to destroy the cysts’ wall. The highest effectiveness of DNA extraction was obtained in case of alternating application of freezing the samples in liquid nitrogen and their incubation in water bath in the temperature of 100 ˚C, and then the extraction with the QIAamp DNA Tissue Mini Kit (QIAGEN – T kit – with an all night long incubation with proteinase K in 56 ˚C. Effectiveness of DNA extraction with the use of each kit after extraction with each treatment was measured by nested PCR product of β-giardin gene fragment and CT values of real time PCR of the SSU rRNA gene of G. intestinalis. The detection limit, defined as the lowest number detected in 100 % cases, was 100 cysts per 200 μl when effectiveness was evaluated with nested PCR and 50 oocysts with real time PCR after extraction DNA with T kit. Results of our comparative studies have shown that all stages preceding the molecular detection of G. intestinalis DNA are equally important, and materially influence on the final effect and this version of method seems to be very useful for the sensitive detection of DNA of G. intestinalis.

  15. Difference in factors associated with low-level viraemia and virological failure: results from the Austrian HIV Cohort Study

    Directory of Open Access Journals (Sweden)

    Gisela Leierer

    2014-11-01

    Full Text Available Introduction: For some patients, it remains a challenge to achieve complete virological suppression which is the goal of antiretroviral therapy (ART. Identifying factors associated with low-level viraemia (LLV and virological failure (VF under ART might help to optimize management of these patients. Materials and Methods: We investigated patients from the Austrian HIV Cohort Study receiving unmodified ART for >6 months with two nucleoside reverse-transcriptase inhibitors (NRTIs with either a non-nucleoside reverse-transcriptase inhibitor (NNRTI or a boosted protease inhibitor (PI or an integrase inhibitor (INSTI between 1 July 2012 and 1 July 2013 with at least one viral load (VL measurement below the limit of detection (BLD or below level of quantification (BLQ in their treatment history. VF was defined as HIV-RNA levels ≥200 copies/mL and all other quantifiable measurements were classified as LLV. Factors associated with LLV and VF compared to BLD and BLQ were identified by using logistic regression models. Results: Of the 2,276 patients analyzed, 1,972 (86.6% were BLD or BLQ, 222 (9.8% showed LLV and 82 (3.6% had VF. A higher risk for LLV and VF was found in patients with ART interruptions and in patients with boosted PI therapy. The risk for LLV and VF was lower in patients from a centre which uses Abbott RealTime HIV-1 assay compared to the other centres measuring VL by the Roche Cobas AmpliPrep/Cobas TaqMan 2.0. A higher risk for LLV but not for VF was found in patients with a higher VL before ART and shorter ART duration. A higher risk for VF but not for LLV was found in patients of younger age, originating from a high prevalence country, with a lower CD4 count and in male injecting drug users. Conclusions: This study of well-defined patients on stable ART over a period of more than six months gives insights into the different factors associated with LLV and VF. In patients with VF, factors associated with adherence play a prominent

  16. Viral escape in the CNS with multidrug-resistant HIV-1

    Directory of Open Access Journals (Sweden)

    Charles Béguelin

    2014-11-01

    Full Text Available Introduction: HIV-1 viral escape in the cerebrospinal fluid (CSF despite viral suppression in plasma is rare [1,2]. We describe the case of a 50-year-old HIV-1 infected patient who was diagnosed with HIV-1 in 1995. Antiretroviral therapy (ART was started in 1998 with a CD4 T cell count of 71 cells/ìL and HIV-viremia of 46,000 copies/mL. ART with zidovudine (AZT, lamivudine (3TC and efavirenz achieved full viral suppression. After the patient had interrupted ART for two years, treatment was re-introduced with tenofovir (TDF, emtricitabin (FTC and ritonavir boosted atazanavir (ATVr. This regimen suppressed HIV-1 in plasma for nine years and CD4 cells stabilized around 600 cells/ìL. Since July 2013, the patient complained about severe gait ataxia and decreased concentration. Materials and Methods: Additionally to a neurological examination, two lumbar punctures, a cerebral MRI and a neuropsycological test were performed. HIV-1 viral load in plasma and in CSF was quantified using Cobas TaqMan HIV-1 version 2.0 (Cobas Ampliprep, Roche diagnostic, Basel, Switzerland with a detection limit of 20 copies/mL. Drug resistance mutations in HIV-1 reverse transcriptase and protease were evaluated using bulk sequencing. Results: The CSF in January 2014 showed a pleocytosis with 75 cells/ìL (100% mononuclear and 1,184 HIV-1 RNA copies/mL, while HIV-1 in plasma was below 20 copies/mL. The resistance testing of the CSF-HIV-1 RNA showed two NRTI resistance-associated mutations (M184V and K65R and one NNRTI resistance-associated mutation (K103N. The cerebral MRI showed increased signal on T2-weighted images in the subcortical and periventricular white matter, in the basal ganglia and thalamus. Four months after ART intensification with AZT, 3TC, boosted darunavir and raltegravir, the pleocytosis in CSF cell count normalized to 1 cell/ìL and HIV viral load was suppressed. The neurological symptoms improved; however, equilibrium disturbances and impaired memory

  17. Relationship between cyclosporine concentrations obtained using the Roche Cobas Integra and Abbott TDx monoclonal immunoassays in pre-dose and two hour post-dose blood samples from kidney transplant recipients.

    Science.gov (United States)

    Garrido, Manuel J; Hermida, Jesús; Tutor, J Carlos

    2002-12-01

    Current evidence suggests that cyclosporine (CsA) concentration in blood samples taken 2 hours after Neoral microemulsion (Novartis Pharmaceuticals; East Hanover, NJ) administration (C2) predicts clinical events in transplant patients better than the pre-dose (trough) concentration (C0). Similarly, previous findings have shown that the metabolites/CsA ratio is substantially lower in C2 than in C0 samples; however the between-monoclonal immunoassay differences for C2 samples have received little attention in the literature. In 56 C samples and 60 C samples from renal transplant patients, CsA levels were determined using the monoclonal fluorescence polarization immunoassay (mFPIA) from Abbott (Abbott Park, IL) and the homogeneous enzyme immunoassay technique (HEIT) from Roche Diagnostics (Basel, Switzerland). In both cases a high correlation coefficient between the results was obtained (r > or = 0.971), with a linear regression for C0 samples: mFPIA = 1.47 HEIT + 22.0 and for C2 samples: mFPIA = 1.11 HEIT + 71.96. The difference between the linear regression slopes was statistically significant (P < 0.001), and the mFPIA/HEIT ratio was significantly higher for C than for C samples (P < 0.001).

  18. UJI COBA STRAIN LOKAL BACILLUS THURINGIENSIS H-14 YANG DITUMBUHKAN DALAM MEDIA AIR KELAPA TERHADAP JENTIK NYAMUK ANOPHELES ACONITUS DAN CULEX PIPIENS QUINQUEFASCIATUS PERANGKAP SENTINEL DI KOLAM KOTAMADIA SALATIGA

    Directory of Open Access Journals (Sweden)

    Blondie Ch. P.

    2012-09-01

    Full Text Available An investigation was conducted on Bacillus thuringiensis H-14 local strain grown in coconut water media and Tryptose Phosphate Broth (TPB. The two media were tested against Anopheles aconitus and Culex p. quinquefasciatus mosquito larvae in the Vector Control Research Station laboratory and in Sentinel traps village Kauman Kidul pool, Salatiga. The pathogenicity test on B. thuringiensis H-14 local strain grown in coconut water and TPB against An. aconitus and Cx. p. quinquefasciatus larvae in the laboratory was done according to WHO procedure. This was conducted to determine the LC50 and LC90 which is counted according to the probit analysis. The pathogenicity test result of B. thuringiensis H-14 local strain cultured in coconut water against An. aconitus and Cx. p. quinquefasciatus larvae at 24 hours of exposure, showed the LC50 were 0,04 ml/100ml and 0,10 ml/100ml with LC90 were 0,15 ml/100ml and 0,20 ml/100ml respectively. The pathogenicity test at 48 hours of exposure showed the LC50 were 0,01 ml/100ml and 0,02 ml/100ml with LC90 were 0,04 ml/100 ml and 0,13 ml/100 ml respectively. The pathogenicity test result o/B. thuringiensis H-14 local strain cultured in TPB media against An. aconitus and Cx. p. quinquefasciatus larvae at 24 hours exposure, showed the LC50 were 0,05 ml/100 ml and 0,06 ml/100 ml with LC90 were 0,20 ml/100 ml and 0,15 ml/100 ml respectively. The pathogenicity test at 48 hours of exposure showed the LC50 were 0,02 ml/100 ml and 0,03 ml/100ml with LC90 were 0,10 ml/100 ml and 0,08 ml/100ml respectively. The field trial showed the patogenicity test result of B. thuringiensis H-14 local strain cultured in coconut water media against An. aconitus larvae at Sentinel trap in r icefield pools, showed a mortality of more than 50% for 4 days at the 0,15 ml/100 ml (LC90 aplication dosage. The pathogenicity against Cx. p. quinquefasciatus larvae at Sentinel trap was more than 50% for 3 days at the 0,20 ml/100 ml (LC90 aplication dosage. However B. thuringiensis H-14 cultured in TPB against An. aconitus larvae at a dosage 0,20 ml/100 ml and against Cx. p. quinquefasciatus larvae at a dosage of 0,15 ml/100 ml showed a mortality of more than 50% for 3 days. The coconut water is suitable as a local media for culturing B. thuringiensis H-14.

  19. Comparison of DNA extraction protocols for Mycobacterium Tuberculosis in diagnosis of tuberculous meningitis by real-time polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Rajeev Thakur

    2011-01-01

    Full Text Available Background: Several nucleic acid amplification techniques are available for detection of Mycobacterium tuberculosis (MTB in pulmonary and extrapulmonary samples, but insufficient data are available on the diagnostic utility of these techniques in tubercular meningitis where bacilli load is less. The success of final amplification and detection of nucleic acid depends on successful extraction of DNA from the organism. Aims: We performed this study to compare four methods of extraction of MTB DNA from cerebrospinal fluid (CSF samples so as to select one method of DNA extraction for amplification of nucleic acid from clinical samples. Materials and Methods: Four methods of extracting MTB DNA from CSF samples for testing by real-time polymerase chain reaction (PCR were compared: QIAGEN R protocol for DNA purification using QIAamp spin procedure (manual, AMPLICOR R respiratory specimen preparation kit, MagNA Pure R kit extraction, combined manual DNA extraction with automated extraction by MagNA Pure R . Real-time PCR was performed on COBAS TaqMan 48 Analyzer R with known positive and negative controls. Results: The detection limit for the combined manual and MagNA Pure R extraction protocol was found to be 100 copies of MTB DNA per reaction as against 1,000 copies of MTB DNA per reaction by the QIAGEN R , AMPLICOR R , and the MagNA Pure R extraction protocol. Conclusion: The real-time PCR assay employing the combination of manual extraction steps with MagNA Pure R extraction protocol for extraction of MTB DNA proved to be better than other extraction methods in analytical sensitivity, but could not detect less than 10 2 bacilli /ml.

  20. Prevalence and clinical relevance of occult hepatitis B in the fibrosis progression and antiviral response to INF therapy in HIV-HCV-coinfected patients.

    Science.gov (United States)

    Laguno, Montserrat; Larrousse, Maria; Blanco, José Luis; Leon, Agathe; Milinkovic, Ana; Martínez-Rebozler, Maria; Loncá, Montserrat; Martinez, Esteban; Sanchez-Tapias, Jose Maria; de Lazzari, Elisa; Gatell, José Maria; Costa, Josep; Mallolas, Josep

    2008-04-01

    Occult hepatitis B virus (HBV) infection is diagnosed when HBc antibodies (HBcAb) and HBV DNA are detectable in serum while hepatitis B surface antigen (HBsAg) is not. This situation has been frequently described in patients with chronic hepatitis C virus (HCV) infection. The objective of this study was to evaluate the prevalence of occult hepatitis B in HIV-HCV-coinfected patients and its clinical relevance in liver histology and viral response after interferon therapy for HCV. A total of 238 HIV-HCV-infected patients,negative for HBsAg, were included. Serum samples were analyzed for the presence of HBV DNA and HBcAb.HBV DNA quantification was determined with the Cobas TaqMan HBV Test (detection limit 6 IU/ml). Data from liver biopsy and laboratory tests were also analyzed. HBcAb resulted in 142 (60%) patients, being the independent associated factors: male gender, previous history of intravenous drug use, age, CD4 count,and HAV antibody presence. Among 90 HBcAb patients that we could analyze, HBV DNA was positive in 15 (16.7% of occult hepatitis B infection in this group, and 6.3% in the whole HIV-HCV cohort studied). No baseline factors, liver histology, or HCV therapy response were related to the presence of HBV DNA. We found that occult hepatitis B is a frequent condition present in at least 6.3% of our HCV-HIV patients and in more than 16% of those with HBcAb. Despite the high prevalence, this phenomenon does not seem to affect the clinical evolution of chronic hepatitis C or modify the viral response to interferon-based HCV therapies

  1. [To consider negative viral loads below the limit of quantification can lead to errors in the diagnosis and treatment of hepatitis C virus infection].

    Science.gov (United States)

    Acero Fernández, Doroteo; Ferri Iglesias, María José; López Nuñez, Carme; Louvrie Freire, René; Aldeguer Manté, Xavier

    2013-01-01

    For years many clinical laboratories have routinely classified undetectable and unquantifiable levels of hepatitis C virus RNA (HCV-RNA) determined by RT-PCR as below limit of quantification (BLOQ). This practice might result in erroneous clinical decisions. To assess the frequency and clinical relevance of assuming that samples that are BLOQ are negative. We performed a retrospective analysis of RNA determinations performed between 2009 and 2011 (Cobas/Taqman, lower LOQ: 15 IU/ml). We distinguished between samples classified as «undetectable» and those classified as «<1.50E+01IU/mL» (BLOQ). We analyzed 2.432 HCV-RNA measurements in 1.371 patients. RNA was BLOQ in 26 samples (1.07%) from 23 patients (1.68%). BLOQ results were highly prevalent among patients receiving Peg-Riba: 23 of 216 samples (10.6%) from 20 of 88 patients receiving treatment (22.7%). The clinical impact of BLOQ RNA samples was as follows: a) 2 patients initially considered to have negative results subsequently showed quantifiable RNA; b) 8 of 9 patients (88.9%) with BLOQ RNA at week 4 of treatment later showed sustained viral response; c) 3 patients with BLOQ RNA at weeks 12 and 48 of treatment relapsed; d) 4 patients with BLOQ RNA at week 24 and/or later had partial or breakthrough treatment responses, and e) in 5 patients the impact were null or could not be ascertained. This study suggests that BLOQ HCV-RNA indicates viremia and that equating a BLOQ result with a negative result can lead to treatment errors. BLOQ results are highly prevalent in on-treatment patients. The results of HCV-RNA quantification should be classified clearly, distinguishing between undetectable levels and levels that are BLOQ. Copyright © 2013 Elsevier España, S.L. and AEEH y AEG. All rights reserved.

  2. PENGEMBANGAN MULTIMEDIAPEMBELAJARAN BAHASA JERMAN PELAJARAN PELAYANAN RESTORAN DI YOTABAKTI YOGYAKARTA

    OpenAIRE

    Melly Handayani; Suwardi Endraswara

    2015-01-01

    Tujuan penelitian dan pengembangan ini adalah (1) mengembangkan multimedia yang berisi materi dan latihan untuk keterampilan berbicara bahasa Jerman, (2) mendeskripsikan proses pengem-bangannya, dan (3) mendeskripsikan hasil evaluasi dan hasil uji coba lapangan. Uji coba terbatas dilakukan pada satu orang pengajar dan empat orang peserta didik. Uji coba lapangan melibatkan 2 orang pengajar dan 10 orang peserta didik. Data dianalisis secara deskriptif kuantitatif. Langkah pengembangan multimed...

  3. Insulin and Breast Cancer Risk

    Science.gov (United States)

    2002-06-01

    of three standard samples. Serum glucose was determined on a Cobas Mira automated chemistry analyzer (Roche Diagnostic Systems, Indianapolis, IL). The...was determined on a Cobas Mira automated chemistry analyzer (Roche Diagnostic Systems, Indianapolis, IL). The intrabatch coefficient of variation

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

    NARCIS (Netherlands)

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

    2010-01-01

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

  5. 库京病毒TaqMan荧光RT-PCR检测方法的建立%Development of TaqMan real-time RT-PCR for detection of the Kunjin virus

    Institute of Scientific and Technical Information of China (English)

    刘梦莹; 许士奇; 孙肖红

    2015-01-01

    目的 库京病毒(Kunjin virus,KUNV)为黄病毒属病毒,是西尼罗病毒(West Nile virus,WNV)的亚型.由于我国存在其流行的生态学条件,传入的风险很大.因此,亟待建立快速、灵敏、高效的TaqMan荧光RT-PCR方法用于国境口岸KUNV的监测,防止其传入. 方法 从GenBank 中检索KUNV全基因序列,通过MEGA 4软件进行序列比对和Blast进行保守序列搜索分析,选取E基因片段为模板.利用Beacon Designer 7.0软件针对模板设计引物和探针,进行TaqMan荧光RT-PCR检测并优化反应条件和引物浓度,退火温度设定为50、55、60℃,引物终浓度设定为200、400、800 nmol/L,探针终浓度设定为100、200、400 nmol/L,通过扩增曲线与Ct值分析确定最佳退火温度、最佳引物及探针终浓度并验证该方法的敏感性和特异性. 结果 通过分析比较,确定KUNV荧光RT-PCR检测方法最佳退火温度为55℃,引物终浓度为400 nmol/L,探针终浓度为200 nmol /L.该方法检测KUNV核酸的敏感度达1.83×102 cop-ies/μl,且与正布尼亚病毒属拉克罗斯病毒(La Crosse virus,LACV)、雪靴野兔病毒(Snowshoe hare virus,SSHV),甲病毒属巴马哈森林病毒(Barmah Forest virus,BFV)、马雅罗病毒(Mayaro virus,MAYV),黄病毒属乙脑病毒(Japanese en-cephalitis virus,JEV)、黄热病毒(Yellow fever virus,YFV),东南亚十二节段RNA病毒属版纳病毒(Banna virus,BAV)均无交叉反应. 结论 建立的TaqMan荧光RT PCR方法灵敏度高、特异性好,适合于KUNV的快速检测,具有应用价值.

  6. Smallpox and pan-Orthodox Virus Detection by Real-Time 3’-Minor Groove Binder TaqMan Assays Oil the Roche LightCycler and the Cepheid Smart Cycler Platforms

    Science.gov (United States)

    2007-11-02

    Bacillus anthracis BA0068 Ames Sterne SPS 97.13.213 Bacillus cereus Bacillus coagulans Bacillus licheniformis Bacillus macerans Bacillus ...megaterium Bacillus polymyxa Bacillus sphaericus Bacillus stearothermophilus Bacillus subtilis subsp. niger Bacillus thuringiensis Bacillus popilliae...HA-MGB assay presented here has been used to monitor the viral load in monkey blood and tissues after infection with

  7. Application of real-time PCR for quantitative detection of Campylobacter jejuni by TaqMan probe%基于TaqMan探针的Real-time PCR定量检测空肠弯曲杆菌

    Institute of Scientific and Technical Information of China (English)

    阳成波; 蒋原; 黄克和; 祝长青

    2003-01-01

    空肠弯曲杆菌(Campylobacter jejuni,C.jijuni)被认为是人类主要食源性病原菌之一.由于空肠弯曲杆菌特殊的生长条件和容易进入不可培养但存活的状态(Viable but nonculturable,VNC),所以传统的生化鉴定结果并不一定可靠,并且是一项费时而繁琐的工作.核酸检测方法的出现为空肠弯曲杆菌的检测带来了方便.在本文基于Light Cycler为平台,建立一种基于TaqMan探针的Real-time PCR方法来定量检测空肠弯曲杆菌.用该方法检测时,发现所有空肠弯曲杆菌(11株)都呈阳性,所有其它弯曲菌(3株)和其它菌株(5株)都是阴性.整个检测过程60 min内可以完成,检测限度为5 CFU,标准曲线的相关系数为0.988.结果表明荧光定量PCR方法既为空肠弯曲杆菌提供了一种特异、敏感、快速和简洁的定量检测方法,又为研究空肠弯曲杆菌致病机理提供了一种重要方法.

  8. Establishment of TaqMan based real-time PCR assay for detection of Theileria equi%马泰勒虫荧光定量PCR检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    李佳; 张杨; 王振宝; 李永畅; 巴音查汗

    2014-01-01

    为建立马泰勒虫(T.equi)敏感、快速的定量检测方法,本研究根据GenBank中登录的T.equi 18SrRNA保守序列设计一对特异性引物和TaqMan探针,经过反应体系及条件优化,建立了T.equi荧光定量PCR检测方法.结果显示,该方法的灵敏度可达10拷贝/μL,比普通PCR灵敏度高1 000倍;该方法可以特异地检测T.equi,对马驽巴贝斯虫、牛巴贝斯虫、双芽巴贝斯虫与牛环形泰勒虫的检测均为阴性;组内及组间重复试验变异系数均小于3%.对50份临床样品进行检测,荧光定量PCR的阳性检出率为40%,常规PCR的阳性检出率仅为34%.该检测方法的建立为T.equi的流行病学调查和实验研究提供了一种敏感特异的定量检测方法.

  9. Detection of the A2058G and A2059G 23S rRNA gene point mutations associated with azithromycin resistance in Treponema pallidum by use of a TaqMan real-time multiplex PCR assay.

    Science.gov (United States)

    Chen, Cheng-Yen; Chi, Kai-Hua; Pillay, Allan; Nachamkin, Eli; Su, John R; Ballard, Ronald C

    2013-03-01

    Macrolide treatment failure in syphilis patients is associated with a single point mutation (either A2058G or A2059G) in both copies of the 23S rRNA gene in Treponema pallidum strains. The conventional method for the detection of both point mutations uses nested PCR combined with restriction enzyme digestions, which is laborious and time-consuming. We initially developed a TaqMan-based real-time duplex PCR assay for detection of the A2058G mutation, and upon discovery of the A2059G mutation, we modified the assay into a triplex format to simultaneously detect both mutations. The point mutations detected by the real-time triplex PCR were confirmed by pyrosequencing. A total of 129 specimens PCR positive for T. pallidum that were obtained from an azithromycin resistance surveillance study conducted in the United States were analyzed. Sixty-six (51.2%) of the 129 samples with the A2058G mutation were identified by both real-time PCR assays. Of the remaining 63 samples that were identified as having a macrolide-susceptible genotype by the duplex PCR assay, 17 (27%) were found to contain the A2059G mutation by the triplex PCR. The proportions of macrolide-susceptible versus -resistant genotypes harboring either the A2058G or the A2059G mutation among the T. pallidum strains were 35.6, 51.2, and 13.2%, respectively. None of the T. pallidum strains examined had both point mutations. The TaqMan-based real-time triplex PCR assay offers an alternative to conventional nested PCR and restriction fragment length polymorphism analyses for the rapid detection of both point mutations associated with macrolide resistance in T. pallidum.

  10. COMPARISON OF GENETIC METHODS TO OPTICAL METHODS IN THE IDENTIFICATION AND ASSESSMENT OF MOLD IN THE BUILT ENVIRONMENT -- COMPARISON OF TAQMAN AND MICROSCOPIC ANALYSIS OF CLADOSPORIUM SPORES RETRIEVED FROM ZEFON AIR-O-CELL TRACES

    Science.gov (United States)

    Recent advances in the sequencing of relevant water intrusion fungi by the EPA, combined with the development of probes and primers have allowed for the unequivocal quantitative and qualitative identification of fungi in selected matrices.In this pilot study, quantitative...

  11. COMPARISON OF GENETIC METHODS TO OPTICAL METHODS IN THE IDENTIFICATION AND ASSESSMENT OF MOLD IN THE BUILT ENVIRONMENT -- COMPARISON OF TAQMAN AND MICROSCOPIC ANALYSIS OF CLADOSPORIUM SPORES RETRIEVED FROM ZEFON AIR-O-CELL TRACES

    Science.gov (United States)

    Recent advances in the sequencing of relevant water intrusion fungi by the EPA, combined with the development of probes and primers have allowed for the unequivocal quantitative and qualitative identification of fungi in selected matrices.In this pilot study, quantitative...

  12. Development of a duplex real-time TaqMan PCR assay with an internal control for the detection of Mycoplasma gallisepticum and Mycoplasma synoviae in clinical samples from commercial and backyard poultry.

    Science.gov (United States)

    Sprygin, A V; Andreychuk, D B; Kolotilov, A N; Volkov, M S; Runina, I A; Mudrak, N S; Borisov, A V; Irza, V N; Drygin, V V; Perevozchikova, N A

    2010-04-01

    In this study, we report the development and validation of a duplex real-time polymerase chain reaction (PCR) assay with an internal control using TaqMan-labelled probes for the detection of Mycoplasma gallisepticum and Mycoplasma synoviae (duplex MGMS PCR). The MGMS PCR was highly specific with a sensitivity of 7 and 1 colony-forming units/ml for M. gallisepticum and M. synoviae, respectively, using dilution of pure culture that corresponds to 34 and 29 DNA copies per reaction. Validation of the assay was completed with 260 and 27 pooled samples (tracheal swabs) from commercial chickens and turkeys, respectively, with potential M. gallisepticum and M. synoviae involvement and 42 samples (palatine cleft swabs) from backyard geese and ducks. Using isolation as the gold standard, the MGMS PCR was more sensitive than isolation and the analytical sensitivity was 0.944 and 0.958 for M. gallisepticum and M. synoviae, respectively. In comparison with a gapA-based assay (gapA PCR) and a 16S rRNA-based assay (16S PCR) for M. gallisepticum and M. synoviae, respectively, the results agreed for 94.5% and 96.6%, respectively. The use of the internal control allowed monitoring of proper extraction and inhibition of amplification that was detected in 12 samples. The duplex MGMS PCR was shown to be superior to the presently reported real-time PCR assays in terms of combination of sensitivity, specificity and capacity of detection of more than one target in a single tube. In conclusion, the duplex MGMS PCR was highly specific, sensitive, and reproducible and could be used on clinical samples from commercial chickens, turkeys and backyard poultry including ducks and geese.

  13. Novel real-time PCR assays using TaqMan minor groove binder probes for identification of fecal carriage of Streptococcus bovis/Streptococcus equinus complex from rectal swab specimens.

    Science.gov (United States)

    Lopes, Paulo Guilherme Markus; Cantarelli, Vlademir Vicente; Agnes, Grasiela; Costabeber, Ane Micheli; d'Azevedo, Pedro Alves

    2014-03-01

    Real-time PCR based on the recN and gyrB genes was developed to detect four Streptococcus bovis/Streptococcus equinus complex (SBEC) subspecies from rectal swab specimens. The overall prevalence was 35.2%: Streptococcus gallolyticus subsp. gallolyticus (11.1%), S. gallolyticus subsp. pasteurianus (13%), Streptococcus infantarius subsp. coli (20.4%), and S. infantarius subsp. infantarius (11.1%). To conclude, these real-time PCR assays provide a reliable molecular method to detect SBEC pathogenic subspecies from rectal swab specimens.

