Development of a TaqMan qPCR for the Simultaneous Detection of the TuMV and BBWV2 Viruses Responsible for the Viral Disease in Pseudostellaria heterophylla.

Abstract

Pseudostellaria heterophylla (Miq.) Pax, a highly valued Chinese medicinal plant, is experiencing a notable decline in yield and quality due to viral diseases, particularly caused those by TuMV and BBWV2. Currently, the absence of a quantitative detection method for these viruses in P. heterophylla impedes the accurate diagnosis. The development of an accurate quantitative detection method is thus essential for effectively managing viral diseases in this plant. In this study, singleplex and duplex TaqMan qPCR were developed for the detection of the two viruses, based on two viral cloning vectors. Concurrently, the levels of both viruses were examined in the main produced regions of P. heterophylla. Furthermore, the levels of BBWV2 were examined during its infection of P. heterophylla. The optimal singleplex qPCR employed 0.1 μM of hydrolysis probe and 0.1 μM of primer for TuMV, while 0.2 μM of hydrolysis probe and 0.1 μM of primer were utilised for BBWV2. In contrast, the duplex qPCR employed the use of 0.1 μM of the upstream/downstream primer from each primer pair, with 0.2 μM of the respective hydrolysis probes. The 95% limit of detection (LOD) for singleplex qPCR was 734 copies for TuMV and 20 copies for BBWV2, while the 95% LOD for duplex qPCR was 945 copies for TuMV and 47 copies for BBWV2. Furthermore, the intra- and inter-assay coefficients of variation were found to be less than 1.2% for both singleplex and duplex qPCR. Of the P. heterophylla sampled 60 sites, 96% were found to be infected by one of two viruses. The levels of BBWV2 in N. benthamiana and P. heterophylla demonstrated an initial increase, followed by a subsequent decrease. The TaqMan qPCR methods provide a technical foundation for the monitoring of virus infections in P. heterophylla.