Rapid and real-time detection of Porcine Sapelovirus by reverse transcription loop-mediated isothermal amplification assay

Chunyan Wang; Dayi Yu; Li Cui; Xiuguo Hua; Congli Yuan; Huan Sun; Yuxiao Liu

doi:10.1016/j.jviromet.2014.03.011

Rapid and real-time detection of Porcine Sapelovirus by reverse transcription loop-mediated isothermal amplification assay

J Virol Methods. 2014 Jul:203:5-8. doi: 10.1016/j.jviromet.2014.03.011. Epub 2014 Mar 22.

Authors

Chunyan Wang¹, Dayi Yu², Li Cui³, Xiuguo Hua⁴, Congli Yuan⁵, Huan Sun⁶, Yuxiao Liu⁷

Affiliations

¹ Shanghai Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, China. Electronic address: wcy_an@sina.cn.
² Animal Disease Control Center of Minhang District, 1633 Humin Road, Shanghai, China. Electronic address: mhqsys@126.com.
³ Shanghai Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, China. Electronic address: lcui@sjtu.edu.cn.
⁴ Shanghai Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, China. Electronic address: hxg@sjtu.edu.cn.
⁵ Shanghai Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, China. Electronic address: ycl@sjtu.edu.cn.
⁶ Shanghai Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, China. Electronic address: sunhuan198419@163.com.
⁷ Shanghai Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, China. Electronic address: 313081543@qq.com.

Abstract

The present study describes the development and validation of a one-step, single-tube, and real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) detecting Porcine Sapelovirus. RT-LAMP characterized by one strand displacement reaction with the specific stem-loop structure and Bst DNA polymerase could be finished in 60 min under isothermal condition at 63 °C. RT-LAMP assay showed higher sensitivity with 10(1) copies/μL than RT-PCR for the detection of Sapelovirus. The specificity of RT-LAMP assay was validated by the absence of any cross-reaction with other closely related virus in Picornaviridae group and other common virus causing porcine diarrhea. 7 positive Sapelovirus infection out of 63 fecal samples were identified using RT-LAMP, while 5 positive samples were determined by a conventional RT-PCR. A cost-effective method for Saplovirus detection with high sensitivity and specificity was developed and evaluated.

Keywords: Detection; Picornaviridae; Porcine Sapelovirus; RT-LAMP.

Publication types

Evaluation Study

MeSH terms

Animals
Costs and Cost Analysis
Feces / virology
Nucleic Acid Amplification Techniques / methods*
Picornaviridae / isolation & purification*
Picornaviridae Infections / diagnosis
Picornaviridae Infections / veterinary*
Picornaviridae Infections / virology
Reverse Transcription
Sensitivity and Specificity
Swine
Swine Diseases / diagnosis*
Swine Diseases / virology*
Temperature
Time Factors