摘要
茉莉生长过程中普遍受到多种病毒的复合侵染.由于缺乏病毒与症状关系的系统研究,仅通过症状很难准确分辨茉莉被何种病毒侵染,所以建立快速精准诊断多种茉莉病毒病的检测技术具有重要应用价值.本研究建立了一种可同时检测 4 种茉莉病毒——茉莉 T 病毒(jasmine virus T,JaVT)、茉莉 C 病毒(jasmine virus C,JaVC)、茉莉 H 病毒(jasmine virus H,JaVH)和茉莉 A 病毒(jasmine virus A,JaVA)——的一步法多重逆转录 PCR(one-step multiplex reverse transcription-PCR,RT-PCR)体系,其最优反应体系为:在总体系20.0 μL中,JaVT、JaVC、JaVH和JaVA的上下游引物浓度分别为200、150、100 和 150 nmol-L-1,One-step Enzyme Mix:0.2 μL,2×反应缓冲液:10.0 μL,模板量:1.0 μg;多重 RT-PCR 参数设定为:RNA反转录50 ℃ 30 min;预变性94 ℃ 2 min;94 ℃变性30 s;55 ℃退火30s;72 ℃延伸45 s,30个循环;72 ℃整体延伸10 min.研究结果表明建立的一步法多重RT-PCR可同时高效精准地检测4种茉莉病毒,极大地提高了检测效率,可被广泛应用于实验室精准、高效检测和田间茉莉病毒病的检测.
Abstract
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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