猪伪狂犬病毒荧光重组酶介导核酸扩增快速检测方法的建立与应用

A Rapid Real Time Fluorescence Recombinase-aided Amplification Method for Detecting Porcine Pseudorabies Virus

【目的】基于荧光重组酶介导核酸扩增(Recombinase-aided amplification,RAA)技术,建立一种猪伪狂犬病毒(Porcine pseudorabies virus,PRV)快速检测方法。【方法】根据PRV gE基因序列,设计特异性引物及探针,优化扩增体系,建立PRV荧光重组酶介导核酸扩增检测方法,检验其特异性、敏感性和重复性,应用该方法对临床样品进行检测。【结果】该方法在43℃恒温反应23 min即可完成PRV核酸扩增,最低检出限为111 copies·μL^(-1);与猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)、猪流行性腹泻病毒(Porcine epidemicdiarrheavirus,PEDV)、猪轮状病毒(Porcinerotavirus,PoRV)、猪传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)、猪圆环病毒2型(Porcine circovirus 2,PCV2)、猪圆环病毒3型(Porcine circovirus 3,PCV3)均无交叉反应。重复性试验显示,组内和组间变异系数均小于5%;40份临床样品检测结果显示PRV阳性率为15%(6/40),检测结果与常规聚合酶链式反应(PCR)一致。【结论】成功建立了简便快速、高效准确的PRV实时荧光RAA检测方法,为PRV的快速检测和流行病学调查提供了新的检测手段。

【Objective】A rapid method applying the recombinase-aided amplification(RAA)method for detecting porcine pseudorabies virus(PRV)was developed.【Methods】Based on the sequence of gE gene in PRV,specific primers and probes were designed.Amplification conditions were optimized,and assay specificity,sensitivity,and reproducibility scrutinized by a verification trial on clinical samples.【Results】The newly developed assay successfully amplified the PRV nucleic acids in merely 23m under the constant temperature of 43℃with a detection limit of 111 copies·μL^(−1).There were no cross reactions with viruses that produced reproductive and respiratory syndromes,epidemic diarrhea,rotavirus,transmissible gastroenteritis,circovirus 2,or circovirus 3 on pigs.The coefficients of variation within a group and between groups on the repeatability test were less than 5%.And,on 40 clinical samples,the positive detection on PRV of the assay was 15%(6/40),which was comparable to that of the conventional PCR.【Conclusion】A simple,rapid,efficient,and accurate method of fluorescence RAA detection on PRV was established for laboratory testing and epidemiological investigation of the disease.