A sensitive nested multiplex RT-PCR assay for the simultaneous detection of three common viruses infecting pear plants

• Published in: Journal of virological methods 2019-01, Vol.263, p.105-110
• Main Authors: Yao, Bingyu, Lu, Yongcan, Li, Liu, Wang, Yanxiang, Li, Qingyu, Wang, Guoping, Hong, Ni
• Format: Article
• Language: English
• Subjects: Apple chlorotic leaf spot virus; Apple stem grooving virus; Apple stem pitting virus; Flexiviridae - classification; Flexiviridae - genetics; Flexiviridae - isolation & purification; NmRT-PCR assay; Pear; Plant Bark - virology; Plant Diseases - virology; Plant Leaves - virology; Pyrus - virology; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral - genetics; Sensitivity and Specificity; Viral Proteins - genetics
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2018.10.021

•A nested multiplex reverse transcription-polymerase chain reaction (nmRT-PCR) assay was developed.•The nmRT-PCR assay was highly sensitive for simultaneous detection of three pear viruses.•The nmRT-PCR was feasible for the detection of the three viruses infecting cultivated and wild pear. A highly sensitive nested multiplex reverse transcription-polymerase chain reaction (nmRT-PCR) assay was developed for the simultaneous detection of Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV) and Apple stem pitting virus (ASPV) infecting pear trees. In the assay, a set of three forward primers specific to each of the three viruses and a universal reverse primer was used as external primers in the first-round PCR, which was followed by a second-round PCR developed previously. The nmRT-PCR assay was 104 times more sensitive than conventional mRT-PCR assay in detecting the three viruses in in vitro pear plantlets. This assay was subsequently used to detect these viruses in leaf and bark samples of cultivated and wild pear trees from orchards and demonstrated to be highly sensitive and reliable. This is the first report describing a use of nmRT-PCR for the sensitive and simultaneous detection of the three viruses infecting pear plants. The assay would be useful for the certification of pear planting materials and surveillance of nursery stocks.