Development and evaluation of a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detection of type II porcine reproductive and respiratory syndrome virus

• Published in: Journal of virological methods 2012-10, Vol.185 (1), p.18-23
• Main Authors: Gao, Ming, Cui, Jin, Ren, Yudong, Suo, Siqingaowa, Li, Guangxing, Sun, Xuejiao, Su, Dingding, Opriessnig, Tanja, Ren, Xiaofeng
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Conventional RT-PCR; Detection; DNA Primers - genetics; double prime M gene; Fundamental and applied biological sciences. Psychology; genes; Genes, Viral; Microbiology; Nucleic Acid Amplification Techniques - methods; Nucleotide sequence; Polymerase chain reaction; Porcine Reproductive and Respiratory Syndrome - diagnosis; Porcine Reproductive and Respiratory Syndrome - virology; Porcine reproductive and respiratory syndrome virus; Porcine respiratory and reproductive syndrome virus; Porcine respiratory and reproductive syndrome virus - isolation & purification; Primers; PRRSV; quantitative polymerase chain reaction; Real-time RT-PCR; reverse transcriptase polymerase chain reaction; Reverse transcription; reverse transcription loop-mediated isothermal amplification; RT-LAMP; Sensitivity and Specificity; sequence alignment; Swine; Techniques used in virology; Veterinary Medicine - methods; Virology; Virology - methods; viruses
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2012.05.016

► A sensitive, specific, fast, and easy to perform assay for detection of porcine reproductive and respiratory syndrome virus (PRRSV) is needed. ► As a detection method the reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was utilized. ► The RT-LAMP assay was more sensitive compared to a conventional RT-PCR and an enzyme-linked immunosorbent assay. ► The RT-LAMP was highly specific. The objective of this study was to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detection of type II porcine reproductive and respiratory syndrome virus (PRRSV). Based on sequence alignment, four primers were designed amplifying the M gene of type II PRRSV and were subsequently utilized in an RT-LAMP assay. The RT-LAMP product had a ladder-like pattern of bands and the optimal reaction condition for this assay was determined to be 40min at 63°C. Comparative analysis indicated that the RT-LAMP method was more sensitive than a conventional RT-PCR assay and comparable to a real-time PCR assay. In addition, the RT-LAMP assay was capable of detecting type II PRRSV in field samples and differentiating type II PRRSV from seven other porcine viruses which are all associated frequently with similar clinical symptoms.