Simultaneous detection and differentiation of three viruses in pear plants by a multiplex RT-PCR

Bingyu Yao; Guoping Wang; Xiaofang Ma; Wenbin Liu; Huihui Tang; Hui Zhu; Ni Hong

doi:10.1016/j.jviromet.2013.11.005

Simultaneous detection and differentiation of three viruses in pear plants by a multiplex RT-PCR

J Virol Methods. 2014 Feb:196:113-9. doi: 10.1016/j.jviromet.2013.11.005. Epub 2013 Nov 20.

Authors

Bingyu Yao¹, Guoping Wang², Xiaofang Ma³, Wenbin Liu³, Huihui Tang³, Hui Zhu³, Ni Hong⁴

Affiliations

¹ National 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.
² National 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; National Indoor Conservation Center of Virus-free Germplasm of Fruit Crops, Huazhong Agricultural University, Wuhan, Hubei 430070, China.
³ The Key Laboratory of Plant Pathology of Hubei Province, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China.
⁴ National 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; National Indoor Conservation Center of Virus-free Germplasm of Fruit Crops, Huazhong Agricultural University, Wuhan, Hubei 430070, China. Electronic address: whni@mail.hzau.edu.cn.

PMID: 24269332
DOI: 10.1016/j.jviromet.2013.11.005

Abstract

A multiplex RT-PCR (mRT-PCR) assay was developed for detection and differentiation of the Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV), which are viruses frequently occurring in pear trees. Different combinations of mixed primer pairs were tested for their specificity and sensitivity for the simultaneous detection of the three viruses. Three primer pairs were used to amplify their fragments of 247bp, 358bp and 500bp, respectively. The primer pair for ASPV was designed in this work, while the primer pairs for ACLSV and ASGV were from previous reports. The sensitivity and specificity of the mRT-PCR assay for the three viruses were comparable to that of each uniplex RT-PCR. The mRT-PCR was applied successfully for the detection of three viruses in leaves of pear and apple plants, but was unreliable in the detection of ASGV in dormant barks. In conclusion, this mRT-PCR provides a useful tool for the routine and rapid detection and the differentiation of three pear viruses.

Keywords: Apple chlorotic leaf spot virus; Apple stem grooving virus; Apple stem pitting virus; Multiplex RT-PCR; Pear.

Publication types

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

MeSH terms

DNA Primers / genetics
Malus / virology
Multiplex Polymerase Chain Reaction / methods*
Plant Diseases / virology*
Plant Leaves / virology
Plant Viruses / genetics
Plant Viruses / isolation & purification*
Pyrus / virology*
RNA, Viral / genetics
RNA, Viral / isolation & purification
Reverse Transcriptase Polymerase Chain Reaction / methods*
Sensitivity and Specificity
Virology / methods*

Substances

DNA Primers
RNA, Viral