Development and validation of a multiplex reverse transcription quantitative PCR (RT-qPCR) assay for the rapid detection of Citrus tristeza virus, Citrus psorosis virus, and Citrus leaf blotch virus

Abstract

A single real-time multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay for the simultaneous detection of Citrus tristeza virus (CTV), Citrus psorosis virus (CPsV), and Citrus leaf blotch virus (CLBV) was developed and validated using three different fluorescently labeled minor groove binding qPCR probes. To increase the detection reliability, coat protein (CP) genes from large number of different isolates of CTV, CPsV and CLBV were sequenced and a multiple sequence alignment was generated with corresponding CP sequences from the GenBank and a robust multiplex RT-qPCR assay was designed. The capacity of the multiplex RT-qPCR assay in detecting the viruses was compared to singleplex RT-qPCR designed specifically for each virus and was assessed using multiple virus isolates from diverse geographical regions and citrus species as well as graft-inoculated citrus plants infected with various combination of the three viruses. 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. Comparison of the viral load for each virus using singleplex and multiplex RT-qPCR assays, revealed no significant differences between the two assays in virus detection. No significant difference in Cq values was detected when using one-step and two-step multiplex RT-qPCR detection formats. Optimizing the RNA extraction technique for citrus tissues and testing the quality of the extracted RNA using RT-qPCR targeting the cytochrome oxidase citrus gene as an RNA specific internal control proved to generate better diagnostic assays. Results showed that the developed multiplex RT-qPCR can streamline viruses testing of citrus nursery stock by replacing three separate singleplex assays, thus reducing time and labor while retaining the same sensitivity and specificity. The three targeted RNA viruses are regulated pathogens for California's mandatory "Section 3701: Citrus Nursery Stock Pest Cleanliness Program". Adopting a compatible multiplex RT-qPCR testing protocol for these viruses as well as other RNA and DNA regulated pathogens will provide a valuable alternative tool for virus detection and efficient program implementation.

Narcissus yellow stripe virus and Narcissus mosaic virus detection in Narcissus via multiplex TaqMan-based reverse transcription-PCR assay

“…Prior to this study, probe‐based detection methods have been described in other studies, including the multiplex simultaneous detection assay for Citrus viruses (Osman et al . ), for diseases and pathogens of potato tubers (Qu et al . ), for viroids and phytoplasmas of pome fruit trees (Malandraki et al .…”

“…This manuscript describes the establishment and application of a reliable and simple multiplex TaqMan RT-qPCR assay for the simultaneous detection of the NYSV and the NMV in narcissus samples. Prior to this study, probe-based detection methods have been described in other studies, including the NYSV 10 À3 10 À8 10 3 10 À8 10 3 NMV 10 À3 10 À8 10 3 10 À8 10 3 multiplex simultaneous detection assay for Citrus viruses (Osman et al 2015), for diseases and pathogens of potato tubers (Qu et al 2011), for viroids and phytoplasmas of pome fruit trees (Malandraki et al 2015), for the Grapevine virus A, B, and D (Osman et al 2013), for the PMTV and for the TRV (Mumford et al 2000). These studies have demonstrated the detection superiority of qPCR over other detection technologies, such as the salient feature of high sensitivity (Fu et al 2015).…”

“…), and citrus with the Citrus tristeza virus , the Citrus psorosis virus and the Citrus leaf blotch virus (Osman et al . ). More recently, a branched, robust and high‐throughput TaqMan method has been described for viral detection in different ornamental crops (Boonham et al .…”

“…Multiplex real-time RT-PCR in particular has been applied to facilitate the detection of target viruses due to high precision and sensitivity, as well as due to fast and simple performance for the detection of several pathogens in a single reaction (Heid et al 1996;Qu et al 2011;Malandraki et al 2015;Wang et al 2016). Accordingly, the common multiplex real-time RT-PCR method was developed for plant material that was infected with multiple viruses, for example, the potato with the Potato mop top virus (PMTV) and the Tobacco rattle virus (TRV) (Mumford et al 2000), and citrus with the Citrus tristeza virus, the Citrus psorosis virus and the Citrus leaf blotch virus (Osman et al 2015). More recently, a branched, robust and high-throughput Taq-Man method has been described for viral detection in different ornamental crops (Boonham et al 2002;Wei et al 2012;Chen et al 2013a,b;Lin et al 2013;Tang et al 2014;Dobhal et al 2016).…”

Pest categorisation of naturally‐spreading psorosis

“…Complete genome sequences of a number of CPsV isolates are available for comparison and a number of molecular tests, hybridisation assays and reverse transcription polymerase chain reaction (RT-PCR) protocols allow a reliable detection of CPsV (Garc ıa et al, 1997;Barthe et al, 1998;Rosa et al, 2007;Osman et al, 2015). A real-time RT-PCR-based assay for simultaneous detection of several citrus viruses, including CPsV, has been developed (Loconsole et al, 2010).…”