Identification of Viruses Infecting Bottle Gourd in Guangdong Province and Establishment of Multiplex RT-PCR Detection Method
[Objective]The objective of this research is to determine the various viruses that infect bottle gourd,establish a multiplex RT-PCR detection method that can simultaneously detect multiple bottle gourd viruses,so as to enhance the efficiency of identifying bottle gourd virus species,and provide a foundation for accurate prevention and control of bottle gourd viral diseases.[Method]From 2018 to 2022,78 bottle gourd samples of suspected virus infection were collected from the main planting areas of bottle gourd in Guangdong Province,including Guangzhou,Huizhou,Qingyuan,Shantou,and Zhanjiang.The collected samples were sorted by region and symptom,and the total RNA of each sample was extracted.The RNA samples with similar symptoms from each region were mixed for small RNA deep sequencing analysis.According to the results of small RNA deep sequencing analysis,specific primers were designed for RT-PCR verification to identify the specific virus species infecting bottle gourd in Guangdong Province.Six viruses with higher incidence and more losses were selected,and multiplex PCR detection primers based on the GenBank database were designed.After optimizing the reaction system and conditions,a method for simultaneous detection of all six viruses via multiplex RT-PCR was established.[Result]A total of 12 viruses were detected in 78 bottle gourd samples of suspected virus infection.These viruses were ranked in order of infection rate,from highest to lowest:Lagenaria siceraria alphaendornavirus(LSEV)(64.1％),cucumber green mottle mosaic virus(CGMMV)(62.8％),zucchini tigre mosaic virus(ZTMV)(51.3％),watermelon green mottle mosaic virus(WGMMV)(43.6％),watermelon virus A(WVA)(32.1％),papaya ringspot virus(PRSV)(19.2％),zucchini yellow mosaic virus(ZYMV)(9.0％),cucurbit aphid-borne yellows virus(CABYV)(7.7％),squash leaf curl China virus(SLCCNV)(7.7％),cucurbit chlorotic yellows virus(CCYV)(5.1％),cucurbit yellow stunting disorder virus(CYSDV)(3.8％),melon yellow spot virus(MYSV)(1.3％).It was found that 80.8％of the 78 samples were infected with multiple viruses.The types of mixed infection included 2 viruses(14.1％),3 viruses(16.7％),4 viruses(23.1％),5 viruses(17.9％),6 viruses(7.7％)and 7 viruses(1.3％),respectively.The WVA+CCYV+ZTMV+PRSV primers were added firstly in the multiplex RT-PCR system,followed by the WGMMV+CGMMV primers after 12 reaction cycles.The volumes of primers were used:WVA 0.5 μL,CCYV 0.5 μL,CGMMV 0.4 μL,WGMMV 0.4 μL,ZTMV 0.6 μL,and PRSV 0.6 μL.Thus,a multiplex RT-PCR assay capable of simultaneously amplifying six viruses was established.[Conclusion]There are 12 primary viruses that infect bottle gourd in Guangdong Province.Among them,CGMMV,ZTMV,WGMMV,and WVA are the most predominant viruses.Mixed infection is a commonly observed phenomenon,with three,four,and five viruses'co-infection being the most prevalent types.A multiplex RT-PCR detection method has been developed to simultaneously identify six viruses in bottle gourd,which improves the detection efficiency of virus species in bottle gourd.
bottle gourdvirus diseasesmall RNA deep sequencingRT-PCR detectionmultiplex PCR method
国家科技期刊平台
NSTL
万方数据
维普
