Establishment And Preliminary Application Of RT-LAMP Method Detecting Porcine Transmissible Gastroenteritis Virus

Transmissible gastroenteritis(TGE) is a highly contagious infectious disease caused by transmissible gastroenteritis virus(TGEV), with clinical manifestations of vomiting, severe diarrhea, and dehydration. It is more serious in TGEV-infected herds for the first-time, and high mortality of 90%-100% could be reached in piglet within 2 weeks old. TGE impaired severely the healthy development of swine industry. However, TGE is really difficult to distinguish from porcine rotavirus infection, porcine epidemic diarrhea and other diarrhetic diseases for their similar clinical symptoms, and mixed infection occurred frequently in clinic. These have brought great difficulties for accurate diagnosis. Routine laboratory testing method is not only time-consuming but also requires special equipment, which is difficult to execute at the production unit. It’s earnestly necessary for them to have a quick and accurate detection method for TGEV.The study established a rapid method to detect TGEV, reverse transcription loop-mediated isothermal amplification(RT-LAMP). The working conditions were optimized, including of temperature and time. And its specificity and sensitivity evaluated. In this study, four RT-LAMP primers were designed according to the sequence of the six TGEV N gene conserved region. RT-LAMP detection method was established for TGEV N gene. The detection system incubated at 63℃ of temperature for 50 min, TGEV N gene was specifically amplified efficiently, and no cross reaction with porcine epidemic diarrhea virus and other swine derived viruses. Meanwhile, the detection method had high sensitivity, could detect minimum 1.314fg/μL of target viral DNA, which was three orders of magnitude higher than the conventional PCR. After completion of the reaction, the fluorescence green color change of positive specimen could be observed under ultraviolet light by the addition of SYBR Green I fluorescent dye, however, the negative sample showed orange of color. 47 clinical samples from swine farms surrounding Harbin were assayed with RT-LAMP method, the results indicated it had 100% coincidence with the validated RT-PCR and ELISA. In generally, RT-LAMP method developed in this study is a rapid, specific, sensitive and easy to operate and low equipment requirements for TGEV field detection.