A developed multiplex RT-PCR assay for simultaneous detection of four main viruses on pepper (Capsicum spp.)

Abstract

Viral diseases cause severe losses commonly and become one of main global limiting factors in pepper production. Chilli veinal mottle virus (ChiVMV), pepper veinal mottle virus (PVMV), cucumber mosaic virus (CMV), and pepper mild mottle virus (PMMoV) are the dominant viruses damaging pepper in South China. To detect these viruses efficiently, a developed multiplex reverse transcription polymerase chain reaction (RT-PCR) method was established. Four pairs of specific primers were used to amplify a 990 bp product for PMMoV, a 923 bp product for ChiVMV, a 823 bp product for PVMV, and a 682 bp product for CMV. The optimal primer concentration, the optimal reaction annealing temperature and cycle number were determined. The developed multiplex RT-PCR could detect PMMoV, ChiVMV, CMV and PVMV from cDNA diluted up to 10–4, 10–4, 10–3 and 10–3, respectively. Then it was sucessfully used to detect virus infection from eight mixed pepper leaf samples. The result were basically consistent with that of monoplex RT-PCR. Additionally, seventy-five diseased samples collected from nine major pepper growing regions in Guangxi were analyzed by the developed multiplex RT-PCR. The results showed that most of samples were coinfected by two or more viruses, and ChiVMV and PVMV had relatively higher total detection rates of 65.33% and 60.00% respectivlely. The results also indicated that the four viruses infecting pepper weren't evenly distributed in Guangxi. In a conclusion, the developed multiplex RT-PCR here will be a efficient tool for routine molecular diagnosis of PMMoV, ChiVMV, PVMV and CMV.