  14. 丙型肝炎患者抗病毒治疗后外周血单个核细胞内病毒 RNA 的检测及其与疗效的关系%Detection of hepatitis C virus RNA in the peripheral blood mononuclear cells in patients with chronic hepatitis C and its relationship with effect of anti-viral treatment

    Institute of Scientific and Technical Information of China (English)

    聂静敏; 胡凤玉; 许敏; 陈伟烈; 何浩岚; 李凌华; 蔡卫平; 唐小平

    2016-01-01

    目的:观察慢性丙型肝炎患者在聚乙二醇干扰素α-2a (Peg IFNα-2a)联合利巴韦林(RBV)抗病毒治疗前、后血清及 PBMC 内 HCV RNA 的变化,初步探讨 PBMC 内 HCV RNA 检测的临床意义。方法2013年6月至2014年12月在广州市第八人民医院感染科就诊的慢性丙型肝炎患者20例,在治疗前、后的不同时间点(0、2、4、12、24、36、48周)采集患者外周血,分离血浆和 PBMC;采用精确荧光定量方法(Cobas TaqMan 实时 PCR)检测丙型肝炎患者血浆 HCV RNA;采用实时-PCR、套式PCR 两种方法分别检测 PBMC 内 HCV RNA。计数资料采用χ2检验。结果 Cobas 精确定量检测各时间点血浆中 HCV RNA 分别为(5.92±0.86)、(2.77±1.32)、(1.30±1.72)、(0.15±0.47)、0、0和(0.06±0.26)lgIU/mL;各时间点差异有统计学意义(F =148.06,P <0.01)。12周时有18例患者血浆中 HCV RNA 完全清除。套式 PCR 较实时 PCR 阳性率高(均 P <0.01);20例患者血浆病毒载量及PBMC 中 HCV RNA 检测结果发现,PBMC 中 HCV RNA 清除速率较血浆明显延迟,其中2例 PBMC中 HCV RNA 持续性存在并在治疗结束24周后复发。结论慢性丙型肝炎患者 PBMC 中可以检测到HCV RNA,且套式 PCR 检测较实时 PCR 检测阳性率高;抗病毒治疗对 PBMC 内、外的 HCV 均有效,但 PBMC 中 HCV RNA 清除速率较血浆明显延迟;干扰素抗病毒过程中 PBMC 内 HCV 清除缓慢,预示存在治疗后复发的风险。%Objective To detect the change of hepatitis C virus (HCV)RNA in the peripheral blood mononuclear cells (PBMC)and serum of patients with chronic hepatitis C (CHC)during treatment with peg-interferon α-2a (Peg IFNα-2a)plus ribavirin (RBV),and to analyze the clinical significance of HCV RNA detection in PBMC.Methods The peripheral blood samples of 20 CHC patients who visited Department of Infectious Diseases in

  15. 国产和进口荧光定量PCR试剂检测HBV-DNA的比对分析%Comparative analysis on domestic and imported fluorescent quantitative PCR reagents for detecting HBV-DNA

    Institute of Scientific and Technical Information of China (English)

    张玥; 田文君; 刘义庆; 张炳昌; 张庆; 渠滕; 刘春梅

    2015-01-01

    Objective To analyze the correlation between the domestic and imported real‐time fluorescent quantitative PCR reagents for detecting HBV‐DNA and to explore their difference in clinical application .Methods The domestic and imported reagents were used to parallelly detect the plasma HBV‐DNA content in 262 cases of hep‐atitis B .The domestic reagent adopted the sample extracting by adopting the manual method and the amplification for nucleic acid was performed by the Roche LightCycler480 Ⅱ (LC480 Ⅱ );the imported reagent used the Roche CO‐BAS AmpliPrep (CAP) and COBAS Taqman48(CTM ) for conducting the sample extracting and amplification;the HBV DNA viral load data (log10) was performed the logarithmic transformation and the 2 sets of data were conduc‐ted the correlation analysis .Results The detection results of the domestic and imported reagents were performed the linear fitting ,R2 =0 .814 3 .The sample with the concentration range of 10 -103 IU/mL ,R2 = 0 .300 6 ;the sample with the concentration range of 104 -105 IU/mL ,R2 = 0 .411 8 ;the sample with the concentration range of 106 -108 IU/mL ,R2 =0 .801 7 .The positive detection rate of the imported reagent was 75 .57% (198/262) ,which was signifi‐cantly higher than 65 .65% (172/262) of the domestic reagent .Conclusion There is a good correlation between the domestic reagent and imported reagent .The correlation is the highest when the HBV‐DNA concentration in the range of 106 -108 IU/mL ;the correlation is general when the concentration in the range of 104 -105 IU/mL ;the correlation is lowest when the concentration in the range of 10-103 IU/mL ,there is a significant difference between the domes‐tic reagent and imported reagent and the sensitivity of the domestic reagent remains to be further improved .%目的:分析国产和进口实时荧光定量聚合酶链反应(PCR)试剂检测乙型肝炎病毒(HBV)‐DNA的相关性,探讨国产和进口试剂在临床应用中的差

  16. HBV and neurological impairment in HIV-infected patients

    Directory of Open Access Journals (Sweden)

    L Manolescu

    2012-11-01

    Full Text Available Objective: HIV can affect CNS in early stages of disease and determine neurological impairment. HBV DNA was found in CSF of HIV co-infected patients, but little is known about the neurotropic character of this virus. Here we assessed the degree of association between HBV infection and neurological impairment in a large cohort of long-term survivors, HIV-infected patients that experienced multiple therapeutic schemes over time. Methods: A total of 462 HIV-1-infected patients were retrospectively followed up for 10 years for HBV infection and neurological impairment. The patients were tested for immune (flow cytometry and virological parameters of HIV infection (Roche Amplicor, version 1.5/ COBAS AmpliPrep/COBAS TaqMan HIV-1 test and for HBV infection markers (HBsAg, anti HBc: Murex Biotech ELISA tests. Many of these patients have experienced between one and six regimens such as: 2 NRTIs, 3 NRTIs, 2 NRTIs+1 NNRTI, 1 NRTI+1 NNRTI+1 PI, 2 NRTIs+2 PIs. Results: After 10 years 29.87% of the patients presented neurological impairment. Out of them 56.52% were HBV-infected. The prevalence of HIV encephalopathy (HE in our studied cohort was 22.7% and 50.4% of these patients were HBV-infected. The median HIV diagnosis age was 7 and the median age of HE diagnosis was 10. In order to establish a possible correlation between HBV infection and HE we first reviewed and excluded the main risk factors associated with HE at the moment of diagnosis: low weight, anemia, constitutional symptoms, low CD4+count, high plasma HIV-RNA load. No patient was infected with HCV. The groups of patients that presented HE and HBsAg and HE without HBsAg were balanced regarding sex, number of deceased patients, number of class C3 patients, but the patients in first group presented lower CD4 values at HE diagnosis vs patients from second group 2: 44.5 vs 95 cells/µL, p=0.3; lower nadir CD4 count: 38 vs 51 cell/µL, p=0.1; and slightly higher HIV viral load: 5.2 vs 5 log10 copies

  17. Application of a newly developed high-sensitivity HBsAg chemiluminescent enzyme immunoassay for hepatitis B patients with HBsAg seroclearance.

    Science.gov (United States)

    Shinkai, Noboru; Matsuura, Kentaro; Sugauchi, Fuminaka; Watanabe, Tsunamasa; Murakami, Shuko; Iio, Etsuko; Ogawa, Shintaro; Nojiri, Shunsuke; Joh, Takashi; Tanaka, Yasuhito

    2013-11-01

    We modified and automated a highly sensitive chemiluminescent enzyme immunoassay (CLEIA) for surface antigen (HBsAg) detection using a combination of monoclonal antibodies, each for a specific epitope of HBsAg, and by improving an earlier conjugation technique. Of 471 hepatitis B virus (HBV) carriers seen in our hospital between 2009 and 2012, 26 were HBsAg seronegative as determined by the Abbott Architect assay. The Lumipulse HBsAg-HQ assay was used to recheck those 26 patients who demonstrated seroclearance by the Abbott Architect assay. The performance of the Lumipulse HBsAg-HQ assay was compared with that of a quantitative HBsAg detection system (Abbott Architect) and the Roche Cobas TaqMan HBV DNA assay (CTM) (lower limit of detection, 2.1 log copies/ml) using blood serum samples from patients who were determined to be HBsAg seronegative by the Abbott Architect assay. Ten patients had spontaneous HBsAg loss. Of 8 patients treated with nucleotide analogues (NAs), two were HBsAg seronegative after stopping lamivudine therapy and 6 were HBsAg seronegative during entecavir therapy. Eight acute hepatitis B (AH) patients became HBsAg seronegative. Of the 26 patients, 16 were HBsAg positive by the Lumipulse HBsAg-HQ assay but negative by the Abbott Architect assay. The differences between the two assays in terms of detectable HBsAg persisted over the long term in the spontaneous loss group (median, 10 months), the NA-treated group (2.5 months), and the AH group (0.5 months). In 9 patients, the Lumipulse HBsAg-HQ assay detected HBsAg when HBV DNA was negative by the CTM assay. HBsAg was also detected by the Lumipulse HBsAg-HQ assay in 4 patients with an anti-HBs concentration of >10 mIU/ml, 3 of whom had no HBsAg escape mutations. The automatic, highly sensitive HBsAg CLEIA Lumipulse HBsAg-HQ is a convenient and precise assay for HBV monitoring.

  18. Early predictive efficacy of core antigen on antiviral outcomes in genotype 1 hepatitis C virus infected patients

    Directory of Open Access Journals (Sweden)

    Bo Feng

    Full Text Available Response-guided therapy is of limited use in developing countries because hepatitis C virus RNA detection by sensitive molecular methods is time- and labor-consuming and expen- sive. We evaluated early predictive efficacy of serum hepatitis C virus core antigen kinetics on sustained virologic response in patients with genotype 1 hepatitis C virus during pegylated interferon plus ribavirin treatment. For 478 patients recruited, hepatitis C virus RNAs were detected at baseline, and at weeks 4, 12, 24, 48, and 72 using Cobas TaqMan. Architect hepatitis C virus core antigen was performed at baseline, and weeks 4 and 12. Predictive values of hepatitis C virus core antigen on sustained virologic response were compared to hepatitis C virus RNA. In the first 12 weeks after treatment initiation the dynamic patterns of serum hepatitis C virus core antigen and hepatitis C virus RNA levels were similar in sustained virologic response, relapse, and null response patients groups. Although areas under the receiver operating characteristics curves of hepatitis C virus core antigen were lower than those of hepatitis C virus RNA at the same time points, modeling analysis showed that undetectable hepatitis C virus core antigen (rapid virological response based on hepatitis C virus core antigen had similar positive predictive value on sustained virologic response to hepatitis C virus RNA at week 4 (90.4% vs 93.3%, and hepatitis C virus core antigen decrease greater than 1 log10 IU/mL (early virological response based on hepatitis C virus core antigen had similar negative predictive value to hepatitis C virus RNA at week 12 (94.1% vs 95.Z%. Analysis on the validation group demonstrated a positive predictivevalue of 97.5% in rapid virological response based on hepatitis C virus core antigen and a negative predictive value of 100% in early virological response based on hepatitis C virus core antigen. In conclusion, hepatitis C virus core antigen is comparable to

  19. The Distribution of Hepatitis C Virus Genotypes in Patients with Chronic Hepatitis C Infection

    Directory of Open Access Journals (Sweden)

    Sevin Kırdar

    2015-12-01

    Full Text Available Objective: Hepatitis C virus (HCV infection represents a major public health problem worldwide. HCV can cause chronic hepatitis infection which may ultimately result in cirrhosis and hepatocellular carcinoma. Seven major genotypes and more than 100 subtypes of HCV are shown by sequence analysis. Genotype 1 is associated with more severity of liver disease than genotypes 2 and 3 and sustained response totreatment is known to be less. In this study, we aimed to determine the HCV genotype distribution in chronic hepatitis C patients. Materials and Methods: A total of 50 patients with chronic HCV infection who attended the Microbiology Laboratory at Adnan Menderes University Hospital between August 2007 and December 2010 found to be positive for anti-HCV and HCV-RNA were included in the study. Anti-HCV testing was performed using microparticle Enzyme-Linked immunosorbent assay test kit (Murex Anti-HCV version 4, UK with autoanalyser (Grifols Triturus, Spain. The quantification of serum HCV-RNA was carried out by a realtime polymerase chain reaction method with two different systems (Cobas TaqMan HCV, Roche Diagnostics, Germany and RotorGene 6000,Corbett Research, USA. HCV genotype analysis was performed by using a kit (HCV-TS; AB Analitica, Italy based on the reverse hybridization of 5’-untranslated region and amplified products with genotype-specific probes. Results: The mean age of the 50 chronic hepatitis C patients [27 (54% female, and 23 (46% male] was 57.1±14.3 years. Genotype 1b was found in 36 (72% subjects, genotype 1a in nine (18%, genotype 2b in one (2%, genotype 3 in one (2%, and genotype 1a/1b was found in three (6% patients. No statistically significant difference was detected in HCV-RNA quantities and anti-HCV index between HCV genotypes (p>0.05. Conclusion: Compatible with the previous data obtained in Turkey, genotype 1b was found to be the most common HCV genotype in patients with chronic hepatitis C followed in our hospital.

  20. Service impact of a change in HIV-1 viral load quantification assay

    Directory of Open Access Journals (Sweden)

    Craig Tipple

    2014-11-01

    Full Text Available Introduction: Due to discontinuation of the Siemens Versant HIV-1 RNA (bDNA assay in the UK, our laboratory switched to the Roche Cobas Ampliprep/Taqman HIV-1 viral load (VL assay (Roche in April 2013. This assay has a lower cut-off of 20 RNA copies/mL (compared with <50 for the Siemens assay. Our laboratory demonstrated previously that a significant proportion (18% of patients undetectable using bDNA HIV-1 RNA quantification exhibited low level viraemia (LLV using the new assay. Local guidelines recommend that patients stable on therapy receive twice-yearly VLs. We evaluated the impact of the introduction of the new assay on our clinical service. Methods: A retrospective cohort analysis of treated patients with stable undetectable VL by bDNA (<50 copies/mL followed by ≥ one low-level (<400 copies/mL VL with the Roche assay. Demographic data were collected in addition to frequency of VL testing and genotypic resistance assays. Referrals to virtual clinic (VC were recorded. Patients were identified using laboratory data and information collected from electronic patient records. Results were analyzed with SPSS v18. Results: One hundred and ninety patients were included. Demographics: 79.5% male; 60.6% homosexual; mean age of 46 years. Duration on stable treatment was 46.35 (std. dev. 38.15 months. Current treatment regimens were 43.3% PI-based; 43.3% NNRTI-based and 13.7% other. Patients were stratified into VL 20–49 copies/mL (n=109; VL 50–199 copies/mL (n=71 and VL 200–399 copies/mL (n=10. In total, there were 471 VLs measured of which 274 were additional as a result of the assay switch. This resulted in six HIV-1 genotype requests and 16 VC discussions (Table 1. Longer duration on HAART was associated with reduced frequency of VL testing. The relative risk of ongoing detectability according to drug class are: PI 1.62 (95% CI 1.18–2.21; NNRTI 0.507 (95% CI 0.30–0.85 and other 1.09 (95% CI 0.48–2.43. Conclusions: Changes in assay

  1. PENGEMBANGAN MODUL SISTEM KEAMANAN JARINGAN BERBASIS SIMULASI CISCO

    Directory of Open Access Journals (Sweden)

    Zulkipli Zulkipli

    2016-03-01

    Tujuan penelitian adalah untuk menghasilkan dan menguji kelayakan modul sistem keamanan jaringan berbasis simulasi Cisco Paket Tracer untuk peserta didik SMK. Model pengembangan yang digunakan adalah model Dick, Carey & Carey dengan sembilan langkah. Pengembangan produk ini divalidasi oleh ahli materi dengan tingkat kevalidan 96%, ahli media dengan tingkat kevalidan 92.8%, ahli desain pembelajaran dengan tingkat kevalidan 83%, uji coba perorangan dengan tingkat kevalidan 92.3%, uji coba kelompok kecil dengan tingkat kevalidan 92% dan uji coba lapangan dengan tingkat kevalidan 89% dengan kualifikasi sangat layak tidak perlu revisi.

  2. InfluenCe of HeliCobaCter Pylori on Motor Symptoms in Patients with Parkinsonˊs Disease%幽门螺杆菌感染对帕金森病患者运动症状的影响研究

    Institute of Scientific and Technical Information of China (English)

    王晏雯; 乔松; 刘小利; 蔡苗; 李雅国

    2015-01-01

    [AbstraCt] ObjeCtive To investigate influence of Helicobacter pylori( Hp)on motor symptoms in patients with Parkinsonˊs disease( PD)and to learn the function of Hp eliminated therapy on the patientsˊ motor fluctuation. Methods Eighty _ nine patients diagnosed with PD from January 2011 to December 2013 in Department of Neurology,Zhejiang Hospital were selected and divided into Hp infection group and Hp noninfection group according to the results of 14 C _ urea breath tests ( 14 C _ UBT). The patients of Hp noninfection group were given Madopar treatment,and Hp eradication therapy was used in Hp infection group besides Madopar. The score of Unified Parkinsonˊs Disease Rating Scale(UPDRS)Ⅲ,the duration of on time and off time and Hoehn _ Yahr staging were used to evaluate the severity of motor symptoms before and after the therapy of Hp eradication. The UPDRS Ⅳ was used to evaluate the change of motor complications before and after treatment. Results The score of UPDRS Ⅳ in Hp infection group was statistically different from that in the group of Hp noninfection(p 0. 05),while they were all significantly different among Hp infection group(p 0.05);Hp 组治疗前后 UPDRS Ⅲ评分、UPDRS Ⅳ评分及“开”“关”期时间比较,差异有统计学意义(p <0.05);治疗后,两组“开”“关”期时间比较,差异有统计学意义(p <0.05)。结论 Hp 感染可以增加 PD 患者运动并发症的发生。对 Hp 感染的 PD 患者行 Hp 根除治疗可以改善其运动症状,减少运动并发症的发生。

  3. Study on the adsorption capacity for mercury (Ⅱ) with chromium/cobaIt-doped graphene oxide materiaIs%负载铬钴石墨烯基材料对汞(Ⅱ)的吸附性能研究

    Institute of Scientific and Technical Information of China (English)

    王卓; 邓娟; 朱君妍; 周超; 周晓吉; 郭永福; 白仁碧

    2016-01-01

    The chromium and cobalt doped reduced graphene oxide (RGO/Cr,RGO/Co) composite materials have been synthesized by making use of the reducibility of RGO and the oxidability of transition metallic salts ,and app-lied to the adsorption for Hg2+. The results show that the specific surface area of RGO/Cr is higher than that of RGO/Co and RGO. The hydrophilicity of RGO is effectively improved after having been doped with chromium or cobalt. The theoretical monolayer adsorption capacities of RGO,RGO/Cr and RGO/Co fitting Langmuir model are 128.98,181.86, and 146.86 mg/g,respectively,and the adsorption processes of these three materials complies with the pseudo-first-order and pseudo-second-order kinetic model.%利用石墨烯的还原性与过渡金属盐类的氧化性,制备得到分别负载有铬和钴的还原氧化石墨烯(RGO)复合材料RGO/Cr和RGO/Co,并将其应用于对Hg2+的吸附。结果表明:RGO/Cr比RGO/Co及RGO具有更大比表面积,负载铬和钴后RGO的亲水性能显著增加。 Langmuir拟合RGO、RGO/Cr、RGO/Co对Hg2+的理论单层最大吸附容量分别为128.98、181.86、146.86 mg/g,同时吸附过程均符合伪一级和伪二级动力学模型。

  4. PENGEMBANGAN MODEL KOLABORASI JIGSAW ROLE PLAYING SEBAGAI UPAYA PENINGKATAN KEMAMPUAN BEKERJASAMA SISWA KELAS V SD PADA PELAJARAN IPS

    Directory of Open Access Journals (Sweden)

    Ika Ari Pratiwi

    2015-11-01

    Full Text Available Penelitian bertujuan untuk mengembangkan model kolaborasi jigsaw, role playing untuk meningkatkan kemampuan bekerjasama siswa yang valid, efektif dan praktis. Metode penelitian adalah penelitian dan pengembangan (R&D. Tahap uji coba pengembangan terdiri atas uji coba ahli, uji coba skala terbatas dan uji coba skala luas. Keefektifan model kolaborasi jigsaw role playing  diperoleh rata-rata 51,83 dalam kategori baik diterapkan dalam pelajaran IPS, peningkatan kemampuan bekerjasama siswa hasil N-gain = 0,56 dengan kategori sedang, peningkatan hasil belajar IPS N-gain = 0,50 dengan kategori sedang dan hasil ketuntasan klasikal pembelajaran IPS 97,14%.  Hasil respon guru dan siswa terhadap model yang digunakan adalah berkriteria baik. Model final penelitian ini menghasilkan model kolaborasi jigsaw role playing yang dikemas dalam suatu buku pedoman.

  5. Human Papillomavirus Assays and Cytology in Primary Cervical Screening of Women Aged 30 Years and Above

    DEFF Research Database (Denmark)

    Rebolj, Matejka; Bonde, Jesper; Preisler, Sarah

    2016-01-01

    In women aged ≥30 years, Human Papillomavirus testing will replace cytology for primary cervical screening. We compared Hybrid Capture 2 (HC2), cobas, CLART, and APTIMA HPV assays with cytology on 2869 SurePath samples from women undergoing routine screening at 30-65 years in Copenhagen, Denmark....... Women with cytological abnormalities were managed according to routine recommendations, with 92% completeness. Those with cytology-normal/HPV-positive samples (on any of the four assays) were invited for repeated cytology and HPV testing in 1.5 year, and 58% had additional testing. HPV testing detected...... more ≥CIN3 than cytology (HC2: 35, cobas, CLART: 37, APTIMA: 34, cytology: 31), although statistically the differences were not significant. Cobas and CLART detected significantly more ≥CIN2 than cytology (cobas, CLART: 49, cytology: 39). The proportion of women with false-positive test results...

  6. IMPLEMENTASI METODE MULTIPLE KERNEL SUPPORT VECTOR MACHINE UNTUK SELEKSI FITUR DARI DATA EKSPRESI GEN DENGAN STUDI KASUS LEUKIMIA DAN TUMOR USUS BESAR

    OpenAIRE

    2012-01-01

    Pada penelitian ini mengimplementasikan metode multiple kernel support vector machine untuk seleksi fitur. Multiple kernel merupakan metode modifikasi fungsi kernel yang mengalikan tiap elemen dari data. Metode ini melakukan seleksi fitur terhadap fitur yang kurang penting dengan tingkat akurasi lebih baik daripada metode dasar support vector machine. Uji coba dilakukan dengan menggunakan dataset ekspresi gen leukimia dan tumor usus besar. Hasil uji coba dibandingkan dengan tingkat akurasi me...

  7. Multicenter Evaluation of a New High-Throughput HbA1c Testing Platform.

    Science.gov (United States)

    Imdahl, R; Roddiger, R; Casis-Saenz, E

    2016-12-01

    This non-interventional, multicenter study with anonymized leftover patient samples was performed to evaluate the reliability and analytical performance of the novel high-throughput HbA1c cobas c 513 analyzer. A performance evaluation was carried out at three sites to validate the overall system functionality, user interaction and analytical performance of the new cobas c 513 analyzer using the Tina-quant® HbA1c Gen. 3 assay. HbA1c applications for both whole blood and hemolysate samples show a high precision using both quality control materials and pools of whole blood or hemolysates. The method comparison of HbA1c Gen. 3 on the cobas c 513 with HbA1c Gen. 2 on the Menarini HA-8180V using 249 whole blood samples shows high concordance. Moreover, analyte concentrations as measured by the cobas c 513 and Tosoh G8 and HbA1c Gen. 2 on COBAS INTEGRA® 800 CTS are comparable. The cobas c 513 has proven to be a reliable system with excellent analytical performance of the Tinaquant® HbA1c Gen. 3 assay in high throughput laboratories.

  8. THE DEVELOPMENT OF COGNITIVE TEST DEVICES FOR COMPETENCE MEASUREMENT OF FUTURE SENIOR VOCATIONAL SCHOOL TEACHERS IN FISHING VESSEL NAUTICAL EXPERTISE

    Directory of Open Access Journals (Sweden)

    Eliza Merina dkk

    2015-06-01

    Abstrak. Penelitian ini dilakukan dengan tujuan untuk mengembangkan sebuah perangkat tes kognitif yang valid dan reliabel untuk pengukuran kompetensi profesional calon-calon guru SMK Keahlian NKPI melalui prosedur yang benar. Metode penelitian yang digunakan dalam penelitian ini adalah riset dan pengembangan. Prosedur yang dilalui dalam penelitan ini yaitu : identifikasi tujuan pengukuran, pengembangan spesifikasi tes, penulisan butir soal, penelaahan butir tes (uji validitas, uji coba tes 1, analisa butir tes 1, uji reliabilitas, uji coba tes 2, analisa butir tes 2, dan perakitan bentuk akhir tes. Pada awal pembuatan spesifikasi tes dan penulisan butir, jumlah butir yang dikembangkan berjumlah 65 butir tes. Seiring proses pengembangan tes dilakukan, jumlah butir tes mengalami degradasi (penurunan. Setelah melalui tahap telaah butir (uji validitas yang dilakukan oleh 3 orang ahli di bidang NKPI, butir tes mengalami pengurangan 5 butir tes sehingga tersisa 60 butir tes untuk dirakit menjadi perangkat tes uji coba 1. Setelah uji coba 1 dilakukan dilanjutkan dengan analisis butir 1 yang dilakukan sesuai teori ujian klasik yang terdiri dari tingkat kesukaran, daya diskriminasi butir, dan efektivitas distraktor. Dari analisis tersebut, jumlah butir soal pun mengalami pengurangan hingga tersisa 29 butir tes untuk dirakit menjadi perangkat tes uji coba 2. Dari hasil uji coba 1 pun dilakukan uji reliabilitas menggunakan pendekatan konsistensi internal (internal consistency yaitu dengan perhitungan reliabilitas Kuder Richardson (KR20. Diperoleh nilai koefisien reliabilitas sebesar 0,71 yang menunjukkan bahwa perangkat tes tergolong reliabel. Uji coba 2 dilakukan dan diteruskan dengan analisa butir 2, kembali lagi butir soal mengalami pengurangan hingga tersisa 19 butir tes untuk dirakit menjadi perangkat tes bentuk akhir. Kata Kunci : pengembangan tes, pengukuran kompetensi, nautika kapal penangkaan ikan

  9. Assembling a dual purpose TaqMan-based panel of single-nucleotide polymorphism markers in rainbow trout and steelhead (Oncorhynchus mykiss) for association mapping and population genetics analysis

    DEFF Research Database (Denmark)

    Hansen, Mette H H; Young, Sewall; Jørgensen, Hanne Birgitte Hede;

    2011-01-01

    We establish a TaqMan-based assay panel for genotyping single-nucleotide polymorphisms in rainbow trout and steelhead (Oncorhynchus mykiss). We develop 22 novel single-nucleotide polymorphism markers based on new steelhead sequence data and on assays from sister taxa. Additionally, we adapt 154...... previously developed markers to the TaqMan platform. At the beginning of this study, 59 SNPs with TaqMan assays were available to the scientific community. By adding 176 additional TaqMan assays to this number, we greatly expand the biological applications of TaqMan genotyping within both population genetics...... and quantitative genetics...

