【摘要】 为了建立一种可检测牛诺如病毒(Bovine norovirus,BNo V)的重组酶介导等温扩增-核酸外切酶(recombinase aided amplification-exonuclease,RAA-EXO)探针方法，试验根据RAA引物探针设计原则，针对BNo V RdRp基因设计引物和探针，通过优化反应条件及特异性、敏感性、重复性试验建立BNo V RAA-EXO探针检测方法，并采用该方法对168份临床样本进行检测。结果表明:该方法在41℃的恒温条件下反应16 min即可完成检测，可特异性地检测BNo V，与牛冠状病毒(Bovine coronavirus,BCo V)、牛轮状病毒(Bovine rotavirus,BRV)、牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)和牛传染性鼻气管炎病毒(Infectious bovine rhinotracheitis virus,IBRV)无交叉反应，最低检测限为5.27×10~2copies/μL,3次重复的Ct值的变异系数均小于10%。168份临床样本中，检出BNo V阳性样本85份，阳性率为50.60%，与普通PCR方法的符合率为91.07%，与实时荧光定量PCR方法的符合率为100%。说明建立的BNo V RAA-EXO探针检测方法特异性强、敏感性高、重复性好，且操作简便、短时间内即可完成检测，可用于临床筛查与流行病学研究。更多还原

【Abstract】 In order to establish a recombinase aided amplification-exonuclease( RAA-EXO) probe method for detecting Bovine norovirus( BNoV),the experiment designed primers and probes targeting the BNoV RdRp gene in accordance with the principles of RAA primer and probe design. The RAA-EXO probe detection method for BNoV was established by optimization of the reaction conditions,and sensitivity,specificity and repeatability test,and was used to detect 168 clinical samples.The results showed that this method could complete the detection within 16 minutes under a constant temperature of 41 ℃ ． It could specifically detect BNoV and had no cross-reaction with Bovine coronavirus( BCoV),Bovine rotavirus( BRV),Bovine viral diarrhea virus( BVDV),and Infectious bovine rhinotracheitis virus( IBRV),with a minimun detection limit of 5. 27 × 10~2 copies/μL. The variable coefficient of Ct values of 3 repetitions were all less than 10%. Among 168 clinical samples,85 were detected as BNoV-positive,with a positive rate of 50. 60%. The coincidence rate with the ordinary PCR method was91. 07%,and the coincidence rate with the real-time fluorescent quantitative PCR method was 100%. These results indicated that the established BNoV RAA-EXO probe detection method had strong specificity,high sensitivity,and good repeatability. Additionally,it was easy to operate,could complete the detection in a short time,and could be used for clinical screening and epidemiological research.更多还原