鸽微RNA病毒实时荧光定量RT-PCR检测方法的建立  

Establishment of a Real-time Fluorescent RT-PCR Assay for Detection of Pigeon Megrivirus

作　　者：张靖鹏 陈翠腾[1] 林琳[1] 付环茹 李兆龙[1] 江斌[1] 黄瑜[1] 万春和[1] ZHANG Jinpeng;CHEN Cuiteng;LIN Lin;FU Huanru;LI Zhaolong;JIANG Bin;HUANG Yu;WAN Chunhe(Institute of Animal Husbandry and Veterinary Medicine/Fujian Key Laboratory for Avian Diseases Control and Prevention/Fujian Animal Diseases Control Technology Development Center,Fuzhou 350013,China)

机构地区：[1]福建省农业科学院畜牧兽医研究所/福建省禽病防治重点实验室/福建省畜禽疫病防治工程技术研究中心,福州350013

出　　处：《畜牧兽医学报》2024年第2期860-866,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：福建省公益类项目(2021R1026001,2021R1026006,2023R1076,2023R1077);福建省自然科学基金项目(2020J06029);现代农业产业体系项目(CARS-42);福建省农业科学院科技计划项目(CXPT202108,CXTD2021005,YCZX202412,DWHZ2024-13)。

摘　　要：旨在建立鸽微RNA病毒(pigeon megrivirus,PiMeV)实时荧光定量RT-PCR检测方法。本研究根据GenBank中PiMeVs序列特征设计特异性检测引物,从信鸽粪便中检测到PiMeV阳性(命名为PiMeV-CHN001株),并对其3 C基因进行核苷酸同源性比较和遗传进化分析,明确其基因特征后,设计特异性实时荧光定量RT-PCR检测(RT-qPCR)引物组,建立检测PiMeV的RT-qPCR方法。结果显示:PiMeV-CHN001株3 C基因全长为591 bp,编码197个氨基酸,和其他2株野鸽源PiMeV(MeV-B1株和MeV-B2株)核苷酸相似性分别为89.5%和92.0%。建立的检测PiMeV的RT-qPCR方法的标准曲线Y轴截距为37.93,斜率为-3.335,相关系数为1.00,扩增效率为99.4%。特异性强,仅PiMeV出现特异性扩增信号和特异性峰值[Tm值为(81.69±0.22)℃],对鸽源禽流感病毒(avian influenza virus,AIV)、鸽源禽I型副黏病毒(pigeon paramyxovirus type I,PPMV-1)、鸽输血传播病毒(pigeon torque teno virus,PTTV)、鸽腺病毒(pigeon adenovirus,PiAd)及鸽圆环病毒(pigeon circovirus,PiCV)检测均未见特异性扩增信号;敏感性优,最低检测限为54.0拷贝·μL^(-1);重复性好,批内和批间变异系数均低于1.5%。用建立的检测方法对42份信鸽粪便样品进行检测,发现2份阳性样品(阳性率为4.76%)。本研究首次证实我国大陆地区信鸽中存在PiMeV,丰富了PiMeV宿主谱信息;建立的RT-qPCR方法为后续开展PiMeV流行病学研究提供支撑。The present study aimed to establish a novel real-time fluorescent RT-PCR assay for detection of pigeon megrivirus(PiMeV).Specific primers were designed based on PiMeVs sequences download from the GenBank,and positive fragments(designated as PiMeV-CHN001 strain)were amplified from racing pigeon feces.Then the 3 C gene of PiMeV-CHN001 strain was obtained and analyzed.Based on the 3C molecular characterization,a real-time fluorescent RT-PCR method(RT-qPCR)was developed using the specific primers.The results demonstrated the 3C gene of PiMeV-CHN001 strain had 591 bp(coding 197 amino acids),with the nucleic acid sequence homology with other wild urban pigeons(Columba livia)(MeV-B1 strain and MeV-B2 strain)was 89.5%and 92.0%,respectively.The RT-qPCR assay standard curve had the axial intercept of standard curve was 37.93 and the slope was-3.335,with a linear correlation(R2)of 1.00 and efficiency of 99.4%.The methods were specificity,no-cross amplification signal was found from other pigeon viruses(such as avian influenza virus,pigeon paramyxovirus type I,pigeon torque teno virus,pigeon adenovirus,and pigeon circovirus).Only one specific peaked with a melting temperature(Tm)was(81.69±0.22)℃form PiMeV-CHN001,with no primer-dimers peak represent.The lowest limit of detection concentration was 54.0 copies/μL.The intra-and inter-assay were less than 1.5%according to the repeatability test.The developed qPCR was used for PiMeV detection of 42 racing pigeon feces.2 positive(positive rate was 4.76%)signals were found.In conclusion,we firstly confirmed the presence of PiMeV in racing pigeons in China's Mainland,and the data can enrich Megrivirus host spectrum.Moreover,the developed RT-qPCR assay also lays good foundation for further PiMeV epidemiological investigation.

关 键 词：信鸽 鸽微RNA病毒 3 C基因 序列分析 实时荧光定量RT-PCR方法 

分 类 号：S852.659.6[农业科学—基础兽医学]