  10. 结核病与BCG疫苗接种个体TaqMan探针荧光定量PCR诊断方法的建立及初步应用%Development of TaqMan real-time PCR assays for the detection of Tuberculosis in BCG-vaccinated population

    Institute of Scientific and Technical Information of China (English)

    王春雨; 杨莉; 王振国; 刘金华; 宋占昀; 周亮; 王宝任; 王全凯

    2010-01-01

    为快速鉴别诊断结核病(TB),本研究以GenBank登录的致病性结核分枝杆菌复合群、人型结核杆菌 和牛分枝杆菌特有基因为对象,设计并合成引物及探针,建立TaqMan探针荧光定量PCR检测方法.实验结果表明,该方法对标准质控菌株反应呈阳性,对卡介苗(BCG)及其他微生物样品反应呈阴性;对结核分枝杆菌或牛分枝杆菌标准菌株的检测灵敏度可达单个茵细胞水平.对45份结核菌素PPD皮肤试验结果为阳性的临床样本进行TaqMan探针荧光定量PCR检测,36份为阳性;而对PPD检测为阴性的50份临床样本进行检测时,7份为阳性.本研究结果表明,所建立的方法可用于TB的鉴别诊断,可对由BCG接种或环境中分枝杆菌引起的PPD检测假阳性样本进行鉴别,对TB的快速检测和早期诊断具有重要意义.

  11. Application of real-time PCR TaqMan assay for rapid detection of Campylobacter jejuni from stool samples%应用TaqMan实时荧光-聚合酶链反应方法快速检测粪便标本空肠弯曲菌的研究

    Institute of Scientific and Technical Information of China (English)

    曲梅; 黄芳; 刘园; 窦相峰; 刘桂荣; 严寒秋; 高志勇; 王全意

    2009-01-01

    目的 建立空肠弯曲菌TaqMan实时荧光-PCR方法,用于粪便标本的直接检测.方法 根据空肠弯曲菌特异性基因hipO和mapA分别设计引物和探针,在对2组引物和探针进行灵敏度、特异性和重复性评价的基础上,对45例临床腹泻患者粪便标本提取DNA之后,荧光PCR检测,同时进行分离培养.结果 两组引物和探针能准确检测空肠弯曲菌菌株2株,检测限可达到10~20 cfu/ml,并与其他肠道致病菌无交叉反应.检测45份腹泻病例粪便标本,该方法检测到3份为阳性,同时进行的传统培养方法仅从该3份标本中的两份中分离到空肠弯曲菌.结论 本研究建立的TaqMan荧光PCR检测粪便标本中所携带的空肠弯曲菌灵敏度高,特异性好,能够提高粪便中空肠弯曲菌的阳性检出率和缩短检测时限.

  12. 基于TaqMan探针三重荧光PCR检测沙门菌、肠炎沙门菌和鼠伤寒沙门菌%Detection of salmonella, salmonella enteritidis and salmonella typhimurium by TaqMan real-time PCR

    Institute of Scientific and Technical Information of China (English)

    袁慕云; 刘二龙; 谢力; 柯碧霞; 曹际娟; 许龙岩

    2015-01-01

    目的 建立基于TaqMan探针三重荧光PCR检测沙门菌(salmonella,Sa)、肠炎沙门菌(salmonella enteritidis,SE)和鼠伤寒沙门菌(salmonella typhimurium,ST)的方法.方法 根据沙门菌aceA基因、肠炎沙门菌特异序列(GenBank:AF370707.1)、鼠伤寒沙门菌的STM4599序列(GenBank:AERV01000023.1),分别设计引物和TaqMan探针,在探针的5'端分别标记FAM、VIC、cy5,建立基于TaqMan探针三重实时荧光PCR检测沙门菌的方法.结果 29种不同血清型沙门菌均扩增出aceA基因,肠炎沙门菌和鼠伤寒沙门菌的引物和探针分别特异性地扩增出15株肠炎沙门菌和11株鼠伤寒沙门菌,而其他血清型沙门菌和17株非沙门菌扩增结果阴性.aceA、肠炎沙门菌、鼠伤寒沙门菌的三重荧光PCR扩增效率分别为89%、87%、90%,最低检测浓度分别达到280 cfu/ml、260 cfu/ml、300 cfu/ml.结论 本研究建立的方法特异性好、灵敏度高,可用于食品中沙门菌、肠炎沙门菌和鼠伤寒沙门菌的特异性检测.

  13. Establishment and application of TaqMan real-time fluorescence quantitative RT-PCR for detection of bovine parainfluenza virus type 3%牛副流感病毒3型TaqMan实时荧光定量RT-PCR检测方法的建立及应用

    Institute of Scientific and Technical Information of China (English)

    董秀梅; 朱远茂; 蔡虹; 王姝; 吕闯; 马磊; 薛飞

    2014-01-01

    根据牛副流感病毒3型(bovine parainfluenza virus type 3,BPIV3)膜蛋白M基因设计引物及探针,以体外转录法制备的cDNA标准品为模板,建立了TaqMan实时荧光定量RT-PCR方法,对其特异性、敏感性、重复性进行了测定,并对人工感染BPIV3的牛群临床样本进行了检测.结果显示,建立的TaqMan实时荧光定量RT-PCR的最低检测限为17.6 copies/μL,同时进行批内、批间5次重复性试验,变异系数均小于2.5%.应用建立的方法检测牛传染性鼻气管炎病毒、牛腺病毒3型、牛呼吸道合胞体病毒、牛病毒性腹泻-黏膜病病毒1型,结果均为阴性,说明建立的方法特异性较强.用建立的方法检测人工感染BPIV3的犊牛鼻拭子样本,检测结果与样本TCIDso的测定结果相符合,但比其更敏感.结果表明,本试验中建立的TaqMan实时荧光定量RT-PCR可以作为BPIV3早期诊断及定量分析的有效技术手段.

  14. Expression analyses of multiple DNA repair genes in glioma by TaqMan low-density array%TaqMan低密度表达芯片检测DNA损伤修复基因在原发胶质瘤中的表达研究

    Institute of Scientific and Technical Information of China (English)

    姜政; 胡锦; 李新钢; 卢大儒; 江玉泉; 周伟

    2007-01-01

    目的 运用低密度表达谱芯片检测人脑原发胶质瘤组织中DNA损伤修复基因的表达情况,进一步分析其表达变化的意义.方法 使用TaqMan低密度表达芯片技术检测27个DNA损伤修复基因在40例不同级别原发胶质瘤组织和10例正常脑组织中的表达情况,并通过统计学分析其在不同级别胶质瘤和正常脑组织中的表达差异.结果 与正常脑组织相比较,有13个DNA损伤修复基因在Ⅱ、Ⅲ、Ⅳ级胶质瘤中均表达下调,包括ERCC1、ERCC2、ERCC3、ERCC4、MGMT、MLH1、MLH3、NTHL1、OGG1、RAD50、SMUG1、XRCC4、XRCC5(P<0.05).MSH2、MSH6、NUDT1和XRCC3只在Ⅱ级和Ⅲ级胶质瘤中表达下调;MRE11A和MUS81只在Ⅲ级和Ⅳ级胶质瘤中表达下调.PMS2、RAD52和XRCC1只在Ⅲ级胶质瘤中表达下调,而UNG只在Ⅱ级中表达下调.结论 TaqMan低密度表达芯片为多个基因的同时定量表达提供了一种准确、快速、有效的多变量检测技术,可用于发现新的肿瘤相关基因.大量的DNA损伤修复基因的表达下调,与胶质瘤的发生密切相关.

  15. Establishment and application of the duplex TaqMan real-time RT-PCR with internal control for bovine viral diarrhea virus detection%牛病毒性腹泻病毒内标双重TaqMan荧光RT-PCR方法的建立及初步应用

    Institute of Scientific and Technical Information of China (English)

    季新成; 史茜; 郭春娟; 王科珂; 冉多良

    2014-01-01

    为建立牛病毒性腹泻病毒(BVDV)内标双重TaqMan荧光RT-PCR方法,本研究根据BVDV 5'-UTR区序列设计引物和荧光标记探针,通过重叠延伸PCR (SOE-PCR)扩增获得内标模板,经体外转录得到可以作为内标物的cRNA.经反应条件的优化,建立了BVDV内标双重TaqMan荧光RT-PCR检测体系.结果表明:当cRNA浓度为103拷贝/μL时,二者均有较好的扩增曲线;该方法对Ⅰ型BVDV的检测灵敏度为0.25 TCID50,对Ⅱ型BVDV的检测灵敏度为2.5 TCID50;利用该方法对猪瘟病毒等其他相关核酸的检测结果均为阴性;重复性检测Ct值变异系数为0.54~4.73.对506份临床样品检测,结果有23份样品为阳性,阳性样品检出数量和编号与不含内标的单一荧光RT-PCR方法完全一致,表明该方法可以用来对BVDV进行准确、快速检测,并且可以对检测结果进行质量监控.

  16. Methodological comparison of MALDI-TOF and TaqMan probe technology in SNP genotyping and their application in screening SNP with susceptibility to tuberculosis%飞行时间质谱与TaqMan探针技术在结核病易感性相关基因单核苷酸多态性筛选中的应用

    Institute of Scientific and Technical Information of China (English)

    汪文斐; 张国良; 杨帆; 杨辉; 赵丽芳; 徐发圣; 张明霞; 陈心春

    2014-01-01

    目的 对比分析飞行时间质谱技术(MALDI-TOF)和TaqMan探针筛选与结核病易感性相关单核苷酸多态性(SNP)位点的结果,以及联合应用的方法学和效果评价.方法 选取2010年10月至2011年4月在深圳市第三人民医院收治并确诊的结核病患者400例为结核病组,对照组为同时期收集的健康体检者300名,利用MALDI-TOF对选取的7个SNP位点(rs2227476、rs1800795、rs56077270、rs1800797、rs2227484、rs2227472和rs2227473)同时进行基因分型,初步筛选易感SNP位点;与结核病易感相关的单个SNP位点,采用基于TaqMan探针技术的实时荧光定量PCR对同样的标本再进行基因分型,比较两种方法的准确性与敏感度;以rs2227473位点为实例对分型结果的基因频率进行分析,确定肺结核的易感SNP.结果 MALDI-TOF分型成功率为99.7%(698/700),TaqMan探针技术为98.4%(689/700);在基因分型过程中,MALDI-TOF与TaqMan探针方法对1例标本的分型结果不一致,经过对此分型结果进行了测序验证,MALDI-TOF的分型结果正确,MALDI-TOF在准确性和敏感度比TaqMan法稍高.位点rs2227473基因频率分析中,结核病组G等位基因频率(90.3%,722/800)明显高于对照组(82.5%,495/600)(x2=6.911,P=0.009).结论 上述肺结核易感基因的筛选方法是可行的;实例分析中,将两种方法联合应用,发现了IL-22基因rs2227473位点等位基因G可能与肺结核发病相关,两位点中等位基因A可能为保护性基因.

  17. 未抗病毒治疗人类免疫缺陷病毒感染者隐匿性乙型肝炎的调查及其临床特点%Epidemiological and clinical features of occult hepatitis B in HIV infection without antiretroviral treatment

    Institute of Scientific and Technical Information of China (English)

    张仁芳; 刘莉; 郑毓芳; 沈银忠; 陈军; 顾士民; 王江蓉; 卢洪洲

    2013-01-01

    Objective To investigate and analyze the differential prevalence,as well as the risk factors and clinical features,of occult hepatitis B virus (HBV) infection in the human immunodeficiency virus (HIV)-infeeted population without antiretroviral therapy (ART) as compared to the general (non-HIV-infected) population.Methods Two-hundred-and-forty-eight individuals with confirmed HIV infection but ART naive (males:220,females:28;15-82 years old) were enrolled in the study,along with 121 healthy individuals (confirmed HIV antibody-negative;males:53,females:68;20-88 years old).HBV markers (hepafits B surface antigen (HBsAg);hepatitis B e antigen (HBeAg);anti-HBs,anti-HBe and anti-hepatitis B core (HBc) antibodies) were detected by microparticle enzyme-linked immunosorbent assay (AxSYM immunology analyzer manufactured by Abbott Laboratories);all cases and controls were confirmed negative for hepatitis B surface antigen (HBsAg).Then,the HBV DNA level in serum was detected using nucleic acid amplification assay (COBAS AmpliPrep/COBAS TaqMan HBV test,version 2.0 manufactured by Roche).CD4+ T lymphocytes were measured by flow cytometry,and alanine aminotransferase (ALT,marker of liver function) was measured by enzymatic assay.Results Twenty-four of the HIV cases (9.7%) and four of the healthy controls (3.3%) tested positive for HBV DNA;the amount of individuals with HBV DNA-positivity was significantly higher in the HIV-infected group (P =0.035).Among the 24 cases of HBV DNA(+) HIV-infected individuals,the lowest HBV DNA load was < 20 IU/ml and the highest was 3.22 x 105 IU/ml;nine of the individuals (37.5%) had HBV DNA load > 100 IU/ml,four (16.7%) had 20-99 IU/ ml,and 11 (45.8%) had < 20 IU/ml.Among the total HIV-infected cases with HBV DNA-positivity,7.3%(8/110) were anti-HBe(+)/anti-HBs(+),20.8% (11/53) were anti-HBc(+)/anti-HBs(-),14.3% (3/21) were anti-HBc(-)/anti-HBs(+),and 3.1% (2/64) were anti-HBc(-)/anti-HBs(-).The amount of

  18. Short Communication: Testosterone Measured with an Automatic Immunoassay Compares Reasonbly Well to Results Obtained by LC-MS/MS

    DEFF Research Database (Denmark)

    Knudsen, Cindy Søndersø; Højskov, Carsten Schriver; Møller, Holger Jon

    2016-01-01

    hormonebinding globulin (SHBG), and albumin employing Cobas e601/c501. Testosterone, androstenedione (andro), dehydroepiandrosterone sulphate (DHEAS), and 17-hydroxyprogesterone (17-OHP) concentrations were measured employing LC-MS/MS. We evaluated the difference between testosterone measured by the two methods...... for the difference between results obtained by the two methods and the sample concentration of DHEAS and andro: Diff (Cobas e601 - LC-MS/MS) = 0.116 x DHEAS - 0.396, r = 0.84 and Diff (Cobas e601 - LC-MS/MS) = 0.08 andro - 0.380, r = 0.58. No statistically significant interference was observed for progesterone, 17......-OHP, SHBG, and albumin. Conclusions: We report significant differences between testosterone measurements employing an automatic second generation immunoassay and LC-MS/MS. The difference can be correlated with the measured concentrations of DHEAS and andro, and its magnitude is judged to be of limited...

  19. Comparative evaluation of three commercial systems for detection of high-risk human papillomavirus in cervical and vaginal ThinPrep PreservCyt samples and correlation with biopsy results.

    Science.gov (United States)

    Binnicker, M J; Pritt, B S; Duresko, B J; Espy, M J; Grys, T E; Zarka, M A; Kerr, S E; Henry, M R

    2014-10-01

    Genital human papillomavirus (HPV) is the etiologic agent of more than 99% of all cervical cancers worldwide, with 14 genotypes being considered oncogenic or "high risk" because of their association with severe dysplasia and cervical carcinoma. Among these 14 high-risk types, HPV-16 and -18 account for approximately 70% of cervical cancers. The aim of this study was to evaluate three FDA-approved HPV nucleic acid-based tests for the ability to predict high-grade cervical intraepithelial neoplasias (CIN2 or worse) in corresponding tissue biopsy specimens. Residual specimens (total n = 793, cervical n = 743, vaginal n = 50) collected in ThinPrep PreservCyt medium with a cytologic result of ≥ atypical squamous cells of undetermined significance were tested by the Hybrid Capture 2 (HC2) assay (Qiagen, Gaithersburg, MD), the cobas HPV test (Roche Diagnostics, Indianapolis, IN), and the APTIMA HPV assay (Hologic, San Diego, CA). Genotyping for HPV-16 and HPV-18 was simultaneously performed by the cobas HPV test. Results were compared to cervical or vaginal biopsy findings, when they were available (n = 350). Among the 350 patients with corresponding biopsy results, 81 (23.1%) showed ≥ CIN2 by histopathology. The ≥ CIN2 detection sensitivity was 91.4% by the cobas and APTIMA assays and 97.5% by HC2 assay. The specificities of the cobas, APTIMA, and HC2 assays were 31.2, 42.0, and 27.1%, respectively. When considering only positive HPV-16 and/or HPV-18 genotype results, the cobas test showed a sensitivity and a specificity of 51.9 and 86.6%, respectively. While the HC2, cobas, and APTIMA assays showed similar sensitivities for the detection of ≥ CIN2 lesions, the specificities of the three tests varied, with the greatest specificity (86.6%) observed when the HPV-16 and/or HPV-18 genotypes were detected.

  20. Performance and Verification of a Real-Time PCR Assay Targeting the gyrA Gene for Prediction of Ciprofloxacin Resistance in Neisseria gonorrhoeae.

    Science.gov (United States)

    Hemarajata, P; Yang, S; Soge, O O; Humphries, R M; Klausner, J D

    2016-03-01

    In the United States, 19.2% of Neisseria gonorrhoeae isolates are resistant to ciprofloxacin. We evaluated a real-time PCR assay to predict ciprofloxacin susceptibility using residual DNA from the Roche Cobas 4800 CT/NG assay. The results of the assay were 100% concordant with agar dilution susceptibility test results for 100 clinical isolates. Among 76 clinical urine and swab specimens positive for N. gonorrhoeae by the Cobas assay, 71% could be genotyped. The test took 1.5 h to perform, allowing the physician to receive results in time to make informed clinical decisions.

  1. PENGEMBANGAN BUKU AJAR BIOLOGI SEL DENGAN PENDEKATAN BIOINFORMATIKA

    Directory of Open Access Journals (Sweden)

    Ardini Pangastuti

    2016-02-01

    Buku ajar merupakan buku panduan pembelajaran yang digunakan oleh siswa guna membantu mencapai tujuan pendidikan nasional. Pengembangan buku ajar merupakan salah satu cara yang dilakukan untuk memfasilitasi tercapainya indikator pembelajaran. Pengembangan buku ajar Biologi Sel dengan pendekatan Bioinformatika menggunakan model pengembangan Dick and Carey. Buku ajar yang dikembangkan divalidasi oleh ahli materi, ahli media pembelajaran, 15 mahasiswa uji coba perorangan, dan 15 mahasiswa uji coba kelompok sedang. Hasil validasi ahli materi menyatakan layak sebesar 84% dengan kategori baik. Hasil validasi ahli media pembelajaran menyatakan layak sebesar 82,4% dengan kategori baik.

  2. PENGEMBANGAN MODEL LATIHAN KARATE KIDS PADA ANAK USIA SEKOLAH DASAR KELAS ATAS

    Directory of Open Access Journals (Sweden)

    Widha Srianto

    2014-09-01

    Full Text Available Penelitian ini bertujuan untuk menghasilkan model latihan karate kids pada anak usia sekolah dasar (SD kelas atas (10-12 tahun. Penelitian pengembangan ini dilakukan dengan mengadaptasi langkah-langkah penelitian sebagai berikut: (1 pengumpulan informasi di lapangan, (2 melakukan analisis terhadap informasi yang telah dikumpulkan, (3 mengembangkan produk awal, (4 validasi ahli dan revisi, (5 uji coba lapangan skala kecil dan revisi,  (6 uji coba lapangan skala besar dan revisi, dan (7 pembuatan produk final. Uji coba skala kecil dilakukan di klub Forki Kota Yogyakarta berjumlah 7 anak. Uji coba skala besar di klub Inkanas DIY berjumlah 16 anak. Instrumen pengumpulan data yang digunakan yaitu: (1 pedoman wawancara, (2 skala nilai, (3 pedoman observasi model, (4 pedoman observasi keefektifan model, dan (5 kuesioner untuk siswa. Teknik analisis data yang dilakukan yaitu analisis deskriptif kuantitatif dan analisis deskriptif kualitatif. Hasil penelitian ini berupa model latihan karate kids pada anak usia SD kelas atas (10-12 tahun yaitu: (1 model latihan maegeri, (2 model latihan gyaku tsuki, (3 model latihan mawashigeri, dan (4 model latihan oi tsuki. Dari hasil analisis data penilaian para ahli materi dan kuesioner anak, dapat ditarik kesimpulan bahwa model latihan karate kids pada anak usia SD kelas atas (10-12 tahun ini dinilai baik dan efektif. Kata kunci: model latihan, karate kids

  3. PERANGKAT LUNAK "DIGITAL SIGNAGE MANAGER"

    Directory of Open Access Journals (Sweden)

    Siti Rochimah

    2006-07-01

    Full Text Available Digital signage adalah suatu alat untuk menampilkan konten multimedia kepada umum. Digital signage pada umumnya terdiri dari dua komponen penting, yaitu manager dan player. Digital Signage Manager (DSM adalah suatu perangkat lunak yang mempunyai fungsi mengelola perangkat lunak Digital Signage Player (DSP. Pengelolaan ini menyangkut pengaturan dan pengiriman konten, pengaturan DSP, dan pengaturan konten yang ada di DSP. Pada penelitian ini telah dibangun sebuah perangkat lunak DSM, yang merupakan bentuk pengembangan dari perangkat lunak yang sebelumnya telah ada yaitu BZNP-100, yang tidak lain adalah perangkat lunak untuk mengelola Sony Network Player NSP-100. DSM dibangun dengan tujuan untuk melengkapi kekurangan dan menambah beberapa fitur tambahan yang belum ada pada perangkat lunak sebelumnya, seperti: mendukung material Flash, mengirim Content Delivery Disc (CDD, menjadwalkan playlist, dan mendukung dua layar. BZNP-100 dan Sony Network Player NSP-100 merupakan digital signage yang dibuat oleh perusahaan elektronik Sony Corporation pada tahun 2003.Uji coba perangkat lunak DSM ini dilakukan dengan menjalankan skenario uji coba berdasarkan fungsionalitas masing-masing fitur. Uji coba dilakukan pada masing-masing fitur antara lain: login, konfigurasi, pembuatan material, manajemen DSP, manajemen playlist, memainkan material dan playlist, dan manajemen remote material dan playlist. Hasil uji coba menunjukkan bahwa perangkat lunak DSM telah berfungsi sesuai dengan tujuan yang diharapkan.Kata kunci: Digital signage, Digital Signage Manager (DSM, Digital Signage Player (DSP, Content Delivery Disc (CDD, plasma TV,  LCD.

  4. PENGEMBANGAN MODEL PEMBELAJARAN BERBASIS PERMAINAN TRADISIONAL UNTUK MENINGKATKAN KEMAMPUAN MOTORIK KASAR ANAK TUNAGRAHITA RINGAN

    Directory of Open Access Journals (Sweden)

    Asep Ardiyanto

    2014-09-01

    Full Text Available Penelitian ini bertujuan untuk menghasilkan model pembelajaran berbasis permainan tradisional untuk meningkatkan kemampuan motorik kasar anak tunagrahita ringan yang layak digunakan. Penelitian pengembangan ini dilakukan dengan langkah-langkah sebagai berikut: (1 pengumpulan informasi, (2 analisis hasil informasi, (3 mengembangkan produk awal, (4 validasi ahli dan revisi, (5 uji coba skala kecil, (6 revisi, (7 uji coba skala besar, (8 revisi akhir, (9 pembuatan produk final, dan (10 diseminasi dan implementasi produk final. Uji coba skala kecil dilakukan terhadap 6 siswa tunagrahita ringan SLB Tunas Kasih 2 Turi. Uji coba skala besar dilakukan terhadap 12 siswa tuna-grahita ringan SLB ABCD Tunas Kasih Donoharjo. Teknik analisis data yang digunakan yaitu deskriptif kuantitatif dan deskriptif kualitatif. Penelitian ini menghasilkan model pembelajaran, yaitu: (1 balap sarung, (2 lempar karet, (3 dorong ban, (4 engkling, (5 pukul balon, (6 layang-layang, (7 lompat tali, dan (8 pesawat terbang. Dari hasil analisis data penilaian para ahli materi dan guru SLB, ditarik kesimpulan bahwa pengembangan model pembelajaran ini sangat baik dan efektif. Kata kunci: pengembangan, permainan tradisional, pembelajaran motorik kasar, anak tunagrahita ringan.

  5. Causes and outcome of hospitalization among HIV-infected adults ...

    African Journals Online (AJOL)

    EB

    patients have advanced disease with increased risk ... ART at Mulago National Referral and Teaching. Hospital, Uganda. Methods. Study site. This study ... Practice standards. ... (Cobas Integra 400) and CD4 count measurement ... baseline clinical and laboratory characteristics were ..... western Uganda: implications for HIV.

  6. Comparison of three human papillomavirus DNA assays and one mRNA assay in women with abnormal cytology

    DEFF Research Database (Denmark)

    Rebolj, Matejka; Lynge, Elsebeth; Ejegod, Ditte;

    2014-01-01

    OBJECTIVE: To compare the clinical characteristics of four human papillomavirus (HPV) assays: hybrid capture 2 (HC2), cobas, CLART, and APTIMA in Danish women with abnormal cytology. METHODS: SurePath samples from 367 consecutive women from Copenhagen, with atypical squamous cells of undetermined...

  7. 77 FR 2071 - Medical Devices; Availability of Safety and Effectiveness Summaries for Premarket Approval...

    Science.gov (United States)

    2012-01-13

    ..., 2011. P110020, FDA-2011-M-0601...... Roche Molecular COBAS 4800 BRAF V600 MUTATION August 17, 2011. Systems, Inc.. TEST. P110012, FDA-2011-M-0630...... Abbott Molecular, VYSIS ALK BREAK APART FISH PROBE August 26, 2011. Inc.. KIT; VYSIS PARAFFIN PRETREATMENT IV & POST HYBRIDIZATION WASH BUFFER KIT...

  8. PENGEMBANGAN MOTION GRAPHIC PEMBELAJARAN MATA PELAJARAN PENDIDIKAN KEWARGANEGARAAN KELAS I SEKOLAH DASAR

    Directory of Open Access Journals (Sweden)

    Asih Purwanti

    2015-12-01

    Full Text Available Penelitian ini merupakan penelitian dan pengembangan yang bertujuan untuk menghasilkan motion graphic pembelajaran mata pelajaran Pendidikan Kewarganegaraan pada kelas 1 Sekolah Dasar yang layak dijadikan sebagai sumber belajar. Model penelitian yang digunakan adalah Borg & Gall (1983 dan Dick & Carey (2005. Hasil sebagai berikut. (1 Kelayakan produk pada aspek pembelajaran dan aspek materi/isi dari ahli materi menunjukkan skor 3,56 (baik, aspek media/teknis dari ahli media I menunjukkan skor 3,57 (baik dan aspek media/teknis ahli media II menunjukkan skor 3,62 (baik. Data yang diperoleh melalui uji coba pada siswa pada uji coba satu-satu menunjukkan skor 75%, melalui uji coba kelompok kecil menunjukkan skor 81%, dan melalui uji coba lapangan menunjukkan skor 90%. (2 Hasil belajar siswa pada saat pretes rerata menunjukkan skor 62, sedangkan hasil belajar siswa pada posttes rerata menunjukkan skor 76. Sehingga menghasilkan N-gain dengan skor 36. Berdasarkan N-gain yang tergolong sedang, maka motion graphic pembelajaran yang dikembangkan efektif dan dapat melatih keterampilan berpikir kritis siswa.

