Reverse transcription loop-mediated isothermal amplification assay for rapid detection of Papaya ringspot virus.
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.
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.
Wentao
W
Shen
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.
Decai
D
Tuo
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.
Pu
P
Yan
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.
Yong
Y
Yang
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.
Xiaoying
X
Li
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.
Peng
P
Zhou
D029441:Carica / Q000821:virology*; D002681:China; D003362:Cost-Benefit Analysis; D017931:DNA Primers / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000191:economics / Q000379:methods*; D017800:Potyvirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics; D048348:Reverse Transcription; D012680:Sensitivity and Specificity; D013995:Time; D013997:Time Factors; D014777:Virus Diseases / Q000821:virology*
D017931:DNA Primers; D012367:RNA, Viral
PRSV detection; Papaya ringspot virus; RT-PCR; Reverse transcription loop-mediated isothermal amplification