鹅星状病毒通用型TaqMan探针实时荧光定量RT-PCR检测方法的建立与应用  

Establishment and Application of Universal Taqman-based Real-time Fluorescence Quantitative RT-PCR Method for the Detection of Goose Astrovirus

作　　者：陈立功[1,2] 穆英丽 张诚 潘保革 范乐乐[1] 魏忠华 王学静[3] 刘聚祥[1,2] CHEN Ligong;MU Yingli;ZHANG Cheng;PAN Baoge;FAN Lele;WEI Zhonghua;WANG Xuejing;LIU Juxiang(Hebei Agricultural University,Baoding,Hebei 071001;North China Research Center of Animal Epidemic Pathogen Biology,Ministry of Agriculture and Rural Affairs,Baoding,Hebei 071001;Animal Husbandry and Veterinary Institute of Heibei Province,Baoding,Hebei 071000)

机构地区：[1]河北农业大学,河北保定071001 [2]农业农村部动物疫病病原生物学华北科学观测实验站,河北保定071001 [3]河北省畜牧兽医研究所,河北保定071000

出　　处：《中国家禽》2025年第2期53-59,共7页China Poultry

基　　金：河北省现代农业产业技术体系建设专项资金(HBCT2024270204);河北农业大学大学生创新创业训练计划项目(x2020043)。

摘　　要：为建立用于鹅星状病毒1(GAstV 1)和鹅星状病毒2(GAstV 2)感染快速检测的实时荧光定量RT-PCR方法,试验根据部分GAstV ORF2基因和部分3′非编码区序列设计合成特异性引物和探针,采用矩阵法获得引物和探针的最优浓度,在优化退火温度的基础上,分别建立扩增GAstV 1和GAstV 2的标准曲线,进一步验证该方法的特异性、敏感性和重复性,建立的方法用于临床样品的检测,并与文献报道的常规RT-PCR方法进行比较。结果显示:优化后的反应体系中最优上、下游引物浓度均为0.40(或0.50)μmol/L,探针浓度均为0.50μmol/L,扩增线性范围分别为3.26×10^(3)~3.26×10^(8)拷贝/μL和7.09×10^(3)~7.09×10^(8)拷贝/μL,相关系数均为0.998;该方法可特异性检出两种GAstV,但对新城疫病毒、H9亚型禽流感病毒、鸭坦布苏病毒、新型鸭呼肠孤病毒、鹅细小病毒和血清4型禽腺病毒6种鹅病相关病毒的核酸均无扩增信号;其检测限分别为3.26×10^(2)拷贝/μL和7.09×10^(1)拷贝/μL;组内变异系数和组间变异系数均低于3%。建立的实时荧光定量RT-PCR方法对临床样品的检测结果显示,GAstV阳性率为90.57%(96/106);而文献报道的常规RT-PCR方法对GAstV 1和GAstV 2的混合阳性率为62.26%。研究表明,建立的TaqMan荧光定量RT-PCR检测方法为同时检测GAstV 1和GAstV 2提供了快速、敏感、特异且能满足临床样本需求的检测方法。In order to establish one real-time fluorescence quantitative RT-PCR to detect goose astrovirus 1(GAstV1)and goose astrovirus 2(GAstV2)infection,a pair of specific primers and one TaqMan probe were designed and synthesized according to the sequence between partial ORF2 gene and partial 3′UTR of the two type of goose astrovirus(GAstV).The optimal concentration of primers and probe were determined by matrix method.And based on optimized annealing temperature,the standard amplification curves of GAstV 1 and GAstV 2 were established,respectively.The specificity,sensitivity and repeatibility of the quantitative RT-PCR were further verified,and the established method was applied to the detection of clinical samples compared with the common RT-PCR.The results showed that the optimal concentrations of the forward primer and reward primer were 0.40 or 0.50μmol/L,respectively.The optimal concentration of probe was 0.50μmol/L.The linear range was 3.26×10^(3) to 3.26×10^(8) copies/μL and 7.09×10^(3) to 7.09×10^(8) copies/μL,and the correlation coefficient was 0.998,respectively.The method could specifically amplify for two types of GAstV,but there was no amplification signal for the nucleic acid of Newcastle disease virus,H9 subtype avian influenza virus,duck Tembusu virus,new duck reovirus,goose parvovirus and fowl adenovirus serotype 4.The detection limits was 3.26×10^(2) copies/μL and 7.09×10^(1) copies/μL.The intra-group and inter-group coefficients of variation of this method were both below 3%.The results of clinical samples detected by the quantitative RT-PCR assay was 90.57%(96/106),while the mixed positive rate of GAstV1 and GAstV2 was 62.26%that from the same tissue samples detected by common RT-PCR methods reported in the reference.The results indicated that the established quantitative RT-PCR could provide a rapid,sensitive and specific detection method for the simultaneous detection of GAstV 1 and GAstV 2.

关 键 词：鹅星状病毒 荧光定量RT-PCR TAQMAN探针 

分 类 号：S852.5[农业科学—基础兽医学]