  9. PENGEMBANGAN ASESMEN ALTERNATIF PRAKTIKUM KIMIA DASAR II MELALUI CHEMISTRY FAIR PROJECT (CFP BERBASIS KONSERVASI DENGAN MEMANFAATKAN DAILY CHEMICAL

    Directory of Open Access Journals (Sweden)

    Indah Urwatin Wusqo

    2016-12-01

    Full Text Available Penelitian ini bertujuan untuk (1 Mengembangkan asesmen alternatif pada praktikum kimia dasar II melalui chemistry fair project berbasis konservasi dengan memanfaatkan daily chemical(2 Mengetahui tingkat kevalidan, kepraktisan dan keefektifannya. Penelitian ini merupakan penelitian pengembangan (Development Research Model pengembangan yang diterapkan Dick dan Carey (1985. Subjek uji coba terbatas maupun subjek uji coba lapangan adalah dosen dan mahasiswa Prodi Pendidikan IPA UNNES. Sampel ditentukan secara purposive, yaitu dosen pengampu dan mahasiswa yang menempuh mata kuliah Praktikum Kimia Dasar II. Data yang diperoleh dari uji coba ini adalah: (1 masukan dari pakar, untuk menentukan validitas isi dan konstruk dari fitur asesmen; (2 masukan dari sampel uji coba terbatas, untuk menentukan kepraktisan petunjuk chemistry fair project (CFP berbasis konservasi dengan memanfaatkan daily chemical ; Instrumen pengumpul data berupa angket keterbacaan petunjuk pembuatan chemistry fair project (CFP berbasis konservasi dengan memanfaatkan daily chemical, pedoman penskoran. (3 data hasil belajar siswa untuk mengetahui efektivitas asesmen. Masukan dari pakar angket mahasiswa, dan nilai chemistry fair project (CFP sampel ujicoba terbatas dianalisis secara kualitatif, dan kuantitatif. Asesmen alternative Praktikum Kimia Dasar II yang dikembangkan dikatakan berhasil baik apabila asesmen yang dikembangkan valid, praktis, dan efektif.

  10. Hepatoprotective Effect of Pandanus odoratissimus L Inflorescence ...

    African Journals Online (AJOL)

    Tropical Journal of Pharmaceutical Research February 2016; 15 (2): 259-266. ISSN: 1596-5996 (print); .... Malaysia; known by locals in India as Ketaki, is used in Ayurvedic .... Cobas C 311 analyzer (Roche Diagnostics -. GmbH, D-68298 ...

  11. CLINICAL PERFORMANCE CHARACTERISTICS OF ELECSYS® FREE-ΒHCG AND PAPP-A FOR FIRST TRIMESTER TRISOMY 21 RISK ASSESSMENT IN GESTATIONAL WEEKS 8+0 TO 14+0

    DEFF Research Database (Denmark)

    Tørring, Niels; aulesa, C; Eiben, Bernd;

    2014-01-01

    Background Screening for fetal trisomy 21 (T21) in the first trimester includes analysis of the serological markers pregnancy-associated plasma protein A (PAPP-A) and free beta choriogonadotropin (free βhCG). With the launch of these assays on the cobas e and Elecsys platforms, we investigated...

  12. A daunting challenge

    DEFF Research Database (Denmark)

    Rebolj, Matejka; Bonde, Jesper; Ejegod, Ditte

    2015-01-01

    We compared cytology with Hybrid Capture 2 (HC2), cobas, CLART and APTIMA Human Papillomavirus (HPV) assays in primary cervical screening at age 23-29 years based on data from the Danish Horizon study. SurePath samples were collected from 1278 women undergoing routine cytology-based screening. Ab...

  13. Pengembangan Skala Sikap Diferensial Semantik Terhadap Fisika Mahasiswa Jurusan Teknik Mesin UNJ

    Directory of Open Access Journals (Sweden)

    Ratu Amilia Avianti

    2016-05-01

    Full Text Available Tujuan penelitian ini adalah untuk mengembangkan skala sikap differensial semantic terhadap fisika Jurusan Teknik Mesin. Skala differensial semantik adalah jenis pertanyaan survey dimana responden diminta untuk merata-rata pendapatannya pada skala linier antara 2 titik, yang secara teoritis ada 7 tingkatan. Skala sikap differensial semantic ini mempunyai 3 ukuran yaitu evaluasi, potensi dan aktivitas. Skala ini diujicobakan 2 kali terhadap 116 mahasiswa Teknik mesin Universitas Negeri Jakarta (UNJ. Metode yang dipergunakan dalam penelitian ini adalah metode pengembangan skala dengan menggunakan pendekatan respons. Pada uji coba tahap pertama, hasilnya mengindikasikan bahwa ketiga faktor diintisarikan dari data yang diperoleh menggunakan analisi komponen penting metode eksplorasi, sejalan dengan faktor pengukuran. Metode konfirmasi dari Maximum Likelihood yang mengukur kebaikan dari ketiga faktor, menemukan indeks signifikansi pada tingkat 62, 689. Hal ini mengindikasikan validitas korelasi model ketiga faktor. Untuk menentukan reliabitas instrumen digunakan Theta reliabilitas (o= 0.7965. Pada uji coba kedua, validitas konstruk juga ditentukan dengan menggunakan analisis faktor. Tiga faktor juga diintisarikan dari metode eksplorasi PCA. Metode konfirmasil ML diterapkan untuk menguji kebaikan ketiga faktor dan indeks tes yang diperoleh 60,978 yang juga signifikan. Ketiga faktor dari pengintisarian juga mendukung pengukuran teori seperti pada uji coba pertama. Konsistensi internal Theta yang diperoleh adalah o= 0.7777 dapat disimpulkan bahwa kuesioner yang mengukur sikap mahasiswa terhadap Fisika mempunyai validitas konstruk yang sesuai menggunakan skala yang berbeda. Namun demikian , dibutuhkan uji coba lebih lanjut untuk membakukan instrumen ini.

  14. Aplikasi Belajar Menulis Aksara Jawa Menggunakan Android

    Directory of Open Access Journals (Sweden)

    As'ad Arismadhani

    2013-03-01

    Full Text Available Aksara Jawa atau yang lebih dikenal dengan nama Hanacaraka merupakan salah satu dari sekian warisan budaya leluhur bangsa Indonesia. Dengan seiring perkembangan zaman, Aksara Jawa seolah menjadi salah satu warisan budaya yang terlupakan. Sebagai generasi muda Indonesia, sudah seharusnya kita melestarikan budaya bangsa yang merupakan peninggalan dari leluhur kita. Atas dasar itulah pada penelitian ini dikembangkan suatu media sekaligus alat bantu berupa aplikasi belajar menulis Aksara Jawa pada perangkat Android. Penelitian ini dimulai dengan melakukan perancangan terhadap kebutuhan-kebutuhan yang akan diintegrasikan pada aplikasi Android. Pengembangan dan pembuatan aplikasi menggunakan teknologi bahasa pemrograman Java dan XML. Proses uji coba dilakukan dengan proses memasukkan data ke berkas pustaka yang berupa coretan pada bidang layar sentuh pada perangkat Android. Kemudian uji coba dilakukan dengan proses menghapus data dan yang terakhir yaitu proses uji coba mencocokkan data antara bentuk aksara yang dituliskan pada bidang layar sentuh dengan daftar aksara yang sudah tersimpan pada berkas pustaka. Dalam penelitian ini dapat diketahui bahwa perangkat genggam Android dapat digunakan sebagai media pembelajaran menulis Aksara Jawa. Pola-pola yang digunakan pada proses uji coba dapat dikenali oleh aplikasi Aksara Jawa dan pola-pola tersebut dapat dicocokkan dengan daftar Aksara Jawa yang terdapat pada berkas pustaka.

  15. Detection of expression level of endogenous jaagsiekte sheep retrovirus and its receptor HYAL2 in endometrium of pregnant Mongolian sheep by TaqMan real-time PCR and in situ hybridization%妊娠绵羊子宫内膜组织内源性反转录病毒及其受体HYAL2基因表达水平的检测

    Institute of Scientific and Technical Information of China (English)

    徐萌杰; 陈大勇; 刘淑英

    2011-01-01

    In order to assess the expression of endogenous jaagsiekte sheep retrovirus(enJSRV) and its receptor HYAL2 mRNAs in the endometrium of Mongolian ewes throughout gestation, by fluorescent quantitative RT-PCR,enJSRV and HYAL2 mRNAs were found to be expressed throughout gestation in the endometrium. The mRNA expression level of enJSRV was higher in placenta on day 30 and 50 during pregnancy. The in situ hybridization results showed that enJSRV and HYAL2 mRNAs were specifically expressed in endometrial luminal epithelium and glandular epithelium, endometrial caruncle, trophoblast giant binucleate cells, as well as stroma, and blood vessel endothelial cells endometrial caruncle, placental cotyledon,stroma and trophectoderm. Collectively, little was known about the molecular mechanisms that trophoblast differentiation and multinucleated syncytia formation regulated by enJSRV, however the temporal and spatial reconstitutions of enJSRV expression in the endometrium of Mongolian ewes indicated that enJSRV and its receptor HYAL2 were beneficial to the host and were involved in protection of the uterus from viral infection and regulators of placental morphogenesis and function.%为了探究内源性反转录病毒(enJSRV)及其受体HYAL2在妊娠蒙古绵羊子宫内膜形成和发育过程中的作用,运用TagMan实时荧光定量PCR和组织原位杂交技术对其在不同妊娠时期(30、50、70、90、110和130 d)蒙古绵羊子宫内膜中的表达规律和分布定位进行了研究.实时荧光定量PCR结果显示,enJSRV及其受体HYAL2在蒙古绵羊子宫内膜的不同妊娠时期均有表达,通过SPSS统计学软件分析可以看出,妊娠蒙古绵羊子宫内膜组织中enJSRV mRNA的表达于妊娠第30和50天相对较高,70~130 d均低于对照组(30 d),且差异都极显著,enJSRV与其受体HYAL2 mRNA之间亦无线性相关性.原位杂交结果显示,enJSRV及其受体HYAL2 mRNA在妊娠第30、50和130天蒙古绵羊子宫内膜的腔上皮细胞、腺上皮细胞、血管内皮细胞、间质及滋养层巨型双核细胞中均有阳性信号表达.表明enJSRV及其受体HYAL2表达于上皮细胞及上皮细胞来源的滋养层巨型双核细胞中,揭示enJSRV及其受体HYAL2在子宫形成过程中和系统防御中可能发挥重要作用.

  16. Pengembangan Instrumen Analisis Kompetensi Tutor Pendidikan Anak Usia Dini (PAUD

    Directory of Open Access Journals (Sweden)

    Agus Tiyono Teguh Maryanto

    2005-12-01

    Full Text Available Penelitian ini bertujuan: (1 mengidentifikasi standar kompetensi dan indikator yang dapat dijadikan tolok ukur untuk menyusun standar kompetensi tutor PAUD, dan (2 mengembangkan instrumen untuk uji kompetensi tutor PAUD yang tepat dan handal berdasarkan indikator kompetensi yang telah tersusun. Penelitian ini merupakan penelitian pengembangan yang terdiri dari dua tahap yaitu tahap, pengembangan standar kompetensi dan pengembangan instrumen uji kompetensi tutor PAUD. Teknik pengumpulan data yang digunakan adalah focus group discussion ( FGD dan teknik Delphi 2 x putaran. Subjek uji coba adalah 110 orang tutor yang mengikuti Pelatihan Tenaga Pendidik PAUD Tingkat Provinsi Jawa Tengah Tahun 2004. Subjek uji coba instrumen pertama sebanyak 20 orang dari Kabupaten Magelang, dan subjek uji coba instrumen kedua sebanyak 32 orang dari Salatiga, Kota Magelang, dan Kabupaten Magelang. Temuan penelitian ada dua. (1 standar kompetensi tutor PAUD terdiri atas 4 dimensi dan 50 indikator, meliputi dimensi personal, dimensi sosial, dimensi profesional, dan dimensi akademik; (2 instrumen untuk menguji kompetensi yang berupa inventori tes dengan indeks validitas berupa angka muatan faktor terendah di atas 0,300 (kriteria yang telah ditetapkan. Koefisien reliabilitas tiap faktor pada uji coba pertama adalah . 0,879 dan tertinggi 0,984, sedangkan koefisien reliabilitas tiap faktor pada uji coba kedua terendah 0,889 dan tertinggi 0,995, telah melebihi syarat minimal untuk pengukuran kelompok yaitu 0,65. Koefisien reliabilitas tes uraian ditentukan dengan reliabilitas antar-rater dengan teknik ANAVA, dan diperoleh koefisien reliabilitas dari 3 orang penilai yaitu 0,957, sedangkan estimasi untuk seorang penilai adalah 0,881. Kata kunci: pengembangan instrumen, anahsis kompetensi, tutor paud

  17. Genetic variants involved in gallstone formation and capsaicin metabolism,and the risk of gallbladder cancer in Chilean women

    Institute of Scientific and Technical Information of China (English)

    Sergio; Báez; Yasuo; Tsuchiya; Alfonso; Calvo; Martha; Pruyas; Kazutoshi; Nakamura; Chikako; Kiyohara; Mari; Oyama; Masaharu; Yamamoto

    2010-01-01

    AIM:To determine the effects of genetic variants associated with gallstone formation and capsaicin (a pungent component of chili pepper) metabolism on the risk of gallbladder cancer (GBC).METHODS: A total of 57 patients with GBC, 119 patients with gallstones, and 70 controls were enrolled in this study. DNA was extracted from their blood or paraffi n block sample using standard commercial kits. The statuses of the genetic variants were assayed using Taqman SNP Genotyping Assays or Custom Taqman SNP Genotypi...

  18. Pemanfaatan Augmented Reality Pada Permainan Othello

    Directory of Open Access Journals (Sweden)

    Rendy Layman Aguston

    2017-03-01

    Full Text Available Perkembangan teknologi telah mengubah cara pengerjaan suatu pekerjaan dari cara konvensional menjadi cara yang lebih praktis. Dengan hadirnya teknologi Augmented Reality, cara bermain yang menggunakan pion dan membalikkan pion musuh secara manual menjadi lebih mudah dalam memainkan permainan selain juga dapat berinteraksi langsung. Pembuatan permainan Othello menggunakan program Unity dengan framework Vuforia untuk mewujudkan Augmented Reality pada permainan Othello. Untuk menerapkan Augmented Reality dengan baik, dibutuhkan papan permainan sebagai image target yang sesuai dengan kriteria, jenis kamera yang digunakan, jarak kamera terhadap papan permainan, intensitas cahaya yang ditangkap kamera, serta tingkat sensitivitas tombol virtual. Pada permainan Othello ini tersedia fitur komputer yang menggunakan algoritma Alpha Beta Pruning dengan 3 level kedalaman yang menggunakan perhitungan fungsi evaluasi berupa mobility, potential mobility dan penguasaan corner yang menghasilkan kemenangan mencapai 73,33% dari 15 kali uji coba terhadap aplikasi Othello serupa dan 78,34% dari 37 kali uji coba terhadap user.

  19. IMPLEMENTASI METODE MULTIPLE KERNEL SUPPORT VECTOR MACHINE UNTUK SELEKSI FITUR DARI DATA EKSPRESI GEN DENGAN STUDI KASUS LEUKIMIA DAN TUMOR USUS BESAR

    Directory of Open Access Journals (Sweden)

    Ariana Yunita

    2012-03-01

    Full Text Available Pada penelitian ini mengimplementasikan metode multiple kernel support vector machine untuk seleksi fitur. Multiple kernel merupakan metode modifikasi fungsi kernel yang mengalikan tiap elemen dari data. Metode ini melakukan seleksi fitur terhadap fitur yang kurang penting dengan tingkat akurasi lebih baik daripada metode dasar support vector machine. Uji coba dilakukan dengan menggunakan dataset ekspresi gen leukimia dan tumor usus besar. Hasil uji coba dibandingkan dengan tingkat akurasi metode support vector machine tanpa seleksi fitur. Tingkat akurasi metode multiple kernel support vector machine yang dihasilkan untuk data ekspresi gen leukimia yaitu 85% dan untuk data tumor usus besar sebesar 69%. Sedangkan tingkat akurasi dengan metode dasar support vector machine yaiu sebesar 82% untuk data leukimia dan 59% untuk data tumor usus besar. Seleksi fitur dapat mempersingkat waktu komputasi sehingga dapat dikembangkan untuk banyak aplikasi pengenalan pola.   Kata Kunci: Multiple kernel, support vector  machine, seleksi fitur, data ekspresi gen

  20. Pengembangan Reaktor Fotokatalitik Rotating Drum untuk Pengolahan Air Limbah Industri Tekstil

    Directory of Open Access Journals (Sweden)

    Cholid Syahroni

    2015-11-01

    Full Text Available Reaksi oksidasi fotokatalitik memiliki potensi untuk mendegradasi senyawa organik hingga tingkat mineralisasi, sehingga tidak meninggalkan residu (sludge. Penelitian ini bertujuan membuat reaktor fotokatalitik rotating drum dan mengaplikasikan pada industri tekstil. Langkah percobaan meliputi pembuatan katalis TiO2 secara anodizing serta karakterisasi dengan XRD dan SEM, pembuatan reaktor fotokatalitik  rotating drum dan uji coba degradasi air limbah industri tekstil. Proses anodizing dilakukan dengan bias potensial sebesar 40 volt selama 2 jam menggunakan elektrolit etilen glikol yang mengandung amonium fluorida dan air. Uji karakterisasi secara XRD dan SEM menunjukkan bahwa struktur kristal TiO2 adalah anatase dengan ukuran kristalit 8–19 nm. Bentuk kristal nanotube, dengan diameter 30–110 nm. Hasil uji coba menunjukkan bahwa  degradasi secara fotokatalitik dengan penambahan H2O2 0,15% terhadap air limbah bisa menurunkan COD 72,12% dalam waktu 2 jam. 

  1. Peramalan Kunjungan Wisatawan Mancanegara Menggunakan Generalized Regression Neural Networks

    Directory of Open Access Journals (Sweden)

    Sri Herawati

    2016-05-01

    Full Text Available Peramalan kunjungan wisatawan mancanegara (wisman sangat penting bagi pemerintah dan industri, karena peramalan menjadi dasar dalam perencanaan kebijakan yang efektif. Penelitian ini menggunakan Generalized Regression Neural Network (GRNN untuk meramalkan kunjungan wisman menurut 19 pintu masuk utama dan kebangsaan, seperti: Ngurah Rai, Soekarno-Hatta, Batam, Tanjung Uban, Polonia, Juanda, Husein Sastranegara, Tanjung Balai Karimun, Tanjung Pinang, Tanjung Priok, Adi Sucipto, Minangkabau, Entikong, Adi Sumarmo, Sultan Syarif Kasim II, Sepinggan, Sam Ratulangi, Bandara Internasional Lombok, dan Makassar. GRNN memiliki kelebihan tidak memerlukan estimasi jumlah bobot jaringan untuk mendapatkan arsitektur jaringan optimal, sehingga tidak memerlukan pengaturan parameter bebas. Uji coba penelitian dilakukan dengan menggunakan spread dari 0,1 sampai 1,0. Hasil uji coba menunjukkan bahwa kinerja Peramalan terbaik dengan menggunakan spread 0,1 baik untuk data latih maupun data uji

  2. EDTA interference in electrochemiluminescence ACTH assay.

    Science.gov (United States)

    Toprak, Burak; Yalcin, Hulya; Arı, Elif; Colak, Ayfer

    2016-11-01

    Background As plasma is the recommended sample type for Roche adrenocorticotropic hormone (ACTH) assay, we evaluated the effect of EDTA concentration on Cobas ACTH assay. Methods Samples containing twofold and fourfold higher concentrations of EDTA were prepared by adding plasma to empty K2EDTA tubes and by making under-filled EDTA tubes. All measurements were performed with four replicates. Results Increased EDTA concentration resulted in a significant decrease in ACTH concentration. Fifty-per cent-filled EDTA tube showed 19% decrease in ACTH concentration and 25% filled EDTA tube showed 50% decrease in ACTH concentration. Conclusion We recommend that inadequately filled EDTA specimens should be rejected when using Cobas ACTH assay.

  3. Implementasi Algoritma Particle Swarm untuk Menyelesaikan Sistem Persamaan Nonlinear

    Directory of Open Access Journals (Sweden)

    Ardiana Rosita

    2012-09-01

    Full Text Available Penyelesaian sistem persamaan nonlinear merupakan salah satu permasalahan yang sulit pada komputasi numerik dan berbagai aplikasi teknik. Beberapa metode telah dikembangkan untuk menyelesaikan sistem persamaan ini dan metode Newton merupakan metode yang paling sering digunakan. Namun metode ini memerlukan perkiraan solusi awal dan memilih perkiraan solusi awal yang baik untuk sebagian besar sistem persamaan nonlinear tidaklah mudah. Pada makalah ini, algoritma Particle Swarm yang diusulkan oleh Jaberipour dan kawan-kawan[1] diimplementasikan. Algoritma ini merupakan pengembangan dari algoritma Particle Swarm Optimization (PSO. Algoritma ini meyelesaikan sistem persamaan nonlinear yang sebelumnya telah diubah menjadi permasalahan optimasi. Uji coba dilakukan terhadap beberapa fungsi dan sistem persamaan nonlinear untuk menguji kinerja dan efisiensi algoritma. Berdasarkan hasil uji coba, beberapa fungsi dan sistem persamaan nonlinear telah konvergen pada iterasi ke 10 sampai 20 dan terdapat fungsi yang konvergen pada iterasi ke 200. Selain itu, solusi yang dihasilkan algoritma Particle Swarm mendekati solusi eksak.

  4. Comparison of Three Nucleic Acid Amplification Assays of Cerebrospinal Fluid for Diagnosis of Cytomegalovirus Encephalitis

    Science.gov (United States)

    Bestetti, Arabella; Pierotti, Chiara; Terreni, Mariarosa; Zappa, Alessandra; Vago, Luca; Lazzarin, Adriano; Cinque, Paola

    2001-01-01

    The diagnostic reliabilities of three cytomegalovirus (CMV) nucleic acid amplification assays of cerebrospinal fluid (CSF) were compared by using CSF samples from human immunodeficiency virus-infected patients with a postmortem histopathological diagnosis of CMV encephalitis (n = 15) or other central nervous system conditions (n = 16). By using a nested PCR assay, the quantitative COBAS AMPLICOR CMV MONITOR PCR, and the NucliSens CMV pp67 nucleic acid sequence-based amplification assay, sensitivities were 93.3, 86.6, and 93.3%, respectively, and specificities were 93.7, 93.7, and 87.5%, respectively. The COBAS AMPLICOR assay revealed significantly higher CMV DNA levels in patients with diffuse ventriculoencephalitis than in patients with focal periventricular lesions. PMID:11230445

  5. PENGEMBANGAN MEDIA PEMBELAJARAN PENDIDIKAN JASMANI OLAHRAGA DAN KESEHATAN BERBASIS KOMPUTER UNTUK SMA

    Directory of Open Access Journals (Sweden)

    Suci Cahyati

    2014-04-01

    Full Text Available Penelitian ini bertujuan untuk menghasilkan produk berupa CD pembelajaran pendidikan jasmani olahraga dan kesehatan berbasis komputer untuk SMA yang valid dan efektif. Penelitian ini menggunakan metode Research and Development (R&D. Media yang dikembangkan memuat materi tentang HIV/AIDS. Pengembangan media pembelajaran ini menggunakan software Adobe Flash Cs3 Professional. Media yang telah dikembangkan melalui dua tahap penelitiannya itu validasi ahli dan uji coba lapangan. Berdasarkan validasi ahli dan uji coba lapangan hasil penelitian menunjukkan produk termasuk kriteria “Sangat baik” dengan rerata skor 4,25. Hasil tes siswa mengalami peningkatan dari rerata skor pretest 5,53 dan rerata skor posttest 7,56. Berdasarkan hasil tes tersebut dapat disimpulkan bahwa media pembelajaran pendidikan jasmani olahraga dan kesehatan efektif digunakan dalam pembelajaran di SMA. Kata kunci: Pengembangan media, penjasorkes, SMA

  6. Analisis Arsitektur Aplikasi Web Menggunakan Model View Controller (MVC pada Framework Java Server Faces

    Directory of Open Access Journals (Sweden)

    Gunawan Gunawan

    2016-06-01

    Full Text Available Aplikasi web yang khususnya memiliki kompleksitas besar dalam melakukan transaksi data sehingga konsep arsitektur (pattern perlu menjadi perhatian khusus untuk dapat mengoptimalkan kinerja performansi sistem ketika pengguna (user menggunakan dalam waktu yang bersamaan dengan jumlah yang banyak. Analisis performa arsitektur aplikasi web yang menggunakan model 2 (MVC dengan menggunakan framework Java Server Faces (JSF dan model 1 sebagai pembanding. Metode yang digunakan adalah Load dan Scalability Testing dengan dua cara yaitu uji coba terhadap response time karena peningkatan ukuran dari database dan uji coba terhadap response time karena peningkatan jumlah user yang menggunakan sistem secara bersamaan (concurrent users dan waktu tunggu (ramp-up yang ditentukan menggunakan Apache Jmeter. Analisis menunjukkan bahwa dalam implementasi arsitektur web yang menggunakan model 1 waktu rata-rata yang dibutuhkan untuk merespon permintaan user lebih cepat dan efisien dibanding model 2 (MVC.  

  7. PENGEMBANGAN MODEL PEMBELAJARAN SOFT SKILLS TERPADU DALAM PEMBELAJARAN PENJASORKES PADA SEKOLAH MENENGAH PERTAMA

    OpenAIRE

    Lilik Indriharta; S. Suharjana; M. Marzuki

    2017-01-01

    Tujuan penelitian ini adalah untuk: (1) mengembangkan model pembelajaran soft skills terpadu, (2) menganalisis keberhasilan pelaksanaan pembelajaran soft skills terpadu, dan (3) me-nganalisis dampak penerapan model pembelajaran Soft Skills Terpadu dalam pembelajaran Penjasorkes di Sekolah Menengah Pertama terhadap siswa. Penelitian ini merupakan penelitian pengembangan (Research & Development). Subjek uji coba dalam penelitian ini adalah kelas VII pada 4 SMP Negeri di Kota Yoyakarta. Analisis...

  8. Sleep Deprivation in Humans, Immunodepression and Glutamine Supplementation

    Science.gov (United States)

    2007-11-02

    centrifuged at 5-8˚C, and the supernatant removed and stored at -20˚C. The samples were measured using a quantitative sandwich enzyme immunoassay ...numbers and differentials; platelet counts; haemoglobin ( Hb ), haematocrit (Hct), mean corpuscular volume (MCV); CD19+ B-lymphocyte cell counts...of caffeine were measured using a homogeneous enzyme immunoassay technique and the COBAS Mira Plus Clinical Analyser. Antioxidant capacity: A

  9. MODULAR ANALYTICS: A New Approach to Automation in the Clinical Laboratory

    OpenAIRE

    Horowitz, Gary L.; Zaman, Zahur; Blanckaert, Norbert J. C.; Chan, Daniel W.; Dubois, Jeffrey A.; Golaz, Olivier; Mensi, Noury; Keller, Franz; Stolz, Herbert; Klingler, Karl; Marocchi, Alessandro; Prencipe, Lorenzo; McLawhon, Ronald W.; Nilsen, Olaug L.; Oellerich, Michael

    2005-01-01

    MODULAR ANALYTICS (Roche Diagnostics) (MODULAR ANALYTICS, Elecsys and Cobas Integra are trademarks of a member of the Roche Group) represents a new approach to automation for the clinical chemistry laboratory. It consists of a control unit, a core unit with a bidirectional multitrack rack transportation system, and three distinct kinds of analytical modules: an ISE module, a P800 module (44 photometric tests, throughput of up to 800 tests/h), and a D2400 module (16 photometric tests, throughp...

