鸽微RNA病毒实时荧光定量RT-PCR检测方法的建立

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中文摘要：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Gen-Bank 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3C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3C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(pi-geon 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.

外文标题：Establishment of a Real-time Fluorescent RT-PCR Assay for Detection of Pigeon Megrivirus

外文摘要：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 se-quences download from the GenBank,and positive fragments(designated as PiMeV-CHN001 strain)were amplified from racing pigeon feces.Then the 3C 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 se-quence 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,pi-geon 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-di-mers 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 Main-land China,and the data can enrich Megrivirus host spectrum.Moreover,the developed RT-qPCR assay also lays good foundation for further PiMeV epidemiological investigation.

外文关键词：

racing pigeonpigeon megrivirus3C genesequence analysisreal-time fluorescent quantitative RT-PCR assay

作者：

张靖鹏、陈翠腾、林琳、付环茹、李兆龙、江斌、黄瑜、万春和

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作者单位：

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

关键词：

信鸽鸽微RNA病毒 3C基因序列分析实时荧光定量RT-PCR方法

基金：

福建省公益类项目福建省公益类项目福建省公益类项目福建省公益类项目福建省自然科学基金现代农业产业体系项目福建省农业科学院科技计划项目福建省农业科学院科技计划项目福建省农业科学院科技计划项目福建省农业科学院科技计划项目

项目编号：

2021R10260012021R10260062023R10762023R10772020J06029CARS-42CXPT202108CXTD2021005YCZX202412DWHZ2024-13

出版年：

2024

DOI：

10.11843/j.issn.0366-6964.2024.02.042

畜牧兽医学报

中国畜牧兽医学会

畜牧兽医学报

CSTPCD北大核心

影响因子：0.729

ISSN：0366-6964

年,卷(期)：2024.55(2)

参考文献量24