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Emir Hodzic 1, 1School of Veterinary Medicine, University of California at Davis, United States; |
Abstract
Different instruments and methods were used for tissue homog- enization, RNA extraction and qPCR based detection of grapevine and citrus RNA viruses were evaluated. Semi-automated and auto- mated homogenization techniques were compared to process samples from grapevine and citrus. Four different high throughput automated nucleic acid extraction platforms were compared with the RNeasy plant extraction kit for their capacity and efficiency of extracting viral RNA from grapevine and citrus infected tissues. The RNA prepared from each extraction platform was then used as tem- plate for a comparative analysis. The study showed that a thorough homogenization of grapevine and citrus tissues using the Tissue Lyser as well as DNase digestion of the purified RNA prior to cDNA synthesis improved the virus detection and yielded the lowest Cq values in RT-qPCR. Comparison of different RNA extraction meth- ods showed that methods implementing the magnetic bead-based technology were superior to other methods used.
The development of diagnostic systems to effectively detect multiple targets in a single assay has provided a constant techno- logical challenge. The main goal of this study was to develop a rapid, sensitive and specific multiplex RT-qPCR assays for the detection and quantification of viruses in grapevines and citrus that can be incorporated into routine virus detection protocols. The proposed approach can then be used as a robust diagnostic tool for grapevine and citrus germplasm screening programs and for the certification program to ensure clean plant material for propagation.
A single real-time multiplex 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 con- ventional multiplex RT-PCR. The results showed that the developed multiplex RT-qPCR assay was a cost-effective diagnostic tool that could streamline the testing of grapevine viruses, and replace the singleplex RT-qPCR assays, thus reducing time and labor while retaining the same sensitivity and specificity.
For detection of Citrus tristeza virus (CTV), Citrus psorosis virus (CPsV), and Citrus leaf blotch virus (CLBV) a single multiplex RT-qPCR was developed and validated using the same approach as above. To increase the detection reliability, coat protein from large number of different isolates of CTV, CPsV and CLBV were sequenced and multi- ple sequence alignments were generated. No significant difference in detection limits was found and specificity was not affected by the inclusion of the three assays in a multiplex RT-qPCR reaction.
Adopting compatible multiplex RT-qPCR testing protocols for grapevine and citrus viruses as well as other RNA and DNA regulated pathogens will provide a valuable alternative tool for pathogen detection and efficient program implementation.
http://dx.doi.org/10.1016/j.bdq.2017.02.084
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