  10. Pengembangan Modul Fisika Pokok Bahasan Hukum Newton bagi Anak Berkebutuhan Khusus (Tunanetra Di Kelas Inklusi SMA/MA Kelas X

    Directory of Open Access Journals (Sweden)

    Fitriany Yudistia R

    2014-04-01

    Full Text Available Siswa tunanetra SMA Muhammadiyah 4 Yogyakarta, MAN Maguwoharjo dan SMAN 1 Sewon belum memiliki sumber belajar mandiri berupa modul Braille khususnya pada materi Hukum Newton. Berdasarkan kenyataan ini maka dibutuhkan sebuah sumber belajar yang didesain khusus bagi siswa tunanetra di kelas Inklusi yakni modul Braille pada pokok bahasan Hukum Newton.Telah dilakukan penelitian yang bertujuan untuk: (1 mengembangkan modul fisika pokok bahasan Hukum Newton untuk siswa tunanetra SMA/MA kelas X sebagai sumber belajar mandiri, (2 mengetahui kualitas modul fisika Braille materi Hukum Newton untuk siswa tunanetra SMA/MA kelas X, (3 mengetahui respon siswa terhadap modul fisika Braille yang telah dikembangkan.Penelitian ini merupakan penelitian R & D dengan model prosedural yang mengadaptasi dari pengembangan perangkat model 4-D, yakni Define, Design, Develop, and Disseminate. Instrumen penelitian berupa angket kualitas modul yaitu menggunakan skala Likert yang dibuat dalam bentuk checklist. Instrumen untuk siswa berupa angket respon siswa yaitu menggunakan skala Guttman yang dibuat dalam bentuk checklist. Modul dinilai kualitasnya oleh 3 ahli materi, 1 ahli media, dan 2 guru fisika SMA/MA. Kelayakan modul berdasarkan respon siswa pada uji coba terbatas sebanyak 2 siswa dan uji coba luas sebanyak 8 siswa.Hasil penelitian berdasarkan penilaian dari ahli materi, ahli media dan guru fisika SMA/MA modul memiliki kategori sangat baik (SB. Persentase keidealan menurut ahli materi adalah 87,88%; persentase keidealan menurut ahli media adalah 90,00% dan persentase keidealan menurut guru Fisika SMA/MA adalah 75,00%. Respon siswa terhadap modul fisika Braille pada uji coba terbatas diperoleh persentase 97,22%; sedangkan pada uji coba luas diperoleh persentase 89,58%. Hasil penelitian ini menunjukkan bahwa modul layak dijadikan sebagai salah satu sumber belajar mandiri bagi siswa tunanetra.

  11. Systemic and Pulmonary Hypertension After Resuscitation with Cell-Free Hemoglobin

    Science.gov (United States)

    1992-07-01

    according to published standards (24). Inorganic phosphate was measured on a blood chemistry analyzer (Cobas-Fara; Roche Diagnostic Systems, Nutley, NJ...alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) on the blood chemistry analyzer . The final 4 ml of blood was heparinized, centrifuged...milliliter of blood was placed in 2 ml of ice-cold perchioric acid (70% wt/vol), which was used to determine whole blood lactate in the blood chemistry

  12. Comparison of three real-time PCR assays for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in young pregnant women.

    Science.gov (United States)

    Peuchant, Olivia; de Diego, Sabrina; Le Roy, Chloé; Frantz-Blancpain, Sandrine; Hocké, Claude; Bébéar, Cécile; de Barbeyrac, Bertille

    2015-12-01

    We compared 3 commercial real-time PCR assays, the Abbott RealTime CT/NG, the cobas® 4800 CT/NG, and the Cepheid Xpert® CT/NG, for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in vaginal swabs collected prospectively from pregnant women aged gonorrhoeae, the overall agreement was 100%. All kits allowed prompt and specific results for C. trachomatis and N. gonorrhoeae in young pregnant women.

  13. Blood donor screening for West Nile virus (WNV) revealed acute Usutu virus (USUV) infection, Germany, September 2016

    Science.gov (United States)

    Cadar, Daniel; Maier, Philipp; Müller, Susanne; Kress, Julia; Chudy, Michael; Bialonski, Alexandra; Schlaphof, Alexander; Jansen, Stephanie; Jöst, Hanna; Tannich, Egbert; Runkel, Stefan; Hitzler, Walter E; Hutschenreuter, Gabriele; Wessiepe, Martina; Schmidt-Chanasit, Jonas

    2017-01-01

    Between 1 June and 31 December 2016, 13,023 blood donations from the University Hospital Aachen in Germany were routinely screened for West Nile virus (WNV) RNA using the cobas TaqScreen WNV Test. On 28 September 2016, one blood donor was tested positive. Subsequent analysis revealed an acute Usutu virus (USUV) infection. During the ongoing USUV epizootics in Germany, blood transfusion services, public health authorities and clinicians should be aware of increased human USUV infections. PMID:28422005

  14. Safety and Immunogenicity of a Live-Attenuated Junin (Argentine Hemorrhagic Fever) Vaccine in Rhesus Macaques

    Science.gov (United States)

    1993-01-01

    immunization of the MATERIALS AND METHODS at-risk population offers the only practical so- Iltron for control of the disease. Attempts to develop a...saphenous or femoral veins using a 23- or 21 -gauge butterfly Materials for direct quantitative viral titration needle. Venipuncture sites were cleansed...21702-5011. 14. Contigiani MS. Sabattini MS. 1977. Virulencia diferencial de cepas de virus Junin per marca- dores biologicos en ratones % coba’.os

  15. PENGEMBANGAN INSTRUMEN ASESMEN BERPIKIR KRITIS UNTUK SISWA SMP KELAS VII PADA MATERI INTERAKSI MAKHLUK HIDUP DENGAN LINGKUNGAN

    Directory of Open Access Journals (Sweden)

    Dharmawati Dharmawati

    2016-08-01

    Full Text Available This study aims to generate critical thinking assessment instrument for seven graders within organism interaction in an environment with an appropriate validity and reliability level. The design of this instrument is developed using stages suggested by Borg and Gall. The stages are researching and collecting information, planning, developing a preliminary product, conducting the limited examination, revising product from limited examination, field testing, revising product from field testing, and finalizing product. The result of content and construction validation shows that the level of feasibility is 88,35% and categorized as very feasible. While the degree of readability of assessment items is 93,51% and it is categorized as excellent. The coefficient of inter-rater reliability of assessment items is 0,951 and categorized as excellent. The instrument acquires reliability coefficient 0,792 for multiple choice and 0,753 for essay items. This study shows that the content, construction, and items in assessment are feasible. Penelitian ini bertujuan untuk menghasilkan instrumen asesmen berpikir kritis untuk siswa SMP kelas VII pada materi interaksi makhluk hidup dengan lingkungan dengan tingkat validitas dan reliabilitas yang memadai. Rancangan penelitian menggunakan model pengembangan menurut Borg & Gall, yang meliputi langkah-langkah: penelitian dan pengumpulan informasi, perencanaan, pengembangan produk awal, uji coba terbatas, revisi produk uji coba terbatas, uji coba lapangan, revisi uji coba produk lapangan, dan penyempurnaan produk akhir. Berdasarkan hasil validasi isi dan konstruk diperoleh tingkat kelayakan produk sebesar 88,35% berada pada kriteria sangat layak. Tingkat keterbacaan soal asesmen sebesar 93,51% dengan kategori sangat baik. Koefisien inter-rater reliability pada soal asesmen bentuk penugasan sebesar 0,951 dengan kategori sangat baik. Instrumen tersebut mempunyai koefisien reliabilitas sebesar 0,792 (soal pilihan ganda dan

  16. Blood-Banking Techniques for Plateletpheresis in Swine

    Science.gov (United States)

    2014-05-01

    mL Fatal Plus, Vortech Pharmaceuticals , Dearborn, MI) while under surgical anesthesia. Validation procedures. Plateletpheresed platelets. The AABB...blood gas analysis (COBAS b221 Blood Analyzer System, Roche Diagnostics, Indianapolis, IN), assessment of platelet function by using...an in vivo energy substrate. Biochim Biophys Acta 842:214–224. 43. Yuasa T, Ohto H, Yasunaga R, Kai T, Shirahama N, Ogata T. 2004. Improved

  17. Benefit of Hepatitis C Virus Core Antigen Assay in Prediction of Therapeutic Response to Interferon and Ribavirin Combination Therapy

    OpenAIRE

    Takahashi, Masahiko; Saito, Hidetsugu; Higashimoto, Makiko; Atsukawa, Kazuhiro; Ishii, Hiromasa

    2005-01-01

    A highly sensitive second-generation hepatitis C virus (HCV) core antigen assay has recently been developed. We compared viral disappearance and first-phase kinetics between commercially available core antigen (Ag) assays, Lumipulse Ortho HCV Ag (Lumipulse-Ag), and a quantitative HCV RNA PCR assay, Cobas Amplicor HCV Monitor test, version 2 (Amplicor M), to estimate the predictive benefit of a sustained viral response (SVR) and non-SVR in 44 genotype 1b patients treated with interferon (IFN) ...

  18. Analytical case reports: major discrepancy between digoxin immunoassay results in a context of acute overdose

    Institute of Scientific and Technical Information of China (English)

    OlivierTRIBUT; HerveALLAIN; DanieleBENTUE-FERRER

    2004-01-01

    AIM: Digoxin, a heteroglycoside drug with cardiotonic effects,has a narrow therapeutic range, so therapeutic drug monitoring has been very helpful in improving patient management and preventing toxic effects. We report two cases of digoxin intoxication where the immunoassay techniques used for drug monitoring produced very different values with important clinical implications. METHODS: Serum levels of digoxin were determined with several analytical immunoassay techniques [Cobas

  19. Development of Quantitative Real-time Polymerase Chain Reaction for the Detection of Vibrio vulnificus Based on Hemolysin (vvhA) Coding System

    Institute of Scientific and Technical Information of China (English)

    ZENG-HUI WU; YONG-LIANG LOU; YI-YU LU; JIE YAN

    2008-01-01

    To establish a TaqMan real-time fluorescent quantitative PCR to detect Vibrio vulnificus based on the hemolysin gene (vvhA) coding cytolysin. Methods Primers and probes in the conserved region of the vvhA gene sequence were designed for the TaqMan real-time PCR to detect 100 bp amplicon from V. vulnificus DNA. Recombinant plasmid pMD19-vvhA100 was constructed and used as a positive control during the detection. Minimal amplification cycles (Ct value) and fluorescence intensity enhancement (△Rn value) were used as observing indexes to optimize the reaction conditions of TaqMan real-time PCR. The TaqMan assay for the detection of Vbirio vulnificus was evaluated in pure culture, mice tissue which artificially contaminated Vibrio vulnificus and clinical samples. Results The established TaqMan real-time PCR showed positive results only for Vibrio vulnificus DNA and pMD19-vvhA100. The standard curve was plotted and the minimum level of the vvhA target from the recombinant plasmid DNA was 10 copies with a Ct value of 37.94±0.19, as the equivalent of 0.01 ng purified genomic DNA of Vibrio vulnificus. The results detected by TaqMan PCR were positive for the 16 clinical samples and all the specimens of peripheral blood and subcutaneous tissue of mice which were infected with Vibrio vulnificus. Conclusion TaqMan real-time PCR is a rapid, effective, and quantitative tool to detect Vibro vulnificus, and can be used in clinical laboratory diagnosis of septicemia and wound infection caused by Vibrio vulnificus.

  20. PENGEMBANGAN MULTIMEDIA PEMBELAJARAN INTERAKTIF MENGENAL ANGKA DAN HURUF UNTUK ANAK USIA DINI

    Directory of Open Access Journals (Sweden)

    Lovandri Dwanda Putra

    2015-12-01

    Full Text Available Penelitian ini bertujuan untuk menghasilkan produk multimedia pembelajaran Mengenal Angka dan Huruf untuk Anak Usia Dini yang layak ditinjau dari aspek materi, aspek pembelajaran, aspek tampilan dan aspek pemrograman. Penelitian ini dilaksanakan di TK Aisyiyah Bustanul Athfal, Pringwulung, Yogyakarta dengan menggunakan metode penelitian dan pengembangan (R&D. Model pengembangan yang digunakan dalam penelitian ini adalah model Lee dan Owens. Tahapan dalam penelitian ini terdiri dari: (1 tahap perencanaan, (2 tahap desain dan (3 tahap pengembangan. Hasil penelitian menunjukkan: produk multimedia yang dihasilkan adalah multimedia pembelajaran mengenal angka (1-10 dan huruf (A-Z untuk anak usia dini, produk multimedia yang dihasilkan layak sebagai media pembelajaran mengenal angka dan huruf berdasarkan validasi oleh ahli materi dan ahli media. Kelayakan produk berdasarkan validasi ahli materi dengan hasil penilaian sangat baik (4,66, validasi oleh ahli media dengan hasil penilaian sangat baik (5 dan penilaian oleh anak pada uji coba satu-satu, uji coba kelompok kecil dan uji coba operasional dengan hasil sangat baik, hal ini dibuktikan dengan rerata masing-masing aspek yang dicapai adalah di atas 81%. Hasil penilaian oleh anak mencakup aspek kemenarikan, kemudahan dan kejelasan petunjuk materi.

  1. Pengembangan Program Macromedia Flash 8 untuk Pembelajaran Fisika di SMA

    Directory of Open Access Journals (Sweden)

    Wiji Susilowati

    2007-12-01

    Full Text Available Tujuan penelitian adalah membuat dan mengevaluasi media pembelajaran fisika SMA menggunakan program Macromedia Flash 8. Subjek coba pada penelitian ini berjumlah 40 orang yang terdiri dari 10 siswa untuk uji coba instrumen penelitian, 10 siswa untuk ujicoba kelompok kecdl, dan 20 siswa untuk uji coba lapangan. Pengumpulan data dilakukan dengan mengguna­kan kuesioner dan tes fisika (pre-test dan post-test. Evaluasi terhadap program ditinjau dari aspek pembelajaran dan aspek media. Kritik dan saran digunakan untuk memperbaiki program. Hasil penelitian menunjukkan bahwa program Macromedia Flash 8 untuk pembelajaran fisika di SMA: (1 dirancang sesuai dengan prinsip-prinsip desain pembelajaran, (2 terbukti mampu menaikkan skor rerata tes fisika siswa sebesar 55,42%, (3 memiliki efisiensi waktu yang tinggi, (4 ditinjau dari aspek media Hinilai cukup bagus/menarik (dinilai/dikategorikan sedang oleh siswa, (5 ditinjau dari aspek pembelajaran secara keseluruhan program ini dinilai tinggi/jelas dimengerti oleh siswa, (6 ditinjau dari aspek media dinilai menarik (bagus/kategori tinggi oleh guru fisika, dan (7 ditinjau dari aspek pembelajaran juga dinilai menarik (bagus/kategori tinggi oleh guru fisika. Kata kunci: media pembelajaran fisika diSMA.

  2. PENGEMBANGAN PERANGKAT PEMBELAJARAN BERBASIS MASALAH UNTUK PENINGKATAN CAPAIAN KOMPETENSI FISIKA UMUM II MAHASISWA PRODI PENDIDIKAN FISIKA FMIPA UNIVERSITAS NEGERI MEDAN

    Directory of Open Access Journals (Sweden)

    Jurubahasa Sinuraya

    2014-06-01

    kegiatan mahasiswa berbasis masalah (LKMBM Fisika Umum II. Penelitian ini termasuk jenis penelitian R & D, namun untuk menghasilkan perangkat pembelajaran LKMBM digunakan model disain Dick dan Carey. Struktur isi LKMBM Fisika Umum II ini merupakan implementasi dari konsep pembelajaran berbasis masalah, dan konsep strategi pemecahan masalah. Pelaksanaan penelitian ini dibagi atas tiga tahapan yaitu tahap perencanaan, pengembangan, dan evaluasi formatif. Pelaksananaan evaluasi formatif dilaksanakan melalui tiga tahapan, yaitu: uji validasi oleh 3 orang reviewer, uji coba satu satu melibatkan 3 orang mahasiswa, uji coba kelompok kecil melibatkan 15 orang mahasiswa. Berdasarkan hasil analisis data dan pembahasan, hasil penelitian ini adalah: (1 seperangkat lembar kegiatan mahasiswa berbasis masalah (LKMBM Fisika Umum II yang ditata dalam 10 komponen, yaitu: (a judul kegiatan, (b tujuan pembelajaran, (c permasalahan, (d hipotesis, (e pengumpulan data, (f pembahasan, (g simpulan, (h daftar pustaka, (i lampiran, dan (j tes kompetensi; 2 hasil uji coba LKMBM beserta perangkat perangkat pendukungnya dengan melibatkan para ahli dan mahasiswa sebagai pengguna LKMBM, secara keseluruhan sudah memberikan penilaian yang baik, layak digunakan dalam perbaikan pembelajaran fisika umum.

  3. PENGEMBANGAN SSP TEMATIK INTEGRATIF UNTUK MEMBANGUN KARAKTER KEJUJURAN DAN KEPEDULIAN SISWA SD KELAS II

    Directory of Open Access Journals (Sweden)

    Sri Hariyati Qodriyah

    2015-07-01

    Full Text Available Penelitian ini bertujuan untuk menghasilkan perangkat pembelajaran berupa SSP Tematik yang dapat mengembangkan karakter siswa kelas 2 sekolah dasar, meliputi karakter kejujuran dan kepedulian. Penelitian ini merupakan penelitian dan pengembangan yang terdiri dari tujuh tahap, yaitu studi pendahuluan, perencanaan, mengembangkan produk awal, uji coba awal, revisi produk utama, uji coba lapangan, dan revisi terhadap produk operasional. SSP yang dikembangkan dievaluasi oleh seorang ahli materi dan media untuk mengetahui validitasnya. Subjek uji coba berjumlah 90 siswa terdiri dari 23 siswa SD Sonosewu, Kasihan, Bantul, DIY sebagai subjek uji coba terbatas dan 33 siswa untuk kelas control dan 34 siswa untuk kelas eksperimen SD 1 Kadipiro, Kasihan, Bantul, DIY. Hasil penelitian ini berupa SSP yang meliputi: silabus, RPP, LKS, dan lembar penilaian. Hasil evaluasi dari ahli materi dan ahli media untuk menguji tingkat kevalidan SSP menyatakan bahwa SSP yang dikembangkan adalah valid dan berkategori “baik”. Hasil uji coba menunjukkan bahwa SSP yang dikembangkan dinyatakan layak serta memenuhi kriteria praktis dan efektif dalam mengembangkan karakter siswa. Pembelajaran dengan SSP yang dikembangkan mampu mengembangkan karakter kejujuran dan kepedulian. Kata Kunci: SSP, karakter jujur dan peduli   DEVELOPING THEMATIC INTEGRATIVE SSP FOR BUILDING THE CHARACTERS OF HONESTY AND CARE GRADE II STUDENT OF ELEMENTARY SCHOOL Abstract This study aims to produce integrated learning sets in the form of thematic SSP that can develop the characters of honesty and care of grade II students of elementary school. This was a research and development study consisting of seven steps, i.e. preliminary study, planning, preliminary product development, preliminary tryout, main product revision, field tryout, revision of operational product, operational product revision, and revision of the final product. The developed SSP was evaluated by a science subject expert to assess

  4. Dynamique institutionnelle des transferts de gestion dans le corridor Fandriana-Vondrozo

    Directory of Open Access Journals (Sweden)

    Thierry Ganomanana

    2011-06-01

    Full Text Available Ten years after their creation, the operation of the community-based natural resource management policy named ‘transfert de gestion’ – the 1996 GELOSE law (applied to any kind of natural resources, and the 2001 GCF decree (only applied to forests – remains little understood. The forest corridor linking Ranomafana and Andringitra National Parks has been extended south, and since 2006 the Fandriana-Vondrozo Corridor has been established as a new protected area within the Madagascar Protected Area System. Eighty-two sites of transfert de gestion have been created since 2001 in the Fandriana-Vondrozo Corridor and Randriamahaleo III are managed by local community associations named COBA. Management is determined by law but is locally adapted to each site. We analyze the dynamic of the community forest management system using six variables and 19 modes: year of creation (five modes, legal form of management (two modes, locality (two modes, principal NGO partners (five modes, management objective (two modes, and surface area of the transferred site (three modes. There are four institutions in charge of forest management: local community associations (COBA, which manage the forest in their territory; the Forest Service (Eaux et Forêt representing the State, which controls this management; the commune, the smallest decentralized unit of the State, which manages the whole communal territory including forests; and NGOs, which facilitate the process. In order to investigate institutional tendencies, the sites of transfert de gestion are analyzed using Multiple Correspondence Analysis. The sites are distributed in the first two factorial plans with percentage variance of 18.5 % and 15.8 % according to their objectives: 1 pure conservation, and 2 economic development. Three types of NGO are distinguished by the form and duration of their support to COBA. The NGOs with greatest weight in the process are those which support the transfert de gestion

  5. PENGEMBANGAN PERANGKAT PEMBELAJARAN KALKULUS UNTUK MENCAPAI KETUNTASAN DAN KEMANDIRIAN BELAJAR SISWA

    Directory of Open Access Journals (Sweden)

    Raekha Azka

    2015-05-01

    Full Text Available Penelitian ini bertujuan untuk (1 mengembangkan perangkat pembelajaran kalkulus MA yang meliputi silabus, RPP, LKS, dan instrumen penilaian untuk mencapai ketuntasan belajar dan keman-dirian belajar; (2 mendeskripsikan kualitas hasil pengembangan perangkat pembelajaran kalkulus MA untuk mencapai ketuntasan belajar dan kemandirian belajar. Penelitian ini merupakan penelitian pengembangan yang mengembangkan perangkat pembelajaran kalkulus dengan menggunakan model pengembangan yang diadaptasi dari model pengembangan Thiagarajan, Semmel, dan Semmel. Tahap-tahap yang dilalui sampai diperoleh perangkat pembelajaran kalkulus yang valid, praktis, dan efektif meliputi: (1 tahap pendefinisian, (2 tahap perencanaan, dan (3 tahap pengembangan. Uji coba yang dilakukan meliputi uji coba ahli/validasi ahli, uji coba terbatas, dan uji coba lapangan. Uji coba lapangan dilakukan di MA PK Ma’arif 01 Kebumen pada dua kelas XI IPS. Penelitian ini menghasil-kan perangkat pembelajaran kalkulus untuk MA kelas XI IPS terdiri atas silabus, RPP, LKS, dan THB yang berkualitas dan layak digunakan dalam proses pembelajaran. Masing-masing komponen perangkat pembelajaran yang terdiri atas silabus, RPP, LKS, dan THB telah memenuhi kriteria valid, praktis, dan efektif untuk mencapai ketuntasan dan kemandirian belajar siswa. Kata kunci: pengembangan, perangkat pembelajaran, kalkukus, ketuntasan belajar, kemandirian belajar.   DEVELOPING A CALCULUS TEACHING PACKAGE TO ACHIEVE MASTERY AND SELF-REGULATED LEARNING Abstract This research aims to: (1 develop a calculus teaching package consisting of syllabus, lesson plan, worksheet, and test of Islamic high school to achieve mastery and self-regulated learning; (2 describe the quality of the calculus teaching in Islamic high school to achieve mastery and self-regulated learning. This research is a developmental research which develops calculus teaching package using the development model adapted from Thiagarajan, Semmel, and Semmel

  6. Development and evaluation of a Quadruplex Taq Man real-time PCR assay for simultaneous detection of clinical isolates of Enterococcus faecalis, Enterococcus faecium and their vanA and vanB genotypes.

    Directory of Open Access Journals (Sweden)

    Taghi Naserpour Farivar

    2014-10-01

    Full Text Available We developed and evaluated the utility of a quadruplex Taqman real-time PCR assay that allows simultaneous identification of vancomycin-resistant genotypes and clinically relevant enterococci.The specificity of the assay was tested using reference strains of vancomycin-resistant and susceptible enterococci. In total, 193 clinical isolates were identified and subsequently genotyped using a Quadruplex Taqman real-time PCR assay and melting curve analysis. Representative Quadruplex Taqman real-time PCR amplification curve were obtained for Enterococcus faecium, Enterococcus faecalis, vanA-containing E. faecium, vanB-containing E. faecalis.Phenotypic and genotypic analysis of the isolates gave same results for 82 enterococcal isolates, while in 5 isolates, they were inconsistent. We had three mixed strains, which were detected by the TaqMan real-time PCR assay and could not be identified correctly using phenotypic methods.Vancomycin resistant enterococci (VRE genotyping and identification of clinically relevant enterococci were rapidly and correctly performed using TaqMan real-time multiplex real-time PCR assay.

  7. Detection and differentiation of Cryptosporidium by real-time polymerase chain reaction in stool samples from patients in Rio de Janeiro, Brazil

    Directory of Open Access Journals (Sweden)

    Roberta Flávia Ribeiro Rolando

    2012-06-01

    Full Text Available This study reports the first genetic characterisation of Cryptosporidium isolates in Brazil using real-time polymerase chain reaction (RT-PCR. A total of 1,197 faecal specimens from children and 10 specimens from human immunodeficiency virus-infected patients were collected between 1999-2010 and screened using microscopy. Forty-eight Cryptosporidium oocyst-positive isolates were identified and analysed using a generic TaqMan assay targeting the 18S rRNA to detect Cryptosporidium species and two other TaqMan assays to identify Cryptosporidium hominis and Cryptosporidium parvum. The 18S rRNA assay detected Cryptosporidium species in all 48 of the stool specimens. The C. parvum TaqMan assay correctly identified five/48 stool samples, while 37/48 stool specimens were correctly amplified in the C. hominis TaqMan assay. The results obtained in this study support previous findings showing that C. hominis infections are more prevalent than C. parvum infections in Brazil and they demonstrate that the TaqMan RT-PCR procedure is a simple, fast and valuable tool for the detection and differentiation of Cryptosporidium species.

  8. 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...

  9. 荧光定量PCR检测细菌NDM-1基因方法的探讨%Application of real-time fluorescent quantitative PCR in NDM-1 detection of bacteria

    Institute of Scientific and Technical Information of China (English)

    张建明; 黄吉城; 吴健; 王向阳; 何洪涛; 陈姗; 师永霞; 李小波; 洪烨

    2013-01-01

    Objective:In this study, TaqmanRT - PCR and SYBRGreenRT - PCR were applied for quantification of NDM - 1 gene in bacteria. Methods: After detection of NDM - 1 gene with Taqman RT - PCR and SYBRGreenRT - PCR, the sensititity was compared. Results: TaqmanRT - PCR was more sensitive and the resultant concentration of RNA level was up to 7.5 × 10 copies/μ1. Compared with SYBR Green RT - PCR, it was up to 7. 8 - fold higher when determined by TaqMan RT - PCR. Conclusion: The detection of NDM - 1 gene in bacteria with TaqmanRT - PCR was proved to be more sensitive and accurate, so Taqman RT - PCR could be suggested as a way to prevent and control super bacteria.%目的:将Taqman荧光定量PCR技术与SYBRGreen染料RT-PCR技术比较后,应用于检测细菌NDM-1基因.方法:使用Taqman荧光定量PCR方法与SYBRGreen染料RT-PCR对已知特性的标准株中NDM-1基因检测,对比其扩增灵敏度.结果:Taqman荧光定量PCR技术灵敏度高于SYBRGreen染料RT-PCR法,所测标准株中NDM-1基因转录水平可高达7.5×104拷贝/μl,比SYBRGreen染料RT-PCR技术检测灵敏度高7.8倍.结论:Taqman荧光PCR技术对超级耐药菌NDM-1基因具有较高的敏感性和准确性,可以作为对超级细菌进行监控与防控的方法.

