Gu Li, Liu Chensi, Yao Shuting, Wu Jiaxin, Wang Lianghong, Mu Jing, Wang Yankun, Wang Jianming, Zhang Zhongyi, Li Mingjie
College of Bee Science and Biomedicine, Fujian Agriculture and Forestry University, Fuzhou 350002, China.
Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, Fujian Agriculture and Forestry University, Fuzhou 350002, China.
Microorganisms. 2024 Dec 22;12(12):2663. doi: 10.3390/microorganisms12122663.
(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 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 . Furthermore, the levels of BBWV2 were examined during its infection of . 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 sampled 60 sites, 96% were found to be infected by one of two viruses. The levels of BBWV2 in and demonstrated an initial increase, followed by a subsequent decrease. The TaqMan qPCR methods provide a technical foundation for the monitoring of virus infections in .
（米q.）白前，一种备受珍视的中药材，由于病毒病，尤其是芜菁花叶病毒（TuMV）和蚕豆萎蔫病毒2型（BBWV2）引起的病毒病，其产量和品质正在显著下降。目前，缺乏针对这些病毒的定量检测方法阻碍了准确诊断。因此，开发一种准确的定量检测方法对于有效管理该植物的病毒病至关重要。在本研究中，基于两种病毒克隆载体，开发了用于检测这两种病毒的单重和双重TaqMan定量聚合酶链反应（qPCR）。同时，在白前的主要产区检测了两种病毒的含量。此外，还检测了BBWV2在白前感染过程中的含量。最佳单重qPCR用于TuMV的水解探针浓度为0.1μM，引物浓度为0.1μM，而用于BBWV2的水解探针浓度为0.2μM，引物浓度为0.1μM。相比之下，双重qPCR使用每个引物对的0.1μM上游/下游引物以及0.2μM各自的水解探针。单重qPCR的95%检测限（LOD）对于TuMV为734拷贝，对于BBWV2为20拷贝，而双重qPCR的95% LOD对于TuMV为945拷贝，对于BBWV2为47拷贝。此外，单重和双重qPCR的批内和批间变异系数均小于1.2%。在采样的60个位点中，96%被发现感染了两种病毒中的一种。BBWV2在白前和其他样本中的含量呈现出先升高后降低的趋势。TaqMan qPCR方法为监测白前的病毒感染提供了技术基础。
