摘要
为建立简捷、灵敏的猪急性腹泻综合征冠状病毒(SADS-CoV)检测方法,本研究针对SADS-CoV N基因片段保守区域设计特异性的反转录环介导等温扩增(RT-LAMP)引物,经条件优化后显示建立的SADS-CoV RTLAMP检测方法最佳反应条件为64℃,50 min,初步建立SADS-CoV的RT-LAMP快速检测方法。利用该方法检测SADS-CoV、猪流行性腹泻性病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪德尔塔冠状病毒(PDCoV)、猪轮状病毒(PoRV)和猪肠道病毒9型(PEV9),评估其特异性,结果显示除SADS-CoV外,该方法与其它病原无交叉反应,特异性较强;将病毒RNA 10倍倍比稀释后作为模板,利用该RT-LAMP与普通RT-PCR同时检测,结果显示,RTLAMP方法比普通RT-PCR方法的敏感性高103倍,敏感性较高。以同一批次或不同批次的SADS-CoV核酸为模板进行RT-LAMP试验,结果显示RT-LAMP批内批间均可稳定的检出阳性样本,表明该方法具有良好的重复性和稳定性。对经口服感染SADS-CoV和未感染仔猪的临床样本进行检测,结果显示RT-LAMP与普通RT-PCR检测结果高度一致。本实验首次建立的SADS-CoV可视化RT-LAMP检测方法具有特异性强、敏感性高,操作简便、快速和高通量检测的优点,为临床SADS-CoV感染的快速诊断和综合防控提供检测手段。
To development a rapid and sensitive detection method for swine acute diarrhea syndrome coronavirus(SADSCoV),we designed specific reverse transcription-loop-mediated isothermal amplification(RT-LAMP)primers targeting the conserved regions of SADS-CoV N gene,and optimized the reaction conditions of 64℃,54 minutes.The developed method has high specificity and no cross-amplifications with porcine epidemic diarrhea viruses(PEDV),transmissible gastroenteritis virus(TGEV),porcine delta coronavirus(PDCoV),porcine rotavirus(PoRV)and porcine enterovirus 9(PEV9).The method also has high sensitivity,which was 103-fold higher than conventional RT-PCR method.Using the same batch or different batches of SADS-CoV nucleic acid as templates for RT-LAMP test,the results show that RT-LAMP can stably detect positive samples,indicating that the method has good reproducibility and stability.The clinical samples of oral challenged SADS-CoV and unchallenged piglets were tested by both the RT-LAMP and conventional RT-PCR,the results showed that the coincidence rate of the two methods was 100%.The SADS-CoV visual RT-LAMP detection method established for the first time in this experiment has the advantages of strong specificity,high sensitivity,simple operation,rapid detection and high throughput detection,which can be used for clinical rapid diagnosis to preventive and control the epidemic diseases caused by SADS-CoV.
作者
韩郁茹
石达
张记宇
时洪艳
陈建飞
张鑫
刘建波
冯力
HAN Yu-ru;SHI Da;ZHANG Ji-yu;SHI Hong-yan;CHEN Jian-fei;ZHANG Xin;LIU Jian-bo;FENG Li(Swine Digestive System Infectious Diseases Division,State Key Laboratory of Veterinary Biotechnology,HarbinVeterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2021年第1期35-39,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
黑龙江省自然科学基金(C2017079)
中国博士后科学基金(2017M610136)。