  10. PENGEMBANGAN MODEL PEMBELAJARAN MALCOLM’S MODELING UNTUK MENINGKATKAN KETERAMPILAN BERPIKIR KRITIS DAN MOTIVASI BELAJAR SISWA

    Directory of Open Access Journals (Sweden)

    Syarifah Syarifah

    2015-10-01

    Full Text Available Penelitian ini bertujuan untuk: (1 menghasilkan model pembelajaran fisika berbasis Malcom’s Modeling Method yang layak digunakan di sekolah, dan (2 mengetahui apakah model pembelajaran fisika berbasis Malcom’s Modeling Method dapat meningkatkan keterampilan berpikir kritis dan motivasi belajar siswa. Penelitian ini termasuk dalam ranah penelitian dan pengembangan (R&D. Prosedur pengembangan mengadaptasi dari prosedur pengembangan yang dikembangkan oleh Borg & Gall dengan langkah-langkah meliputi (1 penelitian dan pengumpulan data, (2 perencanaan, (3 pengembangan bentuk awal produk, (4 uji coba lapangan awal, (5 revisi hasil uji coba lapangan awal, (6 uji coba lapangan, (7 revisi hasil uji coba lapangan dan (8 diseminasi. Subjek uji coba lapangan awal terdiri atas 36 siswa kelas X MIA 6 di SMA N 7 Yogyakarta. Subjek uji coba lapangan pada kelas ekperimen terdiri atas 36 orang siswa kelas X MIA 1 dan pada kelas kontrol terdiri atas 34 orang siswa kelas X MIA 5 di SMA N 7 Yogyakarta. Instrumen pengumpulan data menggunakan soal untuk mengukur keterampilan berpikir kritis, angket untuk mengukur motivasi belajar, angket respon siswa dan lembar observasi keterlaksanaan pembelajaran. Teknik analisis data menggunakan uji MANOVA dengan taraf signifikansi 5%. Hasil penelitian menunjukkan bahwa model Malcom’s Modeling Method ditinjau dari sintaks, sistem sosial, prinsip reaksi, sistem pendukung dan dampak instruksional dan pengiring layak digunakan di sekolah dengan kategori sangat baik. Hasil uji MANOVA menunjukkan model Malcom’s Modeling Method dapat meningkatkan keterampilan berpikir kritis dan motivasi belajar siswa pada taraf signifikansi 5 %. Kata Kunci: Malcom’s Modeling Method, keterampilan berpikir kritis, motivasi belajar.   DEVELOPING A PHYSICS INSTRUCTION MODEL BASED ON MALCOLM’S MODELING TO IMPROVE CRITICAL THINKING SKILLS AND LEARNING MOTIVATION Abstract This research aims to (1 develop a physics instruction model based on

  11. MODEL PERMAINAN AKTIVITAS LUAR KELAS UNTUK MENGEMBANGKAN RANAH KOGNITIF, AFEKTIF, DAN PSIKOMOTORIK SISWA SMA

    Directory of Open Access Journals (Sweden)

    Hendra Setyawan

    2015-10-01

    Full Text Available Penelitian ini bertujuan menghasilkan model-model permainan Aktivitas Luar kelas (ALK yang layak digunakan sebagai materi pembelajaran penjas, serta efektif untuk mengembangkan ranah kognitif, afektif, dan psikomotorik siswa SMA. Penelitian ini dilakukan dengan langkah-langkah sebagai berikut: (1 pengumpulan informasi di lapangan, (2 menganalisis informasi yang telah dikumpulkan, (3 mengembangkan produk awal, (4 validasi ahli dan revisi, (5 uji coba skala kecil dan  revisi, (6 uji coba skala besar dan revisi, dan (7 pembuatan produk final. Uji coba skala kecil dilakukan terhadap siswa kelas X-D dan XI-IPA 4 dari SMA N I Prambanan berjumlah 66 orang. Uji coba skala besar dilakukan terhadap siswa kelas X-G, X-H, XI-IPS 3, dan XI-BHS dari SMA N I Prambanan berjumlah 132 orang. Instrumen pengumpulan data yang digunakan yaitu; (1 pedoman wawancara, (2 skala nilai, (3 pedoman observasi permainan, (4 pedoman observasi keefektifan permainan, (5 pedoman observasi terhadap guru pelaku uji coba, dan (6 rubrik penilaian siswa. Teknik analisis data dengan analisis deskriptif kuantitatif dan kualitatif. Hasil penelitian berupa tujuh model permainan yang layak dan efektif digunakan dalam pembelajaran penjas, hal ini dibuktikan dengan perolehan persentase sebesar 100% dari data hasil kuesioner skala nilai, observasi model permainan, observasi keefektifan permainan, dan observasi guru pelaku uji coba. Model permainan juga efektif untuk mengembangkan ranah kognitif, afektif, dan psikomotorik, yang dibuktikan dengan terjadinya peningkatan persentase nilai rata-rata siswa sebesar 21%. Kata kunci: model, permainan, aktivitas luar kelas, kognitif, afektif, psikomotorik THE OUTDOOR ACTIVITY GAME MODEL TO DEVELOP THE ASPECT OF COGNITIVE, AFFECTIVE, AND PSYCHOMOTOR OF THE SENIOR HIGH SCHOOL STUDENTS Abstract This study aims at producing outdoor activity game models which are proper to be used for physicall education learning and developing cognitive, affective, and

  12. Outliers as a cause of false cardiac troponin results: investigating the robustness of 4 contemporary assays.

    Science.gov (United States)

    Pretorius, Carel J; Dimeski, Goce; O'Rourke, Peter K; Marquart, Louise; Tyack, Shirley A; Wilgen, Urs; Ungerer, Jacobus P J

    2011-05-01

    It is important that cardiac troponin be measured accurately with a robust method to limit false results with potentially adverse clinical outcomes. In this study, we characterized the robustness of 4 analytical platforms by measuring the outlier rate between duplicate results. We measured cardiac troponin concurrently in duplicate with 4 analyzers on 2391 samples. The outliers were detected from the difference between duplicate results and by calculating a z value: z = (result 1 - result 2) ÷ √(SD1(est)² + SD2(est)²), with z > 3.48 identifying outliers with a probability of 0.0005. The outlier rates were as follows: Abbott Architect i2000SR STAT Troponin-I, 0.10% (0.01%-0.19%); Beckman Coulter Access2 Enhanced AccuTnI, 0.44% (0.25%-0.63%); Roche Cobas e601 TroponinT hs, 0.06% (0.00%-0.13%); and Siemens ADVIA Centaur XP TnI-Ultra, 0.10% (0.01%-0.19%). The occurrence of outliers was higher than statistically expected on all platforms except the Cobas e601 (χ² = 2.7; P = 0.10). A conservative approach with a constant 10% CV and z > 5.0 identified outliers with clear clinical impact and resulted in outlier rates of 0.11% (0.02%-0.20%) with the Architect i2000SR STAT Troponin-I, 0.36% (0.19%-0.53%) with the Access2 Enhanced AccuTnI, 0.02% (0.00%-0.06%) with the Cobas e601 TroponinT hs, and 0.06% (0.00%-0.13%) with the ADVIA Centaur XP TnI-Ultra. Outliers occurred on all analytical platforms, at different rates. Clinicians should be made aware by their laboratory colleagues of the existence of outliers and the rate at which they occur.

  13. DUA PULUH LIMA TAHUN KERJA SAMA DEPKES RI DENGAN NAMRU-2

    Directory of Open Access Journals (Sweden)

    Suriadi Gunawan

    2012-09-01

    Full Text Available Buletin nomor ini diterbitkan untuk memperingati 25 tahun kerja sama antara Departemen Kesehatan RI dan U.S. Naval Medical Research Unit No. 2 (NAMRU-2. Kerja sama ini telah menghasilkan sumbangan yang sangat berharga untuk peningkatan kesehatan baik di Indonesia maupun di dunia, khususnya di negara sedang berkembang. Hasil evaluasi vaksin typhoid dan cara pengobatan malaria serta dehidrasi berat akibat diare adalah beberapa contoh dari kerja sama ini yang sangat bermanfaat untuk mencegah penyakit dan kematian. Hasil kerja sama dalam 20 tahun pertama telah diseminarkan dalam tahun 1990 dan dipublikasikan dalam suatu nomor khusus buletin ini. Dengan staf yang terdiri dari 20 orang asing, dan 140 orang Indonesia, NAMRU-2 telah melaksanakan berbagai penelitian di berbagai daerah bersama peneliti Badan Litbangkes, Universitas, Angkatan Bersenjata serta dinas kesehatan setempat. Lebih dari 400 publikasi ilmiah telah dihasilkan oleh kerja sama ini. Dalam lima tahun terakhir penelitian berbagai aspek malaria di Irian Jaya antara lain telah menghasilkan peta resistensi obat malaria dan penemuan manfaat primakuin sebagai obat profilaksis yang aman dan relatif murah. Uji coba fase III vaksin tifoid oral Ty21a telah dilaksanakan di Sumatra Selatan, sedangkan suatu vaksin kolera oral, CVD 103 HgR, sedang diuji coba fase III di Jakarta. Penelitian genotype HIV di Indonesia yang telah dilaksanakan bersama Universitas Indonesia dan Dinas Kesehatan ABRI telah membantu memperjelas epidemi HIV/AIDS di Indonesia. Penelitian lapangan mengenai hepatitis E di Kalimantan dan Japanese Encephalitis di Bali telah memperjelas penularan dan risiko penyakit tersebut. Badan Litbangkes dan NAMRU akan melanjutkan kerjasama penelitian dan pelatihan di bidang penyakit menular dalam masa lima tahun yang akan datang. Beberapa bidang yang akan mendapat perhatian ialah surveilans berbagai penyakit infeksi yang baru dan timbul kembali (new and reemerging infections, uji coba berbagai

  14. DUA PULUH LIMA TAHUN KERJA SAMA DEPKES RI DENGAN NAMRU-2

    Directory of Open Access Journals (Sweden)

    Suriadi Gunawan

    2012-09-01

    Full Text Available Buletin nomor ini diterbitkan untuk memperingati 25 tahun kerja sama antara Departemen Kesehatan RI dan U.S. Naval Medical Research Unit No. 2 (NAMRU-2. Kerja sama ini telah menghasilkan sumbangan yang sangat berharga untuk peningkatan kesehatan baik di Indonesia maupun di dunia, khususnya di negara sedang berkembang. Hasil evaluasi vaksin typhoid dan cara pengobatan malaria serta dehidrasi berat akibat diare adalah beberapa contoh dari kerja sama ini yang sangat bermanfaat untuk mencegah penyakit dan kematian. Hasil kerja sama dalam 20 tahun pertama telah diseminarkan dalam tahun 1990 dan dipublikasikan dalam suatu nomor khusus buletin ini. Dengan staf yang terdiri dari 20 orang asing, dan 140 orang Indonesia, NAMRU-2 telah melaksanakan berbagai penelitian di berbagai daerah bersama peneliti Badan Litbangkes, Universitas, Angkatan Bersenjata serta dinas kesehatan setempat. Lebih dari 400 publikasi ilmiah telah dihasilkan oleh kerja sama ini. Dalam lima tahun terakhir penelitian berbagai aspek malaria di Irian Jaya antara lain telah menghasilkan peta resistensi obat malaria dan penemuan manfaat primakuin sebagai obat profilaksis yang aman dan relatif murah. Uji coba fase III vaksin tifoid oral Ty21a telah dilaksanakan di Sumatra Selatan, sedangkan suatu vaksin kolera oral, CVD 103 HgR, sedang diuji coba fase III di Jakarta. Penelitian genotype HIV di Indonesia yang telah dilaksanakan bersama Universitas Indonesia dan Dinas Kesehatan ABRI telah membantu memperjelas epidemi HIV/AIDS di Indonesia. Penelitian lapangan mengenai hepatitis E di Kalimantan dan Japanese Encephalitis di Bali telah memperjelas penularan dan risiko penyakit tersebut. Badan Litbangkes dan NAMRU akan melanjutkan kerjasama penelitian dan pelatihan di bidang penyakit menular dalam masa lima tahun yang akan datang. Beberapa bidang yang akan mendapat perhatian ialah surveilans berbagai penyakit infeksi yang baru dan timbul kembali (new and reemerging infections, uji coba berbagai

  15. Evaluation of Abbott RealTime CT/NG assay for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in cervical swabs from female sex workers in China.

    Directory of Open Access Journals (Sweden)

    Yan Han

    Full Text Available BACKGROUND: To evaluate the performance of the Abbott RealTime CT/NG assay for detection of Chlamydia trachomatis (CT and Neisseria gonorrhoeae (NG among female sex workers (FSWs in China. METHODS: Cervical swabs from 997 participants were blindly detected by the Abbott RealTime CT/NG assay on the automated m2000 molecular platform and Roche Cobas Amplicor CT/NG assay. Discrepant analysis were confirmed by the Qiagen care CT PCR assay. The sample was defined as candidate nvCT-positive if it was CT positive in the Abbott m2000 assay, but CT negative in the other two assays. RESULTS: 25 specimens that were discordant for CT and 26 specimens that were discordant for NG between the two assays were resolved by Qiagen care CT & NG PCR assays. The sensitivity and specificity, respectively, for Abbott m2000 assay were 92.59% and 100% for CT and 95.45% and 99.90% for NG. The positive predictive value (PPV and negative predictive value (NPV of Abbott m2000 assay were100% and 98.52% for CT and 95.5% and 99.90% for NG, respectively. No candidate new-variant CT(nvCTspecimens were identified. CONCLUSION: Abbott RealTime CT/NG assay were more specify for CT and NG detection, however, its sensitivity for CT and NG were a little bit lower than Roche Cobas Amplicor CT/NG assay. Abbott RealTime CT/NG assay had higher PPV for NG detection than Roche Cobas Amplicor CT/NG assay; it would be more suitable for screening for population with low-prevalence NG. There is currently no evidence that nvCT is present in FSWs in China.

  16. Reference intervals for C-peptide and insulin derived from a general adult Danish population.

    Science.gov (United States)

    Larsen, Pia Bükmann; Linneberg, Allan; Hansen, Torben; Friis-Hansen, Lennart

    2017-05-01

    Despite international efforts to standardize C-peptide and insulin calibrators and immunoassays, platform dependent differences still exist, and platform specific reference intervals are hence needed for correct interpretation. We therefore wanted to establish traceable reference intervals for C-peptide and insulin. In 623 consecutively recruited participants, insulin and C-peptide were measured using the Cobas e411 (Roche Diagnostics, Switzerland). Participants with diabetes were excluded (fasting Glucose ≥7.0mmol/L or HbA1c≥6.5%/≥48mmol/L) and reference intervals were calculated with and without the inclusion of persons who were prediabetic, according to two definitions (The World Health Organization (WHO) and American Diabetes Association (ADA)). To ensure the correctness of calibration, the control pools were analyzed by a reference laboratory. The reference intervals were calculated according to the IFCC guidelines, using the RefVal software (Solberg, Oslo, Norway). Comparison of our results with those from the reference laboratory revealed equivalence for C-peptide results whereas the insulin determined on the Cobas e411 assay were 15-20% higher. The difference is attributed to an incorrect conversion factor for converting from activity to metric units. The Cobas e411 assay uses the factor 6.945 for converting from U/mL to pmol/L. This is in disagreement with the biological activity of insulin which is 166.8×10(6)IU/mol or 6.00nmol/IU. We successfully established reference intervals for C-peptide and insulin for non-diabetic and prediabetic participants. The reference intervals for fasting C-peptide and fasting insulin are ready for implementation. A recertification of the insulin standards is needed. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  17. 应用CLSI EP15-A2指南验证罗氏Cobase 601电化学发光分析仪检测精密度和准确度

    Institute of Scientific and Technical Information of China (English)

    王宏志; 黄金英; 余敏红; 马海玲; 赵晶

    2013-01-01

    目的:利用EPl5-A2对罗氏cobas e 601电化学发光分析仪检测精密度和准确度进行验证。方法精密度验证选用罗氏公司生产的TM 2个水平的质控品,每天重复测定4次,连续5天,分别计算批内不精密度和总不精密度,并与厂家声明的批内不精密度和总不精密度比较。准确度验证选择卫生部临检中心发放的肿瘤标志物室间质评物作为参考物质,选择其中2个水平质评物进行测定,每批重复2次,连续5天,计算均值、标准差及偏倚,与厂家给定的偏倚范围比较。结果罗氏cobas e 601电化学发光分析仪检测CA19-9的精密度和准确度结果均在厂家给定的范围内。结论罗氏cobas e 601电化学发光分析仪检测的精密度和准确度达到了厂家要求,可以用于临床检测。

  18. Hemoglobin variants detected by hemoglobin A1c (HbA1c) analysis and the effects on HbA1c measurements

    OpenAIRE

    Nasir, Nadzimah Mohd; Thevarajah, M; Yean, Chew Yee

    2010-01-01

    Background: Hemoglobin (Hb) A1c is a tool widely used to monitor long-term glycemic control in diabetic patients. The objective of our study is to compare the HbA1c values measured on high performance liquid chromatography (HPLC) and immunoassay in patients who were detected to have hemoglobin variant after HbA1c analysis. Materials and Methods: We compared the HbA1c values measured using the Arkray Adams A1c HA-8160 (HPLC method) and Roche Cobas Integra (immunoturbidimetric method) from diab...

  19. Reference intervals for C-peptide and insulin derived from a general adult Danish population

    DEFF Research Database (Denmark)

    Larsen, Pia Bükmann; Linneberg, Allan; Hansen, Torben

    2016-01-01

    BACKGROUND: Despite international efforts to standardize C-peptide and insulin calibrators and immunoassays, platform dependent differences still exist, and platform specific reference intervals are hence needed for correct interpretation. We therefore wanted to establish traceable reference...... intervals for C-peptide and insulin. METHODS: In 623 consecutively recruited participants, insulin and C-peptide were measured using the Cobas e411 (Roche Diagnostics, Switzerland). Participants with diabetes were excluded (fasting Glucose ≥7.0mmol/L or HbA1c≥6.5%/≥48mmol/L) and reference intervals were...

  20. CLINICAL PERFORMANCE CHARACTERISTICS OF ELECSYS® FREE-ΒHCG AND PAPP-A FOR FIRST TRIMESTER TRISOMY 21 RISK ASSESSMENT IN GESTATIONAL WEEKS 8+0 TO 14+0

    DEFF Research Database (Denmark)

    Tørring, Niels; Aulesa, C; Eiben, Bernd;

    2014-01-01

    Background Screening for fetal trisomy 21 (T21) in the first trimester includes analysis of the serological markers pregnancy-associated plasma protein A (PAPP-A) and free beta choriogonadotropin (free βhCG). With the launch of these assays on the cobas e and Elecsys platforms, we investigated...... their clinical and analytical performance. Patients and Methods We conducted a multicenter study in 5397 pregnancies including 108 cases with verified fetal T21 at 8 to 14 weeks of gestation. A technical validation of the Roche Elecsys® free βhCG and PAPP-A assays were performed, including method comparisons...

  1. PENGEMBANGAN MULTIMEDIAPEMBELAJARAN BAHASA JERMAN PELAJARAN PELAYANAN RESTORAN DI YOTABAKTI YOGYAKARTA

    Directory of Open Access Journals (Sweden)

    Melly Handayani

    2015-10-01

    Full Text Available Tujuan penelitian dan pengembangan ini adalah (1 mengembangkan multimedia yang berisi materi dan latihan untuk keterampilan berbicara bahasa Jerman, (2 mendeskripsikan proses pengem-bangannya, dan (3 mendeskripsikan hasil evaluasi dan hasil uji coba lapangan. Uji coba terbatas dilakukan pada satu orang pengajar dan empat orang peserta didik. Uji coba lapangan melibatkan 2 orang pengajar dan 10 orang peserta didik. Data dianalisis secara deskriptif kuantitatif. Langkah pengembangan multimedia pembelajaran bahasa Jerman yang digunakan adalah (1 analisis kebutuhan, (2 perencanaan, (3 pengembangan produk, (4 evaluasi produk, (5 revisi produk, (6 uji coba lapangan, dan (7 produk akhir. Kualitas multimedia pembelajaran bahasa Jerman menurut dosen ahli materi adalah sangat baik dan menurut ahli media adalah baik. Kualitas multimedia menurut dua orang pengajar bahasa Jerman adalah sangat baik dan menurut peserta didik adalah baik. Dari 10 orang peserta didik, 80% mencapai nilai KKM. 100% respon peserta didik terhadap multimedia yang dikembangkan adalah sangat baik. Kata Kunci: Pengembangan, multimedia, keterampilan berbicara, pelayanan restoran   DEVELOPING MULTIMEDIA FOR GERMAN LANGUAGE TEACHING MATERIAL FOR RESTAURANT SERVICE LESSONS IN YOTABAKTI YOGYAKARTA Abstract The objectives of this research and development are (1 to develop a learning media for German language teachers, a computer assisted media with learning materials and exercises for speaking skill, (2 to describe the process of developing Multimedia German Language Teaching Material for Restaurant Service Lessons, and ( 3 to describe the result of product evaluation and field try out. The small group tryout involved five participants, a German language teacher and four students of Yotabakti. Field try out involved two German language teachers and ten students. The data of this study were analysed descriptively and quantitatively. The procedures of developing multimedia German language

  2. Calidad y evaluación de la educación superior: situación actual y prospectiva

    OpenAIRE

    Apocada (Coord.), Pedro M.

    2015-01-01

    APORTACIONES:Calidad y evaluación de la educación superior: situación actual y prospectiva.Pedro M. Apodaca.La evaluación de la calidad de las universidades. Eduardo CobaEl marco español y europeo en las políticas de calidad. José Ginés MoraModelos académicos de evaluación y mejora en la enseñanza superior. Mario deMiguel DíazAlgunas cuestiones ante nuevas iniciativas evaluadoras. Sebastián Rodrí...

  3. Aprendizaje cooperativo. Un recurso indispensable en la formación universitaria

    OpenAIRE

    Cobas Cobiella, María Elena; Montes Rodríguez, María Pilar; Alventosa del Río, Josefina; Guillén Catalán, Raquel; Ortega Giménez, Alfonso; Marí Farinós, Jesús; Muñoz Pérez, David; Martínez Evora, Joan; Panadero de la Cruz, Catalina Edeltrudis; Vega Cardona, Raúl José; Company Alcañiz, Mireia; Rivera Rodón, Gretchen; Ordelín Font, Jorge Luis; Febles Pozo, Nayiber; García Juncos, María Antonia

    2016-01-01

    El Libro “Aprendizaje cooperativo Un recurso indispensable en la formación universitaria”, se enmarca dentro del Proyecto de Innovación docente Finestra Oberta UV_ SFPIE GER 15-314671, bajo la dirección de la Profesora María Elena Cobas Cobiella, del Departamento de Derecho Civil, de la Facultad de Derecho de la Universidad de Valencia. Este libro contiene 19 artículos inéditos de un grupo importante de profesores y especialistas en la temática nacionales e internacionales, así como c...

  4. MODEL PEMBELAJARAN NEUROLINGUISTIC PROGRAMMING BERORIENTASI KARAKTER BAGI PENINGKATAN KEMAMPUAN MENULIS SISWA SMP

    Directory of Open Access Journals (Sweden)

    Wikanengsih -

    2014-06-01

    Abstrak: Model Pembelajaran Neurolinguistic Programming Berorientasi Karakter untuk Mening­katkan Kemampuan Menulis Siswa. Penelitian ini bertujuan untuk mengetahui keefektifan model pem­belajaran neurolinguistik programming berorientasi karakter (MPNLPBK terhadap kemampuan menulis siswa. Metode penelitian yang digunakan metode penelitian kombinasi (mixed method jenis sequential exploratory strategy. Hasil penelitian tahap pertama (penelitian kualitatif menghasilkan model pembelajaran hipotetik. Penelitian tahap kedua merupakan uji coba penerapan model hipotetik (penelitian kuantitatif. Hasil pengujian menunjukkan bahwa pembelajaran menulis dengan menggunakan MPNLPBK dapat meningkatkan kemampuan menulis siswa kelompok eksperimen. Selain itu, berdasarkan hasil pengamatan terhadap aspek karakter komunikatif, toleran, tanggungjawab dan kreatif siswa, terdapat perkembangan pada diri siswa untuk setiap aspek tersebut.

  5. Effekten af sund skolekost på udvalgte blodparametre

    DEFF Research Database (Denmark)

    Horn, Peer Bendix; Brandslund, Ivan; Schmedes, Anne;

    2009-01-01

    blood before (week 39) and after the intervention (week 49). The intervention group received a healthy meal for two months (25-30% of the daily intake of calories). Blood samples were analyzed for 17 parameters related to carbohydrate, fat and protein metabolism as well as vitamins and minerals. RESULTS...... nmol/l. In week 49, more than 94% of the pupils were lower than 80 nmol/l, and they generally had low calcium values. CONCLUSION: The intervention group showed significant alterations in TSH, CA, HB, COBA and CREA values from the start to the end of the intervention period compared with the control...

  6. [Pediatric reference intervals : retrospective study on thyroid hormone levels].

    Science.gov (United States)

    Ladang, A; Vranken, L; Luyckx, F; Lebrethon, M-C; Cavalier, E

    2017-01-01

    Defining reference range is an essential tool for diagnostic. Age and sexe influences on thyroid hormone levels have been already discussed. In this study, we are defining a new pediatric reference range for TSH, FT3 and FT4 for Cobas C6000 analyzer. To do so, we have taken in account 0 to 18 year old outclinic patients. During the first year of life, thyroid hormone levels change dramatically before getting stabilized around 3 years old. We also compared our results to those obtained in a Canadian large-scale prospective study (the CALIPER initiative).

  7. PENGEMBANGAN BUKU AJAR BERBASIS PENELITIAN EVOLUSI DAN FILOGENETIK MOLEKULER UNTUK MATAKULIAH EVOLUSI DI UNIVERSITAS JEMBER

    Directory of Open Access Journals (Sweden)

    Ulin Nuha

    2016-09-01

    Filogenetik molekuler merupakan salah satu kajian yang dipelajari pada matakuliah Evolusi untuk jenjang S1 Pendidikan Biologi. Kajian ini memerlukan bahan ajar yang kontekstual dan tetap mengikuti perkembangan IPTEKS. Kendala yang muncul adalah pengetahuan mahasiswa dalam ranah molekuler masih rendah. Salah satu solusi yang dapat dilakukan adalah dengan menyediakan buku ajar berbasis penelitian denan pendekatan molekuler. Buku ajar dikembangkan berdasarkan model pengembangan ADDIE. Produk divalidasi oleh ahli media, materi, praktisi pendidikan dan diujicobakan pada kelompok kecil. Hasil validasi ahli media, ahli materi, praktisi pendidikan, dan uji coba kelompok kecil secara berturut turut adalah 87,14%, 91,00%, 75,78%, dan 82,22%.

