中国动物传染病学报2025，Vol.33Issue(2)：70-79,10.
基于N基因的RT-LAMP技术检测PEDV、TGEV、PDCoV三种猪腹泻冠状病毒的研究
Detection of Three Porcine Diarrhea Coronaviruses PEDV,TGEV and PDCoV by RT-LAMP Based on N gene
摘要
Abstract
This study developed a reverse transcription loop-mediated isothermal amplification(RT-LAMP)assay for the detection of PEDV,TGEV and PDCoV,which provided technical support for rapid on-site diagnosis.Three sets of primers for LAMP detection were designed according to the conserved N gene sequences of PEDV,TGEV and PDCoV published in GenBank as the target sequences.Then,three rapid and sensitive RT-LAMP detection methods for porcine coronavirus were developed through the optimization of reaction conditions and testing of sensitivity,specificity and detection of clinical samples.The results showed that the optimal reaction temperatures of the RT-LAMP assay were 65℃,63℃ and 63℃ for PEDV,TGEV and PDCoV,respectively.All the three detection methods were able to complete the isothermal amplification in an ordinary thermostatic device for about 45 minutes with application of specific stepped-like bands.The detection limits of the RT-LAMP method were 20 copies/μL,23 copies/μL and 10 copies/μL for PEDV,TGEV and PDCoV,respectively,indicating its good sensitivity.There was no cross-reaction with other swine viruses.In addition,the RT-LAMP method was used to test 45 porcine diarrhea samples and the positive rates were 33.33％(15/45),22.22％(10/45)and 13.33％(6/45)for PEDV,TGEV and PDCoV,respectively,which were all higher than the conventional RT-PCR.Taken together,the RT-LAMP method developed in this study was simple,rapid,economical and had a lower threshold for detection equipment,thus it was more suitable for primary clinical diagnosis.关键词
猪/冠状病毒/反转录环介导等温扩增/检测方法Key words
Porcine/Coronavirus/reverse transcriptional loop-mediated isothermal amplification(RT-LAMP)/detection method分类
农业科技引用本文复制引用
邓可辉,陈志飞,俞婷,王志林,王修武,郭智轩,李松倍,魏文康,贺东生..基于N基因的RT-LAMP技术检测PEDV、TGEV、PDCoV三种猪腹泻冠状病毒的研究[J].中国动物传染病学报,2025,33(2):70-79,10.基金项目
广东省科技计划(P20211154-0302) （P20211154-0302）
茂名实验室科研启动项目(2021TDQD002) （2021TDQD002）
科技创新战略专项资金(202131TD) （202131TD）
