Metagenomics-based Plant Virus Detection Method with TMV Model

The tobacco leaves infected with Tobacco mosaic virus (TMV) was positive control, and the dsRNAs were extracted and the sequences analysis were performed by random primers amplification and high throughput sequencing. The PCR products of metagenomic sequencing results showed that the detection rate of TMV was 99.84%, and statistical results of classification and abundance of genus level display Tobamovirus accounted for 99.913%. The experiments proved that the method of dsRNA extraction method of metagenomic sequencing to plant virus were established successfully. According to the method of extracting virus like particles from tobacco infected by to TMV, then extracted RNA from the virus like particles, RT-PCR was conducted using TMV specific primers, and the results showed that the TMV sequence homology was 99%. The results proved that the method of nucleic acid of virus like particles extraction was established successfully and met the requirements of metagenomic analysis. The dsRNA and virus DNA of the like particles of 40 viral samples were constructed by this study. The nucleic acid of the all the samples had good quality. 20 kinds of plant viruses were detected from those samples. This study lays a foundation for the detection of plant virus populations and ecological adaptation in agricultural cultivation and non farming areas.