Detection of tobamoviruses by RT-PCR using a novel pair of degenerate primers

• Published in: Journal of virological methods Vol. 259; pp. 122 - 128
• Main Authors: Li, Yueyue, Tan, Guanlin, Lan, Pingxiu, Zhang, Ansheng, Liu, Yong, Li, Ruhui, Li, Fan
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.09.2018
• Subjects: Generic RT-PCR assay; Tobamovirus; Universal primers; Virus detection; Tobamovirus; Virus detection; Generic RT-PCR assay; Universal primers
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2018.06.012

•A generic RT-PCR assay for the universal detection of tobamoviruses using a novel pair of degenerate primers TobamodF/TobamodR was established.•The sensitivity of the RT-PCR assay was examined, and the detection limits of the assay were at least 10−7 for ToMMV, 10−5 for ORSV and 10−6 for CGMMV.•The RT-PCR detection assay was evaluated using pepper and tomato field samples.•The Tobamovirus generic RT-PCR assay is simple, rapid, specific, sensitive, reliable, and cost-effective for the detection of tobamoviruses. A generic RT-PCR assay was developed for the universal detection of viruses of the genus Tobamovirus using a novel pair of degenerate primers designed based on conserved regions on replicase genes of 32 tobamoviruses. The assay detected nine tobamoviruses, including six Solanaceae-infecting subgroup tobamoviruses of Tobacco mosaic virus (TMV), Tomato mosaic virus (ToMV), Tomato mottle mosaic virus (ToMMV), Tobacco mottle green mosaic virus (TMGMV), Pepper mild mottle virus (PMMoV), Paprika mild mottle virus (PaMMV), one Orchidaceae-infecting tobamovirus of Odontoglossum ringspot virus (ORSV) and two Cucurbitaceae-infecting subgroup tobamoviruses of Cucumber green mottle mosaic virus (CGMMV) and Zucchini green mottle mosaic virus (ZGMMV), with high amplification efficiency, specificity and sensitivity. The assay was applied to detect tobamoviruses in pepper and tomato fields. Five tobamoviruses, PMMoV, TMV, ToMV, ToMMV and TMGMV, were detected from the pepper fields in single and mixed infections. Single infections of PMMoV, ToMV and ToMMV and mix-infection of ToMV + PMMoV were detected from the tomato fields. Among these viruses, PMMoV was first detected from tomato worldwide, while ToMMV was first detected from tomato plants in China. This generic assay is simple, cost-effective and has great potential to detect more tobamoviruses in the field.