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J Virol Methods. 2014 Aug;204:93-100. doi: 10.1016/j.jviromet.2014.04.012. Epub 2014 Apr 24.

Reverse transcription loop-mediated isothermal amplification assay for rapid detection of Papaya ringspot virus.

Author information

1
Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology & Analysis and Testing Center, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China.
2
Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology & Analysis and Testing Center, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China. Electronic address: dnaswt@hotmail.com.

Abstract

Papaya ringspot virus (PRSV) and Papaya leaf distortion mosaic virus (PLDMV), which causes disease symptoms similar to PRSV, threaten commercial production of both non-transgenic-papaya and PRSV-resistant transgenic papaya in China. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to detect PLDMV was developed previously. In this study, the development of another RT-LAMP assay to distinguish among transgenic, PRSV-infected and PLDMV-infected papaya by detection of PRSV is reported. A set of four RT-LAMP primers was designed based on the highly conserved region of the P3 gene of PRSV. The RT-LAMP method was specific and sensitive in detecting PRSV, with a detection limit of 1.15×10(-6)μg of total RNA per reaction. Indeed, the reaction was 10 times more sensitive than one-step RT-PCR. Field application of the RT-LAMP assay demonstrated that samples positive for PRSV were detected only in non-transgenic papaya, whereas samples positive for PLDMV were detected only in commercialized PRSV-resistant transgenic papaya. This suggests that PRSV remains the major limiting factor for non-transgenic-papaya production, and the emergence of PLDMV threatens the commercial transgenic cultivar in China. However, this study, combined with the earlier development of an RT-LAMP assay for PLDMV, will provide a rapid, sensitive and cost-effective diagnostic power to distinguish virus infections in papaya.

KEYWORDS:

PRSV detection; Papaya ringspot virus; RT-PCR; Reverse transcription loop-mediated isothermal amplification

PMID:
24769198
DOI:
10.1016/j.jviromet.2014.04.012
[Indexed for MEDLINE]

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