Establishment of a rapid one-step multiplex RT-PCR assay for simultaneous detec-tion of four viruses infecting jasmine
Jasmine plants often suffer from co-infection of multiple viruses during their growth.Due to the lack of research on the relationship between each virus and its symptom,it is challenging to accurately diagnose which type of virus is present solely based on viral symptoms.Therefore,the development of a rapid and simul-taneous one-step multiplex reverse transcription PCR(RT-PCR)method is crucial for the diagnosis of jasmine virus diseases.This study successfully established a one-step multiplex reverse transcription PCR(RT-PCR)sys-tem capable of simultaneously detecting four types of jasmine viruses:jasmine virus T(JaVT),jasmine virus C(JaVC),jasmine virus H(JaVH)and jasmine virus A(JaVA).In a total reaction volume of 20.0 μL,the opti-mal reaction system is as follows:the final concentrations of upstream and downstream primers for JaVT,JaVC,JaVH,and JaVA were set at 200,150,100 and 150 nmol·L-1,respectively.The optimal quantities of One-step Enzyme Mix,2xES One-Step Reaction Mix,and jasmine RNA were determined to be 0.2 μL,10.0 μL,and 1.0μg,respectively.The RT-PCR parameters were set as follows:RNA reverse transcription at 50 ℃ for 30 min,Pre-denaturation step at 94 ℃ for 2 min,30 cycles of denaturation at 94 ℃ for 30 s,annealing at 55℃for 30 s,extension at 72 ℃ for 45 s,and a final extension at 72 ℃ for 10 min.The results demonstrated that the estab-lished one-step multiplex RT-PCR method can efficiently and accurately detect all four types of jasmine viruses simultaneously,significantly enhancing detection efficiency.This method has broad applications in laboratory-based accurate detection and field-based diagnosis of jasmine virus diseases.
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