  8. Mapas Conceptuales y Aprendizaje Cooperativo : una visión desde la enseñanza universitaria

    OpenAIRE

    Cobas Cobiella, María Elena; Alventosa del Río, Josefina; Catalán Guillén, Raquel; Uriol Egido, Carmen; Angulo Alemán, Tanya; Chaparro Matamoros, Pedro; Marí Farinós, Jesús; Febles Pozo, Nayiber; Company Alcañiz, Mireia; Valero Llorca, Javier; Martín García, Elena; Pérez Fuentes, Gisela María; Cantoral Domínguez, Karla

    2015-01-01

    El libro que se presenta bajo el título “Mapas Conceptuales y Aprendizaje Cooperativo. Una visión desde la enseñanza universitaria, constituye uno de los resultados del trabajo de un grupo de profesores y especialistas suscrito en el Proyecto de Innovación “ Los Mapas Conceptuales como metodología docente activa dentro del Nuevo Espacio de Educación Superior Europeo”, Finestra Oberta.UV-SFPIE_2014_221354, bajo la dirección de la profesora María Elena Cobas Cobiella, del Departamento de Dere...

  9. Analytical performance specifications: relating laboratory performance to quality required for intended clinical use.

    Science.gov (United States)

    Dalenberg, Daniel A; Schryver, Patricia G; Klee, George G

    2013-03-01

    This article proposes analytic performance goals for five quality indicators: precision, trueness, linearity, detection limits, and consistency across instruments and time. We defined our goals using methods linked to clinical practice data. Goals for desirable precision and trueness are based on biological variation. Linearity goals are related to total error recommendations. Detection limit goals are derived from 0.1 percentile of patient values. Goals for consistency are derived from the variability of distributions of patient test values. Data were collected and evaluated for each of these quality indicators for 46 chemistry tests measured on the Roche cobas 8000 analyzer.

  10. PENGEMBANGAN BAHAN AJAR DIGITAL BERLANDASKAN MODEL GUIDED-PROJECT BASED LEARNING

    Directory of Open Access Journals (Sweden)

    Ighfir Rijal Taufiqy

    2016-04-01

    Hasil observasi menunjukkan SMK Negeri 1 Beji Pasuruan sudah menggunakan bahan ajar digital dan project based learning dalam pembelajaran. Tujuan pengembangan menghasilkan bahan ajar digital pada mata pelajaran Teknik Pengambilan Gambar Bergerak berlandaskan model Guided-Project Based Learning di SMK Negeri 1 Beji Pasuruan. Model penelitian dan pengembangan mengadaptasi model Dick & Carey yang dimodifikasi dengan menghilangkan evaluasi formatif. Subjek uji coba ahli materi, ahli media, guru, siswa kelas XII. Rata-rata kelayakan sebesar 89,5% dan pencapaian post-test sebesar 81,77. Saran pengembangan lebih lanjut dengan menambahkan empat proyek sehingga menjadi satu keutuhan bahan ajar dalam periode satu tahun dan perlu uji efektivitas.

  11. BRAF mutation testing with a rapid, fully integrated molecular diagnostics system

    OpenAIRE

    Janku, Filip; Claes, Bart; Huang, Helen J.; Falchook, Gerald S.; Devogelaere, Benoit; Kockx, Mark; Bempt, Isabelle Vanden; Reijans, Martin; Naing, Aung; Fu, Siqing; Piha-Paul, Sarina A.; Hong, David S.; Holley, Veronica R.; Tsimberidou, Apostolia M.; Stepanek, Vanda M.

    2015-01-01

    Fast and accurate diagnostic systems are needed for further implementation of precision therapy of BRAF-mutant and other cancers. The novel IdyllaTM BRAF Mutation Test has high sensitivity and shorter turnaround times compared to other methods. We used Idylla to detect BRAF V600 mutations in archived formalin-fixed paraffin-embedded (FFPE) tumor samples and compared these results with those obtained using the cobas 4800 BRAF V600 Mutation Test or MiSeq deep sequencing system and with those ob...

  12. Interactions between Hepatitis C Virus and the Human Apolipoprotein H Acute Phase Protein: A Tool for a Sensitive Detection of the Virus.

    Science.gov (United States)

    Stefas, Ilias; Tigrett, Sylvia; Dubois, Grégor; Kaiser, Marco; Lucarz, Estelle; Gobby, Delphine; Bray, Dorothy; Ellerbrok, Heinz; Zarski, Jean Pierre; Veas, Francisco

    2015-01-01

    The Hepatitis C virus (HCV) infection exhibits a high global prevalence frequently associated with hepatocellular carcinoma, taking years to develop. Despite the standardization of highly sensitive HCV quantitative RT-PCR (qRT-PCR) detection methods, false-negative diagnoses may be generated with current methods, mainly due to the presence of PCR inhibitors and/or low viral loads in the patient's sample. These false-negative diagnoses impact both public health systems, in developing countries, and an in lesser extent, in developed countries, including both the risk of virus transmission during organ transplantation and/or blood transfusion and the quality of the antiviral treatment monitoring. To adopt an appropriate therapeutic strategy to improve the patient's prognosis, it is urgent to increase the HCV detection sensitivity. Based upon previous studies on HBV, we worked on the capacity of the scavenger acute phase protein, Apolipoprotein H (ApoH) to interact with HCV. Using different approaches, including immunoassays, antibody-inhibition, oxidation, ultracentrifugation, electron microscopy and RT-PCR analyses, we demonstrated specific interactions between HCV particles and ApoH. Moreover, when using a two-step HCV detection process, including capture of HCV by ApoH-coated nanomagnetic beads and a home-made real-time HCV-RT-PCR, we confirmed the presence of HCV for all samples from a clinical collection of HCV-seropositive patients exhibiting an RT-PCR COBAS® TaqMan® HCV Test, v2.0 (COBAS)-positive result. In contrast, for HCV-seropositive patients with either low HCV-load as determined with COBAS or exhibiting HCV-negative COBAS results, the addition of the two-step ApoH-HCV-capture and HCV-detection process was able to increase the sensitivity of HCV detection or more interestingly, detect in a genotype sequence-independent manner, a high-proportion (44%) of HCV/RNA-positive among the COBAS HCV-negative patients. Thus, the immune interaction between ApoH and

  13. Interactions between Hepatitis C Virus and the Human Apolipoprotein H Acute Phase Protein: A Tool for a Sensitive Detection of the Virus.

    Directory of Open Access Journals (Sweden)

    Ilias Stefas

    Full Text Available The Hepatitis C virus (HCV infection exhibits a high global prevalence frequently associated with hepatocellular carcinoma, taking years to develop. Despite the standardization of highly sensitive HCV quantitative RT-PCR (qRT-PCR detection methods, false-negative diagnoses may be generated with current methods, mainly due to the presence of PCR inhibitors and/or low viral loads in the patient's sample. These false-negative diagnoses impact both public health systems, in developing countries, and an in lesser extent, in developed countries, including both the risk of virus transmission during organ transplantation and/or blood transfusion and the quality of the antiviral treatment monitoring. To adopt an appropriate therapeutic strategy to improve the patient's prognosis, it is urgent to increase the HCV detection sensitivity. Based upon previous studies on HBV, we worked on the capacity of the scavenger acute phase protein, Apolipoprotein H (ApoH to interact with HCV. Using different approaches, including immunoassays, antibody-inhibition, oxidation, ultracentrifugation, electron microscopy and RT-PCR analyses, we demonstrated specific interactions between HCV particles and ApoH. Moreover, when using a two-step HCV detection process, including capture of HCV by ApoH-coated nanomagnetic beads and a home-made real-time HCV-RT-PCR, we confirmed the presence of HCV for all samples from a clinical collection of HCV-seropositive patients exhibiting an RT-PCR COBAS® TaqMan® HCV Test, v2.0 (COBAS-positive result. In contrast, for HCV-seropositive patients with either low HCV-load as determined with COBAS or exhibiting HCV-negative COBAS results, the addition of the two-step ApoH-HCV-capture and HCV-detection process was able to increase the sensitivity of HCV detection or more interestingly, detect in a genotype sequence-independent manner, a high-proportion (44% of HCV/RNA-positive among the COBAS HCV-negative patients. Thus, the immune interaction

  14. Development of Targeted Nonionic Surfactant Vesicles for Treatment of Vascular Injury

    Science.gov (United States)

    2008-12-01

    cardiac puncture. Plasma was analyzed for total cholesterol, HDL cholesterol, LDL cholesterol and triglycerides on a Cobas Mira Plus analyzer (Yale...Phenotyping Center, Yale University School of Medicine, New Haven, CT LDL -direct reagents purchased from Roche; triglycerides, total cholesterol, HDL ...787.6 ± 337.7 832.1 ± 517.3 701.4 ± 276.2 N 4 5 6 5 6 5 5 HDL -direct (mg/dl) 15.8 ± 6.1 11.4 ± 4.1 12.4 ± 3.4 11.7 ± 3.5 10.6 ± 4.1

  15. Department of Defense In-House RDT and E Activities. Management Analysis Report

    Science.gov (United States)

    1975-10-30

    PROGRAM SAALL CALIBER AK14UN171UN MODERNIZATION PRUGRAM,SCAMP F-IRE CONTROL EQ FOR AIRCRAFT .AP7I AIRCRAFT,9ARTIL *COMB ViH ,AD SMALL CAL W 4 TEST...XrI9B RETREAO TIRE PROGRAM Nc-CH INF COMBAT VIH IMICV) IMP COBaA ARM SYST (ICAS) A.. FUNCTIONS/EQUIPNENT/CAPABILI[TIES INSTRUMENTED ARTY RANIGE...PERSONNEL MICROORGANISMS IN INDUC8D PERIODONTAL DISEASE BIOCHEMISTRY OF INFECTION FREE. AND PATHOLOGICAL ORAL ENVIRONMENTS BACTERIOLOGY OF ThE ORAL

  16. Detección temprana y altamente sensible de citomegalovirus en muestras de plasma humano VIH-positivas

    OpenAIRE

    2009-01-01

    La detección temprana de citomegalovirus humano puede evitar graves consecuencias en la salud de pacientes inmunocomprometidos; por ello, es importante el empleo de métodos de detección sensibles y precisos. En el presente trabajo comparamos la sensibilidad y la especificidad de detección de tres métodos: 1) COBAS AMPLICOR CMV MONITOR¿ Test (CACM), método comercial validado y semiautomatizado que utiliza la reacción en cadena de la polimerasa (PCR) para detectar la expresión del gen temprano ...

  17. Detection of Bartonella spp. DNA in clinical specimens using an internally controlled real-time PCR assay

    NARCIS (Netherlands)

    Bergmans, Anneke M C; Rossen, John W A

    2013-01-01

    Bartonella henselae is the causative agent of cat-scratch disease (CSD), usually presenting itself as a -self-limiting lymphadenopathy. In this chapter an internally controlled Taqman probe-based real-time PCR targeting the groEL gene of Bartonella spp. is described. This assay allows for the rapid,

  18. Effects of the EVCAM chemical validation library on differentiation using marker gene expression in lmouse embryonic stem cells

    Science.gov (United States)

    The adherent cell differentiation and cytotoxicity (ACDC) assay was used to profile the effects of the ECVAM EST validation chemical library (19 compounds) on J1 mouse embryonic stem cells (mESC). PCR-based TaqMan Low Density Arrays (TLDA) provided a high-content assessment of al...

  19. Development of a non invasion real-time PCR assay for the quantitation of chicken parvovirus in fecal swabs

    Science.gov (United States)

    The present study describes the development of a real time Taqman polymerase chain reaction (PCR) assay using a fluorescent labeled probe for the detection and quantitation of chicken parvovirus (ChPV) in feces. The primers and probes were designed based on the nucleotide sequence of the non struct...

  20. Apolipoprotein(a) Kringle-IV Type 2 Copy Number Variation Is Associated with Venous Thromboembolism

    DEFF Research Database (Denmark)

    Sticchi, Elena; Magi, Alberto; Kamstrup, Pia R

    2016-01-01

    without hereditary and acquired thrombophilia and 1117 healthy control subjects, comparable for age and sex, were investigated. LPA KIV-2 polymorphism, rs3798220 and rs10455872 SNPs were genotyped by TaqMan technology. Concerning rs1853021 and rs1800769 SNPs, PCR-RFLP assay was used. LPA KIV-2 repeat...

  1. Quantitative real-time PCR assay for rapid detection of plant and human pathogenic Macrophomina phaseolina from field and environmental samples.

    Science.gov (United States)

    Babu, Bandamaravuri Kishore; Mesapogu, Sukumar; Sharma, Anu; Somasani, Saida Reddy; Arora, Dilip K

    2011-01-01

    A real-time qPCR assay was developed to detect and quantify Macrophomina phaseolina abundance in rhizosphere soil and plant tissue. Both TaqMan and SYBR green techniques were targeted on ~ 1 kb sequence characterized amplified region (SCAR) of M. phaseolina and two sets of specific primers were designed for SYBR green (MpSyK) and TaqMan (MpTqK) assays. No cross-hybridization and no fluorescent signal exceeding the baseline threshold was observed in TaqMan and SYBR green assays, respectively. The minimum detection limit or sensitivity of TaqMan assay was 30 fg/μL of M. phaseolina DNA and limit of quantification of M. phaseolina viable population was estimated as 0.66 × 10(5) CFU/g soil(-1) equivalent to 10 pg/μL of target DNA. This is the first report which demonstrated real-time qPCR assays with greater specificity and sensitivity to detect M. phaseolina population in soil and plant materials.

  2. Correlation between lipoprotein-associated phospholipase A2 activity and its gene polymorphism in coronary heart disease

    Institute of Scientific and Technical Information of China (English)

    李珊珊

    2012-01-01

    Objective To detect the mutation of A379,we developed a TaqMan fluorogenic probe based amplification refractory mutation system(TaqMan-ARMS) and investigate whether the A379V variant and activity of Lp-PLA2 were the risk factors for CAD. Methods According to the amplification refractory mutation system

  3. Effect of methotrexate combined with ginger, silymarin or propolis on ...

    African Journals Online (AJOL)

    aghomotsegin

    2015-02-16

    Feb 16, 2015 ... effect of methotrexate (MTX) in normal liver cells. TaqMan ... al., 2011). MTX is a structural analogue of folic acid. It ... chemicals within cells (Rhee and Galivan, 1986). The two .... probes were labeled by FAM fluorescent dye. RT-PCR ..... analysis of apoptotic and necrotic outer hair cells after exposure to.

  4. Development and Validation of a Novel Real-time Assay for the Detection and Quantification of Vibrio cholerae

    DEFF Research Database (Denmark)

    Rashid, Ridwan Bin; Ferdous, Jannataul; Tulsiani, Suhella

    2017-01-01

    is rapid since neither lengthy incubation period nor electrophoresis is required. The assay had excellent repeatability (CV%: 0.24–1.32) and remarkable reproducibility (CV%: 1.08–3.7). Amplification efficiencies in the 89–100% range were observed. The assay is more economical than Taqman-based multiplex...

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

    Science.gov (United States)

    2004-06-01

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

  6. BRCA1 Protein Complexes: Dynamic Changes and Functions Important in Breast Cancer

    Science.gov (United States)

    2007-04-01

    performed on separate samples at the Harvard Biopolymers Facility on the Affymetrix (Santa Clara, CA) HG-U133plus2 chip. For TaqMan assays, 10 ng of cDNA...hyperphosphorylated Rpb1. In cells, however, efficient Rpb1 ubiquitination required the car - boxyl terminus of BRCA1, suggesting that interactions mediated by this

  7. EVALUATION OF A RAPID, QUANTITATIVE REAL-TIME PCR METHOD FOR ENUMERATION OF PATHOGENIC CANDIDA CELLS IN WATER

    Science.gov (United States)

    Quantitative Real-Time PCR (QRT-PCR) technology, incorporating fluorigenic 5' nuclease (TaqMan?) chemistry, was developed for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C....

  8. Multiplex real-time PCR assays for the identification of the potato cyst and tobacco cyst nematodes

    Science.gov (United States)

    TaqMan primer-probe sets were developed for the detection and identification of potato cyst nematodes (PCN) Globodera pallida and G. rostochiensis using two-tube, multiplex real-time PCR. One tube contained a primer-probe set specific for G. pallida (pale cyst nematode) multiplexed with another prim...

  9. Effects of the EVCAM chemical validation library on differentiation using marker gene expression in lmouse embryonic stem cells

    Science.gov (United States)

    The adherent cell differentiation and cytotoxicity (ACDC) assay was used to profile the effects of the ECVAM EST validation chemical library (19 compounds) on J1 mouse embryonic stem cells (mESC). PCR-based TaqMan Low Density Arrays (TLDA) provided a high-content assessment of al...

  10. Report for Detection of Biothreat Agents and Environmental Samples using the LLNL Virulence Array for DHS

    Energy Technology Data Exchange (ETDEWEB)

    Jaing, Crystal [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Gardner, Shea [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); McLoughlin, Kevin [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Thissen, James [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Jackson, Paul [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2011-04-18

    The objective of this project is to provide DHS a comprehensive evaluation of the current genomic technologies including genotyping, Taqman PCR, multiple locus variable tandem repeat analysis (MLVA), microarray and high-throughput DNA sequencing in the analysis of biothreat agents from complex environmental samples. This report focuses on the design, testing and results of samples on the Virulence Array.

  11. Evaluation of probe chemistries and platforms to improve the detection limit of real-time PCR

    DEFF Research Database (Denmark)

    Reynisson, E.; Josefsen, Mathilde Hartmann; Krause, Michael

    2006-01-01

    A validated PCR-based Salmonella method targeting a 94-bp sequence of the ttr gene was used as a model to compare six different combinations of reporter and quencher dyes of a TaqMan probe, on three different instruments, to improve the detection limit in a real-time PCR assay with the aim of a s...

  12. Molecular detection of foodborne pathogens

    DEFF Research Database (Denmark)

    Josefsen, Mathilde Hartmann

    ), Scorpion and TaqMan probes. The LNA probe was shown to be the most sensitive probe chemistry in the real-time PCR assay for detection of Campylobacter, producing the highest amplification efficiency. Choice of probe chemistry was found to impact the sensitivity of PCR assays, and should be considered...

  13. Diagnostic PCR: Comparative sensitivity of four probe chemistries

    DEFF Research Database (Denmark)

    Josefsen, Mathilde Hartmann; Löfström, Charlotta; Sommer, Helle Mølgaard

    2009-01-01

    Three probe chemistries: locked nucleic acid (LNA), minor groove binder (MGB) and Scorpion were compared with a TaqMan probe in a validated real-time PCR assay for detection of food-borne thermotolerant Campylobacter. The LNA probe produced significantly lower Ct-values and a higher proportion...

  14. Rapid Detection of KPC, NDM, and OXA-48-Like Carbapenemases by Real-Time PCR from Rectal Swab Surveillance Samples

    Science.gov (United States)

    Lee, Tracy D.; Adie, Kathleen; McNabb, Alan; Purych, Dale; Mannan, Kulvinder; Azana, Robert; Ng, Corrinne; Tang, Patrick

    2015-01-01

    We describe a multiplex real-time PCR assay for use on the ABI 7500 Fast TaqMan platform to detect all currently described Klebsiella pneumoniae carbapenemases (KPC), New Delhi metallo-β-lactamases (NDM), and the OXA-48-like family of carbapenemases from bacterial culture lysates or sample enrichment broth lysates. PMID:26019195

  15. Comparison of Gull Feces-specific Assays Targeting the 16S rRNA Gene of Catellicoccus Marimammalium and Streptococcus spp.

    Science.gov (United States)

    Two novel gull-specific qPCR assays were developed using 16S rRNA gene sequences from gull fecal clone libraries: a SYBR-green-based assay targeting Streptococcus spp. (i.e., gull3) and a TaqMan qPCR assay targeting Catellicoccus marimammalium (i.e., gull4). The main objectives ...

  16. Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages.

    Directory of Open Access Journals (Sweden)

    David Stucki

    Full Text Available There is increasing evidence that strain variation in Mycobacterium tuberculosis complex (MTBC might influence the outcome of tuberculosis infection and disease. To assess genotype-phenotype associations, phylogenetically robust molecular markers and appropriate genotyping tools are required. Most current genotyping methods for MTBC are based on mobile or repetitive DNA elements. Because these elements are prone to convergent evolution, the corresponding genotyping techniques are suboptimal for phylogenetic studies and strain classification. By contrast, single nucleotide polymorphisms (SNP are ideal markers for classifying MTBC into phylogenetic lineages, as they exhibit very low degrees of homoplasy. In this study, we developed two complementary SNP-based genotyping methods to classify strains into the six main human-associated lineages of MTBC, the "Beijing" sublineage, and the clade comprising Mycobacterium bovis and Mycobacterium caprae. Phylogenetically informative SNPs were obtained from 22 MTBC whole-genome sequences. The first assay, referred to as MOL-PCR, is a ligation-dependent PCR with signal detection by fluorescent microspheres and a Luminex flow cytometer, which simultaneously interrogates eight SNPs. The second assay is based on six individual TaqMan real-time PCR assays for singleplex SNP-typing. We compared MOL-PCR and TaqMan results in two panels of clinical MTBC isolates. Both methods agreed fully when assigning 36 well-characterized strains into the main phylogenetic lineages. The sensitivity in allele-calling was 98.6% and 98.8% for MOL-PCR and TaqMan, respectively. Typing of an additional panel of 78 unknown clinical isolates revealed 99.2% and 100% sensitivity in allele-calling, respectively, and 100% agreement in lineage assignment between both methods. While MOL-PCR and TaqMan are both highly sensitive and specific, MOL-PCR is ideal for classification of isolates with no previous information, whereas TaqMan is faster

  17. Quantitative PCR for glucose transporter and tristetraprolin family gene expression in cultured mouse adipocytes and macrophages.

    Science.gov (United States)

    Cao, Heping; Cao, Fangping; Roussel, Anne-Marie; Anderson, Richard A

    2013-12-01

    Quantitative real-time PCR (qPCR) such as TaqMan and SYBR Green qPCR are widely used for gene expression analysis. The drawbacks of SYBR Green assay are that the dye binds to any double-stranded DNA which can generate false-positive signals and that the length of the amplicon affects the intensity of the amplification. Previous results demonstrate that TaqMan assay is more sensitive but generates lower calculated expression levels than SYBR Green assay in quantifying seven mRNAs in tung tree tissues. The objective of this study is to expand the analysis using animal cells. We compared both qPCR assays for quantifying 24 mRNAs including those coding for glucose transporter (Glut) and mRNA-binding protein tristetraprolin (TTP) in mouse 3T3-L1 adipocytes and RAW264.7 macrophages. The results showed that SYBR Green and TaqMan qPCR were reliable for quantitative gene expression in animal cells. This result was supported by validation analysis of Glut and TTP family gene expression. However, SYBR Green qPCR overestimated the expression levels in most of the genes tested. Finally, both qPCR instruments (Bio-Rad's CFX96 real-time system and Applied Biosystems' Prism 7700 real-time PCR instrument) generated similar gene expression profiles in the mouse cells. These results support the conclusion that both qPCR assays (TaqMan and SYBR Green qPCR) and both qPCR instruments (Bio-Rad's CFX96 real-time system and Applied Biosystems' Prism 7700 real-time PCR instrument) are reliable for quantitative gene expression analyses in animal cells but SYBR Green qPCR generally overestimates gene expression levels than TaqMan qPCR.

  18. PEMANFAATAN KONSORSIUM MIKROBIA UNTUK MENINGKATKAN KINERJA SISTEM LUMPUR AKTIF

    Directory of Open Access Journals (Sweden)

    Novarina Irnaning Handayani

    2015-05-01

    Full Text Available Industri tekstil sebagian besar menggunakan mengolah limbah cair pada instalasi pengolahan air limbah dengan menggunakan sistem fisika, kimia, dan biologi. Sistem biologi yang digunakan biasanya adalah lumpur aktif yang terkadang mengalami gangguan. Tujuan penelitian ini adalah membuat konsorsium mikrobia terpilih yang dapat menaikkan kinerja lumpur aktif yang sedang terganggu, diindikasikan dengan turunnya nilai sludge volume dalam reaktor lumpur aktif serta menurunkan COD air limbah terolah. Terpilih 6 (enam jenis bakteri non patogen yaitu Bacillus macerans, Bacillus subtilis, Bacillus thuringiensis, Bacillus sp, Kurthia zopfii,dan Pseudomonas stutzeri untuk digabungkan dalam satu konsorsium. Hasil uji antagonisme antar species terpilih menunjukkan tidak munculnya zone penghambatan, sehingga 6 (enam jenis bakteri tersebut dapat digabungkan menjadi satu kesatuan.Hasil uji coba laboratorium menunjukkan konsorsium yang ditambahkan nutrien berupa 25 gr bekatul dan 50 gram gula per liter air dengan pencapaian sludge volume 30 menit 85% dan setelah diendapkan 24 jam adalah 35% denganpenurunan COD 82% Uji coba lanjutan menunjukkan bahwa konsorsium dalam 6 jenis bakteri ditambah Nitrobacter dan yeast sludge volume 30 menit terbaik mencapai 73% setelah diendapkan 24 jam menjadi 32% dengan penurunan COD mencapai 81%. 

  19. QuickASP: PEMBANGKIT KODE PROGRAM ASP UNTUK APLIKASI BASIS DATA BERBASIS WEB

    Directory of Open Access Journals (Sweden)

    Imam Kuswardayan

    2007-01-01

    Full Text Available Dalam pembuatan sistem aplikasi basis data berbasis web, perancangan antarmuka pengguna (presentation layer dan lapisan bisnis (bussiness layer merupakan tahap yang dilalui setelah pemahaman terhadap kebutuhan pengguna sistem. Adanya pola atau keteraturan dalam implementasi tahap ini menyebabkan pengembangan sistem akan lebih efisien jika menggunakan suatu aplikasi yang dapat menghasilkan kerangka dasar aplikasi web dengan cepat untuk kedua lapisan tersebut dan bahkan beserta kode programnya. Pada penelitian ini telah diimplementasikan suatu perangkat lunak yang selanjutnya disebut QuickASP. QuickASP membangkitkan kode ASP untuk membangun  homepage otomatis. Untuk membangkitkan kode ASP, QuickASP membutuhkan komponen berupa basis data dan file Cascading Style Sheets (CSS. Proses awal yang dilakukan QuickASP dalam membangkitkan kode program ASP adalah membaca informasi basis data berupa tabel-tabel, nama field dan tipe data. Setelah itu QuickASP akan membangkitkan file-file ASP beserta file-file pendukungnya berdasarkan hasil pengaturan tampilan halaman web yang dilakukan oleh pengguna.Uji coba QuickASP dilakukan pada tiga jenis basis data yaitu Microsoft Access, Microsoft SQL Server, dan Oracle. Dari hasil uji coba tersebut, QuickASP terbukti dapat membangkitkan homepage otomatis beserta fungsi–fungsi yang disediakan untuk modifikasi record dan fungsi navigasi.Kata kunci: QuickASP, file cascading style sheets, kode program ASP.

  20. Is 58% sensitivity for detection of cervical intraepithelial neoplasia 3 and invasive cervical cancer optimal for cervical screening?

    Directory of Open Access Journals (Sweden)

    R. Marshall Austin

    2014-01-01

    Full Text Available Recent Food and Drug Administration (FDA approval of a Roche cobas human papillomavirus (HPV test application as a first line primary cervical screening tool in women 25 and older introduces a new era of complex cervical screening choices. Perhaps the most surprising findings in Roche′s supporting ATHENA trial data were the unexpectedly low verification bias-adjusted CIN3+ sensitivities documented by the FDA for both the proposed cobas HPV testing algorithm (58.26% and Pap testing algorithm (42.63%. These unexpectedly low sensitivity estimates suggest intuitively that there is still considerable room for improvement in cervical screening, and available data from large systems point to routine cytology and HPV co-testing as offering the greatest protection against development of cervical cancer. Observational studies of large populations screened over time remain essential to document actual protection from development of cervical cancer with any new cervical screening options, as natural history studies and available data from large systems indicate that most CIN2/3 cases detected in short term clinical trials would not progress to invasive cervical cancer. Interpretation of ATHENA trial data and its application to routine clinical practice is further limited by published studies which document that a significant proportion of CIN2/3 biopsy diagnoses in the ATHENA trial could not be confirmed as accurate when evaluated with p16 immunohistochemistry and that cytology laboratory performance in the trial was notably suboptimal.

