title: Development and validation of a multiplex reverse transcription PCR assay for simultaneous detection of three papaya viruses.
abstract: Papaya ringspot virus (PRSV), Papaya leaf distortion mosaic virus (PLDMV), and Papaya mosaic virus (PapMV) produce similar symptoms in papaya. Each threatens commercial production of papaya on Hainan Island, China. In this study, a multiplex reverse transcription PCR assay was developed to detect simultaneously these three viruses by screening combinations of mixed primer pairs and optimizing the multiplex RT-PCR reaction conditions. A mixture of three specific primer pairs was used to amplify three distinct fragments of 613 bp from the P3 gene of PRSV, 355 bp from the CP gene of PLDMV, and 205 bp from the CP gene of PapMV, demonstrating the assay's specificity. The sensitivity of the multiplex RT-PCR was evaluated by showing plasmids containing each of the viral target genes with 1.44 × 103, 1.79 × 103, and 1.91 × 102 copies for the three viruses could be detected successfully. The multiplex RT-PCR was applied successfully for detection of three viruses from 341 field samples collected from 18 counties of Hainan Island, China. Rates of single infections were 186/341 (54.5%), 93/341 (27.3%), and 3/341 (0.9%), for PRSV, PLDMV, and PapMV, respectively; 59/341 (17.3%) of the samples were co-infected with PRSV and PLDMV, which is the first time being reported in Hainan Island. This multiplex RT-PCR assay is a simple, rapid, sensitive, and cost-effective method for detecting multiple viruses in papaya and can be used for routine molecular diagnosis and epidemiological studies in papaya.
authors.affiliation: Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China. 273986569@qq.com.
authors.forename: Decai
authors.initials: D
authors.lastname: Tuo
authors.affiliation: Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China. dnaswt@hotmail.com.
authors.forename: Wentao
authors.initials: W
authors.lastname: Shen
authors.affiliation: Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China. 340014852@qq.com.
authors.forename: Yong
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China. yanpu@itbb.org.cn.
authors.forename: Pu
authors.initials: P
authors.lastname: Yan
authors.affiliation: Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China. lixiaoying@itbb.org.cn.
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Li
authors.affiliation: Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China. zhoupeng@itbb.org.cn.
authors.forename: Peng
authors.initials: P
authors.lastname: Zhou
mesh_terms: D029441:Carica / Q000821:virology*; D017931:DNA Primers / Q000235:genetics; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D010935:Plant Diseases / Q000821:virology*; D017863:Potexvirus / Q000235:genetics / Q000302:isolation & purification*; D017800:Potyvirus / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D017423:Sequence Analysis, RNA
chemical_list: D017931:DNA Primers; D012367:RNA, Viral