title: A multiple RT-PCR assay for simultaneous detection and differentiation of latent viruses and apscarviroids in apple trees.
abstract: Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), and Apple stem pitting virus (ASPV) are three latent viruses frequently occurring in apple trees worldwide. In field orchards, these viruses are frequently found in a mixed infection with viroids in the genus Apscarviroid, including Apple scar skin viroid, and Apple dimple fruit viroid. Together these viruses and viroids could cause serious damage to apple fruit production worldwide. Rapid and efficient detection methods are pivotal to identify and select the virus-free propagation material for healthy apple orchard management. In this study a multiplex Reverse Transcription-PCR (RT-PCR) was developed and optimized for simultaneous detection and differentiation of the three latent viruses and apscarviroids. With newly designed specific primers for ACLSV, ASGV, APSV, and EF-1α (as an internal control), and a pair of degenerate primers for apscarviroids, optimized parameters for multiplex RT-PCR were determined. The resulting PCR products from each target virus and viroid could be easily identified because their product sizes differ by at least a 100bp. The multiplex RT-PCR method is expected to detect different variants of the viruses as the test results showed that a variety of isolates from different regions in China gave positive results. To the best of our knowledge, this multiplex RT-PCR assay is the first to simultaneously detect multiple viruses and viroids infecting apple trees in a single reaction tube. This assay, therefore, offers a useful tool for routine certification and quarantine programs.
authors.affiliation: State Key Laboratory of Agrobiotechnology and Department of Plant Pathology, China Agricultural University, No. 2 West Yuanmingyuan Road, Beijing 100193, China.
authors.forename: Lu
authors.initials: L
authors.lastname: Hao
authors.affiliation: State Key Laboratory of Agrobiotechnology and Department of Plant Pathology, China Agricultural University, No. 2 West Yuanmingyuan Road, Beijing 100193, China.
authors.forename: Jipeng
authors.initials: J
authors.lastname: Xie
authors.affiliation: State Key Laboratory of Agrobiotechnology and Department of Plant Pathology, China Agricultural University, No. 2 West Yuanmingyuan Road, Beijing 100193, China.
authors.forename: Shanyi
authors.initials: S
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Agrobiotechnology and Department of Plant Pathology, China Agricultural University, No. 2 West Yuanmingyuan Road, Beijing 100193, China.
authors.forename: Shaojie
authors.initials: S
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Agrobiotechnology and Department of Plant Pathology, China Agricultural University, No. 2 West Yuanmingyuan Road, Beijing 100193, China.
authors.forename: Zhuoqun
authors.initials: Z
authors.lastname: Gong
authors.affiliation: U.S. Department of Agriculture, Agricultural Research Service, U.S. Vegetable Laboratory, 2700 Savannah Highway, Charleston, SC 29414, USA.
authors.forename: Kai-Shu
authors.initials: KS
authors.lastname: Ling
authors.affiliation: State Key Laboratory of Agrobiotechnology and Department of Plant Pathology, China Agricultural University, No. 2 West Yuanmingyuan Road, Beijing 100193, China.
authors.forename: Liyun
authors.initials: L
authors.lastname: Guo
authors.affiliation: State Key Laboratory of Agrobiotechnology and Department of Plant Pathology, China Agricultural University, No. 2 West Yuanmingyuan Road, Beijing 100193, China.
authors.forename: Zaifeng
authors.initials: Z
authors.lastname: Fan
authors.affiliation: State Key Laboratory of Agrobiotechnology and Department of Plant Pathology, China Agricultural University, No. 2 West Yuanmingyuan Road, Beijing 100193, China. Electronic address: taozhoucau@cau.edu.cn.
authors.forename: Tao
authors.initials: T
authors.lastname: Zhou
mesh_terms: D002681:China; D017931:DNA Primers; D052677:Flexiviridae / Q000235:genetics / Q000302:isolation & purification; D016679:Genome, Viral; D027845:Malus / Q000821:virology*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D010935:Plant Diseases / Q000821:virology; D010942:Plant Viruses / Q000302:isolation & purification*; D012367:RNA, Viral / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D014772:Viroids / Q000235:genetics / Q000302:isolation & purification*; D017735:Virus Latency
chemical_list: D017931:DNA Primers; D012367:RNA, Viral
keywords: Apple Chlorotic leaf spot virus (ACLSV); Apple scar skin viroid (ASSVd); Apple stem grooving virus (ASGV); Apple stem pitting virus (ASPV); Apscarviroid; Certification program