کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
6134210 | 1593482 | 2013 | 8 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Development and validation of a multiplex quantitative PCR assay for the rapid detection of Grapevine virus A, B and D
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
ایمنی شناسی و میکروب شناسی
ویروس شناسی
پیش نمایش صفحه اول مقاله
![عکس صفحه اول مقاله: Development and validation of a multiplex quantitative PCR assay for the rapid detection of Grapevine virus A, B and D Development and validation of a multiplex quantitative PCR assay for the rapid detection of Grapevine virus A, B and D](/preview/png/6134210.png)
چکیده انگلیسی
A single real-time multiplex quantitative PCR (qPCR) assay for the simultaneous detection of Grapevine virus A, B and D (GVA, GVB and GVD) was developed, using three different fluorescently labeled minor groove binding probes. This multiplex RT-qPCR was compared to singleplex RT-qPCR designed specifically for each virus and a conventional multiplex RT-PCR. The capacity of the multiplex RT-qPCR assay in detecting the three vitiviruses in mixed infections from a range of virus concentrations in the host was assessed. A series of cDNA derived from 48 different grapevine cultivars obtained from diverse geographical regions infected with various isolates and strains of GVA, GVB and GVD were subjected to singleplex, multiplex RT-qPCR, and conventional multiplex RT-PCR testing. The results showed that the developed multiplex RT-qPCR assay was a cost-effective diagnostic tool that could streamline the testing of grapevine vitiviruses, and replace the singleplex RT-qPCR assays, thus reducing time and labor while retaining the same sensitivity and specificity. In particular, no significant differences in detection limits were found between singleplex and multiplex RT-qPCR and specificity was not affected by the inclusion of the three primer/probe combinations within a multiplex RT-qPCR. Comparing the viral load for each virus using singleplex and multiplex RT-qPCR assays revealed no significant differences between the two assays in detecting GVB and GVD. However, while in detecting GVA using singleplex RT-qPCR assay, viral load was higher. Finally, the multiplex RT-qPCR assay was also more sensitive and time efficient than the conventional multiplex RT-PCR that was designed using degenerate primers to detect GVA, GVB and GVD. This multiplex RT-qPCR method could detect viruses in 95.83% of mixed infected samples as compared to 77.08% for multiplex RT-PCR.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Virological Methods - Volume 194, Issues 1â2, December 2013, Pages 138-145
Journal: Journal of Virological Methods - Volume 194, Issues 1â2, December 2013, Pages 138-145
نویسندگان
Fatima Osman, Emir Hodzic, Alicja Omanska-Klusek, Tammi Olineka, Adib Rowhani,