猪流行性腹泻病毒变异毒株一步法RT-PCR鉴别检测方法的建立及临床应用

Establishment and clinical application of a one-step RT-PCR method for differential detection of Porcine epidemic diarrhea virus variants

为了建立一种快速鉴别检测猪流行性腹泻病毒(PEDV)变异毒株的病原学方法和掌握洛阳市PEDV变异毒株的流行规律,试验根据GenBank中PEDV变异毒株的特异性序列设计引物,通过优化退火温度、模板添加量、引物添加量建立PEDV变异毒株一步法RT-PCR鉴别检测方法,并分析了该方法的特异性、敏感性和重复性;同时应用该方法检测采集自洛阳市的251份临床样品,并对不同地区、不同年份、不同养殖模式的检测结果进行比较分析。结果表明:优化后的退火温度为51℃,模板添加量为5μL,引物添加量为0.5μL;该方法对PEDV变异毒株能够扩增出550 bp特异性条带,而检测的PEDV经典毒株、猪传染性胃肠炎病毒(TGEV)、猪轮状病毒(RV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)、猪细小病毒(PPV)、猪伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV-2)均为阴性;对PEDV变异毒株RNA的最低检测限达到0.74 ng,对3份PEDV变异毒株阳性病料和3份PEDV变异毒株阴性病料重复检测3次的结果完全一致;检测采集于洛阳市251份临床样品的平均阳性率为29.48%,其中不同地区阳性率介于13.33%~44.44%之间,2018—2022年阳性率介于28.30%~31.58%之间,散养户和规模化猪场的阳性率分别为38.24%和19.13%。说明试验建立的PEDV变异毒株一步法RT-PCR鉴别检测方法特异、敏感、稳定、准确,洛阳市PEDV变异毒株的流行特点为个别地区较严重的情况、近几年流行率基本持平、散养户流行情况较规模化猪场严重。

In order to establish a rapid pathogenetic method for the differential detection of Porcine epidemic diarrhea virus(PEDV)variants,and to grasp the epidemiological pattern of PEDV variants in Luoyang City,in the experiment,the primers were designed according to the specific sequences of PEDV variants in GenBank,and the one-step RT-PCR differential detection method was established by optimizing the annealing temperature,template content and primer content,and the specificity,sensitivity and reproducibility of the method were analyzed.At the same time,the method was applied to detect 251 clinical samples collected in Luoyang City,and the results were compared and analyzed in different regions,years and breeding modes.The results showed that the optimized annealing temperature was 51℃,the template content was 5μL,and the primer content was 0.5μL.The method was able to amplify a 550 bp specific band for the PEDV variants,while the classical strain of PEDV,Transmissible gastroenteritis virus(TGEV),Porcine rotavirus(RV),Porcine reproductive and respiratory syndrome virus(PRRSV),Classical swine fever virus(CSFV),Porcine parvovirus(PPV),Pseudorabies virus(PRV),and Porcine circovirus type 2(PCV-2)were all negatively detected.The lowest limit of detection for RNA of PEDV variants reached 0.74 ng.The results of three repetitions of the test on three PEDV variants-positive and three PEDV variants-negative samples were completely consistent.The average positive rate of the 251 clinical samples collected in Luoyang City was 29.48%,of which the positive rates in different areas ranged from 13.33%to 44.44%,and the positive rates from 2018 to 2022 ranged from 28.30%to 31.58%,and the positive rates in free-range and large-scale swine farms were 38.24%and 19.13%,respectively.The results indicated that the one-step RT-PCR differential detection method of PEDV variants established in the experiment was specific,sensitive,stable and accurate;and the prevalence of PEDV variants in Luoyang City was characterized by a more serious situation in individual areas,a basically flat prevalence rate in recent years,and a more serious prevalence situation in free-ranging households than that in large-scale pig farms.