  1. Rancang Bangun Permainan Edukasi Matematika dan Fisika dengan Memanfaatkan Accelerometer dan Physics Engine Box2d pada Android

    Directory of Open Access Journals (Sweden)

    Putri Nikensasi

    2012-09-01

    Full Text Available Perkembangan industri permainan mobil yang semakin meningkat memotivasi para pengembang permainan mobil untuk membuat inovasi-inovasi terbaru dalam permainannya. Salah satu inovasi tersebut yaitu permainan edukasi, namun saat ini permainan edukasi kurang diminati karena aturan permainannya yang cenderung membosankan. Pengembangan permainan ini ditujukan untuk membuat sebuah permainan mobil edukasi dengan memanfaatkan teknologi mobil terbaru dalam aturan permainannya sehingga permainan tersebut tidak membosankan. Aplikasi yang dikembangkan merupakan aplikasi permainan mobil edukasi yang mengajarkan ilmu matematika dan fisika kepada pemainnya. Teknologi baru yang digunakan dalam permainan ini yaitu accelerometer pada sistem operasi Android yang diintegrasikan dengan Physics Engine Library Box2D. Selain itu, permainan ini dibangun dengan menggunakan Adobe Flash CS5.5 dan bahasa pemrograman Actionscript 3 (AS3 serta Adobe Air sebagai runtime aplikasinya. Uji coba dilakukan dengan menggunakan perangkat Android versi 2.3. Dari uji coba dapat disimpulkan bahwa Adobe Flash CS5.5 dapat digunakan untuk membuat permainan mobil edukasi pada perangkat Android dan mengakses sensor accelerometer-nya.

  2. RANCANG BANGUN PROGRAM PENGEDITAN KURVA B-SPLINE MULTIRESOLUSI BERBASIS WAVELETS

    Directory of Open Access Journals (Sweden)

    Nanik Suciati

    2002-07-01

    Full Text Available Penelitian ini menyusun representasi multiresolusi untuk kurva B-spline kubik yang menginterpolasi titik-titik ujung dengan basis wavelets. Representasi multiresolusi ini digunakan untuk mendukung beberapa tipe pengeditan kurva, yaitu penghalusan kurva dengan tingkat resolusi kontinyu untuk menghilangkan detail-detail kurva yang tidak diinginkan, pengeditan bentuk keseluruhan kurva dengan tetap mempertahankan detaildetailnya, perubahan detail-detail kurva tanpa mempengaruhi bentuk keseluruhannya, dan pengeditan satubagian tertentu dari kurva melalui manipulasi secara langsung terhadap titik-titik kontrolnya. Untuk menguji kemampuan representasi multiresolusi dalam mendukung empat tipe manipulasi kurva tersebut, disusun program pengeditan kurva dengan menggunakan bahasa pemrograman Visual C++ pada komputer Pentium 133 MHz, memori 16 Mbyte, sistem operasi Windows 95, lingkungan pengembangan Microsoft DevelopmentStudio 97 dan pustaka Microsoft Foundation Class. Dari hasil uji coba program diketahui bahwa representasi multiresolusi memberikan dukungan yang sangat baik terhadap tipe-tipe pengeditan seperti yang disebutkan di atas. Representasi multiresolusi tidak membutuhkan memori penyimpan ekstra selain dari yang digunakan untuk menyimpan titik kontrol. Dari hasil uji coba program menggunakan ratusan titik kontrol, algoritma berjalan cukup cepat dan memadai berkaitan dengan tuntutan komunikasi interaktif antara user dan program.Kata kunci: B-Spline, Wavelet, Multiresolusi

  3. PENDIDIKAN KARAKTER BANGSA BERBASIS STRATEGI PEMBELAJARAN PAKEM MELALUI PERMAINAN CINCIN DI JEMPOL TANGAN (Karya Inovasi Pembelajaran Sekolah Dasar

    Directory of Open Access Journals (Sweden)

    Ani Adibatin

    2016-02-01

    Full Text Available Berhasil tidaknya pencapaian tujuan pembelajaran sangat ditentukan oleh kemampuan guru dalam mengelola kelas, mengelola siswa, memilih strategi pembelajaran, serta kebermaknaan dalam memberikan tugas pada siswa. Berkaitan dengan permasalahan tersebut, penulis sebagai pengawas mengembangkan alat permainan inovatif yang bisa dipakai oleh semua guru, baik guru kelas, guru mata pelajaran, maupun guru ekstra kurikuler. Alat permainan ini bisa dipakai oleh semua siswa dari berbagai tingkatan kelas, berbagai tingkat jenjang sekolah, dan untuk pembelajaran materi yang berbeda. Alat permainan inovatif pembelajaran ini dikembangkan dengan tujuan agar peserta didik bisa belajar sambil bermain dan bermain sambil belajar. Disamping itu, untuk membantu para guru dalam menanamkan pendidikan karakter, sehingga peserta didik menjadi anak yang berkarakter, berwatak sesuai dengan nilai-nilai Pancasila demi terwujudnya tujuan pendidikan nasional. Uji coba alat permainan inovatif ini dilakukan di 9 SD binaan Kecamatan Tuntang. Dari hasil uji coba diperoleh temuan bahwa ada perbedaan yang sangat signifikan antara pendidikan karakter pada pembelajaran tanpa alat permainan inovatif dibandingkan dengan pembela-jaran yang menggunakan alat permainan cincin akik di jempol tangan. Nilai rata-rata pembelajaran tanpa alat inovatif 76,55 %, sedangkan nilai rata-rata dengan menggunakan alat permainan cincin akik di jempol sebesar 86,88 %. Hasil pembelajaran menggunakan alat peraga inovatif ini menunjukkan bah-wa melalui strategi pembelajaran PAKEM dengan bermain cincin akik di jempol tangan dapat membangun karakter peserta didik sampai 11,33 %.

  4. Detection and identification of occult HBV in blood donors in Taiwan using a commercial, multiplex, multi-dye nucleic acid amplification technology screening test.

    Science.gov (United States)

    Lin, K T; Chang, C L; Tsai, M H; Lin, K S; Saldanha, J; Hung, C M

    2014-02-01

    The ability of a new generation commercial, multiplex, multi-dye test from Roche, the cobas TaqScreen MPX test, version 2.0, to detect and identify occult HBV infections was evaluated using routine donor samples from Kaohsiung Blood Bank, Taiwan. A total of 5973 samples were tested by nucleic acid amplification technology (NAT); 5898 in pools of six, 66 in pools of less than six and nine samples individually. NAT-reactive samples were retested with alternative NAT tests, and follow-up samples from the donors were tested individually by NAT and for all the HBV serological markers. Eight NAT-only-reactive donors were identified, and follow-up samples were obtained from six of the donors. The results indicated that all eight donors had an occult HBV infection with viral loads high prevalence of occult HBV infections since the uncertainty associated with identifying samples with very low viremia is removed by the ability of the test to identify the viral target in samples that are reactive with the cobas TaqScreen MPX test, version 2.0. © 2013 International Society of Blood Transfusion.

  5. PENGEMBANGAN MULTIMEDIA PEMBELAJARAN INTERAKTIF KOMPETENSI DASAR PEMASANGAN SISTEM PENERANGAN DAN WIRING KELISTRIKAN DI SMK

    Directory of Open Access Journals (Sweden)

    Nopriyanti Nopriyanti

    2015-06-01

    Full Text Available Penelitian ini bertujuan untuk: (1 menghasilkan multimedia pembelajaran interaktif kompetensi dasar pemasangan sistem penerangan dan wiring kelistrikan; (2 mengetahui kualitas multimedia pembelajaran interaktif; dan (3 mengetahui efektifitas multimedia pembelajaran interaktif kompetensi dasar pemasangan sistem penerangan dan wiring kelistrikan pada kelas XI SMK bidang keahlian Teknik Kendaraan Ringan. Penelitian ini merupakan penelitian dan pengembangan. Validasi multimedia dilakukan oleh ahli materi dan ahli media. Subjek uji coba penelitian ini adalah 32 siswa kelas XI SMKN 2 Depok, Sleman, Yogyakarta bidang keahlian Teknik Kendaraan Ringan. Kesimpulan dari penelitian ini adalah: (1 produk multimedia pembelajaran interaktif kompetensi dasar pemasangan sistem penerangan dan wiring kelistrikan layak digunakan; (2 kualitas multimedia interaktif ini sangat baik, hasil penilaian ahli ditinjau dari pembelajaran 66 (baik, isi sebesar 54 (baik, aspek tampilan 97 (baik, dan program 50 (baik, sedangkan hasil dari penilaian peserta didik uji coba lapangan pada aspek pembelajaran1277 (sangat baik, isi 1195 (sangat baik, tampilan 1562 (sangat baik, dan pemograman 519 (sangat baik; (3 Produk multimedia pembelajaran interaktif kompetensi dasar pemasangan sistem penerangan dan wiring kelistrikan efektif meningkatkan hasil belajar siswa. Rata-rata penilaian hasil belajar siswa ketika pretest adalah 63,75 dengan nilai terendah 50 dan nilai tertinggi 75. Sedangkan rata-rata nilai posttest sebesar 78,75 dengan nilai terendah 65 dan nilai tertinggi 90.

  6. CLINICAL LABORATORY PARAMETERS AMONG ADULT MALES DURING A PRIMAQUINE CHEMOPROPHYLAXIS TRIAL IN IRIAN JAYA, INDONESIA

    Directory of Open Access Journals (Sweden)

    David J. Fryauff

    2012-09-01

    Full Text Available Primakuin yang digunakan sebagai profilaksis malaria terbukti efektif dan diterima dengan baik oleh tubuh manusia yang normal terhadap aktivitas enzim 6 glukosa-6 fosfat dehidrogenase (G-6PD. Pemeriksaan laboratoris klinik adalah bagian dari uji coba secara acak dengan kontrol plasebo dalam rangka mengevaluasi penggunaan primakuin sebagai profilaksis pada penduduk transmigran yang tidak kebal di Irian Jaya. Penelitian ini dilakukan terhadap 129 pria Jawa dewasa yang normal G-6PDnya. Pemeriksaan hematologi, fungsi hati dan ginjal, dan pemeriksaan limfosit dilakukan berulang kali selama waktu penelitian profilaksis dilakukan untuk menjamin keamanan dari sukarelawan tersebut dan mengawasi perubahan yang mungkin terjadi akibat obat profilaksis. Seperti yang diperkirakan, pengguna primakuin tidak menunjukkan gejala peningkatan methemoglobin yang kembali dalam batas normal setelah 7 hari pemberian dosis terakhir. Pada akhir penelitian (12 bulan profilaksis nilai hematologi, fungsi hati dan ginjal, dan nilai limfosit dari kelompok primakuin sebanding dengan kelompok plasebo, dan berada dalam batas nilai normal untuk orang Indonesia.Hasil penelitian ini memberikan masukan adanya keluhan fisik yang sedikit dari sukarelawan pengguna profilaksis primakuin. Untuk membuktikan hasil penelitian ini dan mempersiapkan penggunaan secara umum primakuin untuk profilaksis malaria, perlu dilakukan uji coba lebih lanjut keamanan primakuin. Di Indonesia, primakuin tidak digunakan sebagai profilaksis dan laporan hasil penelitian ini hendaknya tidak ditafsirkan sebagai laporan keamanan dari primakuin.

  7. PENGEMBANGAN INSTRUMEN PENILAIAN KETERAMPILAN TEKNIK FOREHAND DAN BACKHAND DRIVE TENIS MEJA PADA ATLET USIA DINI

    Directory of Open Access Journals (Sweden)

    Verandita Rihtiana

    2014-09-01

    Full Text Available Penelitian ini bertujuan untuk mengembangkan instrumen penilaian keterampilan teknik forehand dan backhand drive tenis meja pada atlet usia dini. Penelitian ini menggunakan metode pene-litian dan pengembangan, dengan langkah-langkah pengembangan sebagai berikut: (1 pengumpulan informasi di lapangan, (2 melakukan analisis terhadap informasi yang dikumpulkan, (3 mengembangkan produk awal (draf model, (4 validasi ahli dan revisi, (5 uji coba skala kecil dan revisi, (6 uji coba skala besar dan revisi, (7 pembuatan produk final. Subjek penelitian atlet tenis meja usia dini. Analisis data untuk uji validitas dilakukan dengan CVR (content validity ratio dan reliabilitas dengan  menggunakan Alpha Crobanch. Penelitian ini menghasilkan buku panduan instrumen penilain keterampilan teknik forehand dan backhand drive tenis meja untuk atlet pemula  yang didalamnya berisi  petunjuk penggunaan, lembar tugas siswa, pedoman penilaian, rubrik penilaian, serta tabel penyekoran, yang memiliki validitas yang tinggi (1 dan reliabilitas yang tinggi¸forehand drive sikap awal: r = 0,975, pelaksanaan: r = 0,961, gerak lanjutan: r = 0, 955. Backhand drive sikap awal: r = 0,961, pelaksanaan r = 0,974, gerak lanjutan: r = 0, 989. Kata Kunci: instrumen penilaian teknik forehand dan backhand drive, tenis meja

  8. Deceased tissue donor serology and molecular testing for HIV, hepatitis B and hepatitis C viruses: a lack of cadaveric validated tests.

    Science.gov (United States)

    Victer, Thayssa Neiva da Fonseca; Dos Santos, Cris Stéphany Rodrigues; Báo, Sônia Nair; Sampaio, Thatiane Lima

    2016-12-01

    Vital to patient safety is the accurate assessment and minimization of risk for human immunodeficiency virus (HIV), Hepatitis C (HCV), and Hepatitis B (HBV) virus transmission by deceased donor organ and tissue transplantation. The pathogens are tested by serological kits based on enzyme-linked immunosorbent assay (ELISA), chemiluminescence (CLIA) and eletrochemiluminescence (ECLIA) immunoassays. Organ transplantation is a highly successful life-saving treatment in Brazil, but the Brazilian Health Surveillance Agency currently mandates that all deceased organ donors are screened for HIV, HCV and HBV following living donor policies. In this review, six ELISA (Wama(®), Bio-Rad(®), Biomerieux(®), DiaSorin(®), Acon Biotech(®) and Biokit(®)), three CLIA (Abbott(®), Siemens(®), Diasorin(®)) and one ECLIA (Roche(®)) were utilized for evaluating the effectiveness of those serological tests for deceased donors in Brazil according to manufacturer's guidelines. NAT for HIV, HCV and HBV can assist with detection of pre-seroconversion for those infections, and only Cobas(®) TaqScreen MPX(®) test, the Tigris System(®) Procleix Ultrio Assay(®) and the Bio-Manguinhos(®) HIV/HCV/HBV NAT are commercially available. Between all the tests, only the manufacturer Abbott(®) and Cobas(®) TaqScreen MPX(®) test are currently validated for cadaver samples.

  9. Rapid Sputum Multiplex Detection of the M. tuberculosis Complex (MTBC) and Resistance Mutations for Eight Antibiotics by Nucleotide MALDI-TOF MS

    Science.gov (United States)

    Su, Kang-Yi; Yan, Bo-Shiun; Chiu, Hao-Chieh; Yu, Chong-Jen; Chang, So-Yi; Jou, Ruwen; Liu, Jia-Long; Hsueh, Po-Ren; Yu, Sung-Liang

    2017-01-01

    The increasing incidence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis (MTB) adds further urgency for rapid and multiplex molecular testing to identify the MTB complex and drug susceptibility directly from sputum for disease control. A nucleotide matrix-assisted-laser-desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based assay was developed to identify MTB (MTBID panel) and 45 chromosomal mutations for resistance to eight antibiotics (MTBDR panel). We conducted a 300 case trial from outpatients to evaluate this platform. An MTBID panel specifically identified MTB with as few as 10 chromosome DNA copies. The panel was 100% consistent with an acid-fast stain and culture for MTB, nontuberculous mycobacteria, and non-mycobacteria bacteria. The MTBDR panel was validated using 20 known MDR-MTB isolates. In a 64-case double-blind clinical isolates test, the sensitivity and specificity were 83% and 100%, respectively. In a 300-case raw sputum trial, the MTB identification sensitivity in smear-negative cases using MALDI-TOF MS was better than the COBAS assay (61.9% vs. 46.6%). Importantly, the failure rate of MALDI-TOF MS was better than COBAS (11.3% vs. 26.3%). To the best of our knowledge, the test described herein is the only multiplex test that predicts resistance for up to eight antibiotics with both sensitivity and flexibility. PMID:28134321

  10. PENGEMBANGAN ENSIKLOPEDI BIOLOGI MOBILE BERBASIS ANDROID DALAM RANGKA IMPLEMENTASI KURIKULUM 2013

    Directory of Open Access Journals (Sweden)

    Dian Noviar

    2016-06-01

    Full Text Available Penelitian pengembangan ini bertujuan untuk menghasilkan Ensiklopedi Biologi Mobile Berbasis Android Untuk Siswa Madrasah Aliyah dalam Rangka Implementasi Kurikulum 2013, dan mengetahui kelayakan produk ensiklopedi biologi mobile yang dihasilkan. Model pengembangan yang digunakan dalam penelitian ini menggunakan model ADDIE. Empat tahapan pada prosedur pengembangan ADDIE yang dilalui dalam penelitian ini adalah Analysis, Design, Develop dan Evaluation. Ensiklopedi Biologi Mobile Berbasis Android dinilai oleh 1 orang Ahli Media, 1 orang Ahli Materi, 1 orang Ahli IT, 9 orang Peer Reviewer, dan 3 orang Guru Biologi. Uji coba terbatas dilakukan pada 25 orang siswa kelas X MA Ali Maksum Yogyakarta dan didukung 5 orang mahasiswa pendidikan biologi untuk mengetahui tanggapan responden terhadap produk ensiklopedi biologi mobile. Instrumen yang digunakan berupa angket check list. Data yang diperoleh berupa data kualitatif yang diubah kuantitatif kemudian ditabulasi dan dianalisis secara deskriptif kuantitatif dan kualitatif untuk mengetahui kualitas produk yang dikembangkan. Berdasarkan hasil penelitian menunjukkan bahwa Ensiklopedi Biologi Mobile Berbasis Android yang telah dikembangkan berdasarkan penilaian keseluruhan reviewer memperoleh kualitas Sangat Baik (87,42%. Hasil uji coba terbatas dari respon siswa dan mahasiswa menunjukkan bahwa Ensiklopedi Biologi Mobile Berbasis Android memiliki kualitas Sangat Baik (89,3% dan Sangat Baik (84,8%.     Kata Kunci: Ensiklopedi Biologi, Android, Mobile, sumber belajar, kurikulum

  11. Implementasi Indoor Positioning System Berbasis Smartphone dengan Penambahan Access Point untuk Studi Kasus Gedung Teknik Informatika ITS

    Directory of Open Access Journals (Sweden)

    Fananda Herda Perdana

    2017-01-01

    Full Text Available Saat ini kebutuhan masyarakat akan informasi lokasi sangat tinggi, terutama dengan memanfaatkan teknologi teknologi GPS. Tetapi untuk di dalam ruangan sistem ini memiliki akurasi yang rendah, apalagi untuk gedung indoor sehingga dikembangkan dengan konsep 3D Indoor Positioning System. Untuk meminimalisir penurunan akurasi Indoor Positioning System karena minimnya jumlah access point atau persebaran access point yang kurang merata, maka dalam penelitian ini akan ditambahkan access point di lokasi yang memiliki akses sinyal Wi-Fi di Gedung Teknik Informatika. Uji coba dilakukan dengan cara melakukan perbandingan hasil akurasi dari pengujian di beberapa lokasi kampus Teknik Informatika ITS ketika sebelum dilakukan penambahan access point dan ketika sesudah dilakukan penambahan access point. Setelah dilakukan penambahan access point di beberapa titik lokasi yang memiliki cakupan sinyal Wi-Fi lemah, dihasilkan akurasi yang meningkat hingga 16,67%, dari akurasi semula 78,70% menjadi  95,36% untuk seluruh titik uji coba pada gedung kampus. Selain itu juga penambahan access point berhasil meningkatkan akurasi 9 ruangan dari total 23 ruangan yang diuji secara signifikan (meningkat di atas 20%, terutama di daerah sekitar penambahan access point.

  12. The method of estimating bisulfite conversion rate in DNA methylation analysis.

    Science.gov (United States)

    Yangyang, Liu; Hengmi, Cui

    2015-09-01

    To establish an effective method to estimate the conversion rate of bisulfite-treated genomic DNA, TaqMan qPCR assay was performed using probes and primers that are specific for bisulfite-converted or -unconverted DNA standard samples separately. Then two linear standard curves were generated by plotting Ct values against logarithm of absolute DNA amount with serial dilutions of the bisulfite-converted or unconverted DNA samples. Based on two standard curves, the unknown bisulfite-treated genomic DNA sample was analyzed using the same TaqMan probes and the bisulfite conversion rate was precisely estimated. This method was further verified to be reliable using known mixed bisulfite-converted and -unconverted DNA templates as well as DNA samples treated with different bisulfite kits. These results showed that this method can effectively estimate bisulfite conversion rate of genomic DNA and thus provides a reliable and quick method for accurate analyses of DNA methylation.

  13. A new QRT-PCR assay designed for the differentiation between elements provided from Agrobacterium sp. in GMOs plant events and natural Agrobacterium sp. bacteria.

    Science.gov (United States)

    Nabi, Nesrine; Chaouachi, Maher; Zellama, Mohamed Salem; Ben Hafsa, Ahmed; Mrabet, Besma; Saïd, Khaled; Fathia, Harzallah Skhiri

    2016-04-01

    The question asked in the present work was how to differentiate between contamination of field samples with and GM plants contained sequences provided from this bacterium in order to avoid false positives in the frame of the detection and the quantification of GMO. For this, new set of primers and corresponding TaqMan Minor Groove Binder (MGB) probes were designed to target Agrobacterium sp. using the tumor-morphology-shooty gene (TMS1). Final standard curves were calculated for each pathogen by plotting the threshold cycle value against the bacterial number (log (colony forming units) per milliliter) via linear regression. The method designed was highly specific and sensitive, with a detection limit of 10CFU/ml. No significant cross-reaction was observed. Results from this study showed that TaqMan real-time PCR, is potentially an effective method for the rapid and reliable quantification of Agrobacterium sp. in samples containing GMO or non GMO samples.

  14. Validation of a fast real-time PCR method to detect fraud and mislabeling in milk and dairy products.

    Science.gov (United States)

    Di Domenico, M; Di Giuseppe, M; Wicochea Rodríguez, J D; Cammà, C

    2017-01-01

    Fast real-time PCR TaqMan assays were developed and validated for species identification in dairy products. Based on the amplification of 12S rRNA and cytB partial genes of mitochondrial DNA, the methods were demonstrated to be sensitive, fast, and species-specific for Bos taurus, Ovis aries, Bubalus bubalis, and Capra hircus. The limit of detection calculated was lower than 1%, and the efficiency was reported to be higher than 96% in every assay. An internal amplification control was used to detect possible false negatives. The method was validated by means of laboratory-prepared samples mixing different species. Moreover, 18 commercial dairy samples were analyzed by both real-time PCR and isoelectric focusing, the official European Union reference method. The 4 TaqMan assays were confirmed to be a useful tool for milk and dairy product authentication.

  15. Development of a real-time PCR method for the identification of Atlantic mackerel (Scomber scombrus).

    Science.gov (United States)

    Velasco, Amaya; Sánchez, Ana; Martínez, Icíar; Santaclara, Francisco J; Pérez-Martín, Ricardo I; Sotelo, Carmen G

    2013-12-01

    A Real Time-PCR method based on TaqMan technology for the identification of Scomber scombrus has been developed. A system of specific primers and a Minor Groove Binding (MGB) TaqMan probe based on sequences of the mitochondrial cytochrome b region was designed. The method was successfully tested in 81 specimens of S. scombrus and related species and validated in 26 different commercial samples. An average Threshold Cycle (Ct) value of 15.3 was obtained with S. scombrus DNA. With the other species tested fluorescence signal was not detected or Ct was significantly higher (P<0.001). The efficiency of the assay was estimated to be 92.41%, with 100% specificity, and no cross reactivity was detected with any other species. These results reveal that the developed method is a rapid and efficient tool to unequivocally identify S. scombrus and may aid in the prevention of fraud or mislabelling in mackerel products.

  16. Interim Report on SNP analysis and forensic microarray probe design for South American hemorrhagic fever viruses, tick-borne encephalitis virus, henipaviruses, Old World Arenaviruses, filoviruses, Crimean-Congo hemorrhagic fever viruses, Rift Valley fever

    Energy Technology Data Exchange (ETDEWEB)

    Jaing, C; Gardner, S

    2012-06-05

    The goal of this project is to develop forensic genotyping assays for select agent viruses, enhancing the current capabilities for the viral bioforensics and law enforcement community. We used a multipronged approach combining bioinformatics analysis, PCR-enriched samples, microarrays and TaqMan assays to develop high resolution and cost effective genotyping methods for strain level forensic discrimination of viruses. We have leveraged substantial experience and efficiency gained through year 1 on software development, SNP discovery, TaqMan signature design and phylogenetic signature mapping to scale up the development of forensics signatures in year 2. In this report, we have summarized the whole genome wide SNP analysis and microarray probe design for forensics characterization of South American hemorrhagic fever viruses, tick-borne encephalitis viruses and henipaviruses, Old World Arenaviruses, filoviruses, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus and Japanese encephalitis virus.

  17. Report for the NGFA-5 project.

    Energy Technology Data Exchange (ETDEWEB)

    Jaing, C; Jackson, P; Thissen, J; Wollard, J; Gardner, S; McLoughlin, K

    2011-11-15

    The objective of this project is to provide DHS a comprehensive evaluation of the current genomic technologies including genotyping, TaqMan PCR, multiple locus variable tandem repeat analysis (MLVA), microarray and high-throughput DNA sequencing in the analysis of biothreat agents from complex environmental samples. To effectively compare the sensitivity and specificity of the different genomic technologies, we used SNP TaqMan PCR, MLVA, microarray and high-throughput illumine and 454 sequencing to test various strains from B. anthracis, B. thuringiensis, BioWatch aerosol filter extracts or soil samples that were spiked with B. anthracis, and samples that were previously collected during DHS and EPA environmental release exercises that were known to contain B. thuringiensis spores. The results of all the samples against the various assays are discussed in this report.

  18. Detection of the free living amoeba Naegleria fowleri by using conventional and real-time PCR based on a single copy DNA sequence.

    Science.gov (United States)

    Régoudis, Estelle; Pélandakis, Michel

    2016-02-01

    The amoeba-flagellate Naegleria fowleri is a causative agent of primary amoebic meningoencephalitis (PAM). This thermophilic species occurs worldwide and tends to proliferate in warm aquatic environment. The PAM cases remain rare but this infection is mostly fatal. Here, we describe a single copy region which has been cloned and sequenced, and was used for both conventional and real-time PCR. Targeting a single-copy DNA sequence allows to directly quantify t