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

Bingyu Yao; Yongcan Lu; Liu Li; Yanxiang Wang; Qingyu Li; Guoping Wang; Ni Hong

doi:10.1016/j.jviromet.2018.10.021

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

J Virol Methods. 2019 Jan:263:105-110. doi: 10.1016/j.jviromet.2018.10.021. Epub 2018 Oct 29.

Authors

Bingyu Yao¹, Yongcan Lu², Liu Li³, Yanxiang Wang³, Qingyu Li⁴, Guoping Wang⁵, Ni Hong⁶

Affiliations

¹ State Key Laboratory of Agromicrobiology, Huazhong Agricultural University, Wuhan, Hubei 430070, China; Key Laboratory of Horticultural Crop (Fruit Trees) Biology and Germplasm Creation of the Ministry of Agriculture, Huazhong Agricultural University, Wuhan, Hubei 430070, China.
² State Key Laboratory of Agromicrobiology, Huazhong Agricultural University, Wuhan, Hubei 430070, China; The Key Laboratory of Plant Pathology of Hubei Province, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China.
³ State Key Laboratory of Agromicrobiology, Huazhong Agricultural University, Wuhan, Hubei 430070, China.
⁴ Yantai Academy of Agricultural Science, Yantai, Shandong 264000, China.
⁵ State Key Laboratory of Agromicrobiology, Huazhong Agricultural University, Wuhan, Hubei 430070, China; The Key Laboratory of Plant Pathology of Hubei Province, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China; Key Laboratory of Horticultural Crop (Fruit Trees) Biology and Germplasm Creation of the Ministry of Agriculture, Huazhong Agricultural University, Wuhan, Hubei 430070, China.
⁶ State Key Laboratory of Agromicrobiology, Huazhong Agricultural University, Wuhan, Hubei 430070, China; The Key Laboratory of Plant Pathology of Hubei Province, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China; Key Laboratory of Horticultural Crop (Fruit Trees) Biology and Germplasm Creation of the Ministry of Agriculture, Huazhong Agricultural University, Wuhan, Hubei 430070, China. Electronic address: whni@mail.hzau.edu.cn.

PMID: 30385299
DOI: 10.1016/j.jviromet.2018.10.021

Abstract

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.

Keywords: Apple chlorotic leaf spot virus; Apple stem grooving virus; Apple stem pitting virus; NmRT-PCR assay; Pear.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Flexiviridae / classification
Flexiviridae / genetics
Flexiviridae / isolation & purification*
Plant Bark / virology
Plant Diseases / virology
Plant Leaves / virology
Pyrus / virology*
RNA, Viral / genetics
Reverse Transcriptase Polymerase Chain Reaction*
Sensitivity and Specificity
Viral Proteins / genetics

Substances

RNA, Viral
Viral Proteins