| 摘要: |
| 【目的】 建立禽坦布苏病毒（Avian Tembusu virus，ATmV）TaqMan实时荧光定量PCR检测方法。【方法】 根据ATmV非结构蛋白NS1的基因特征，设计特异性的引物和探针，建立基于TaqMan探针检测ATmV的实时荧光定量PCR检测方法，对其特异性、重复性进行检测。用建立的TaqMan实时荧光定量PCR检测方法和之前建立的RT PCR方法同时对临床15份疑似ATmV感染的病料进行检测，计算其符合率。【结果】 成功建立了检测ATmV的实时荧光定量PCR检测方法，当NS1基因含量为（2.67×102）～（2.67×107） 拷贝/μL时有良好的线性扩增，其扩增相关系数为0.998，扩增效率为99.9%。建立的ATmV实时荧光定量PCR检测方法敏感度高，最低检测限为2.67×102拷贝/μL；特异性强，对水禽常见病毒（如Ⅰ型鸭肝炎病毒、新型鸭呼肠孤病毒、禽流感病毒、禽Ⅰ型副粘病毒与番鸭细小病毒等）检测均为阴性；重复性好，组内变异系数和组间变异系数分别为0.39%～1.17%和0.51%～1.82%。对临床送检的15份病料，TaqMan实时荧光定量PCR检测方法的阳性率为73.33%（11/15），RT-PCR方法的阳性率为60.0%（9/15），2种方法的符合率为100.0%。【结论】 建立了基于TaqMan探针的ATmV实时荧光定量PCR检测方法，该方法可应用于ATmV感染的早期检测。 |
| 关键词: 禽坦布苏病毒 NS1基因 TaqMan 实时荧光定量PCR方法 |
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| 基金项目:国家现代水禽产业体系项目（CARS-43）；福建省属公益类项目（2015R1023-7）；福建省农业科学院“青年科技英才百人计划”项目（YC2015-12）；福建省农业科学院青年人才创新基金项目（2014QA-7） |
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| Development of a TaqMan-based real-time PCR method for avian Tembusu virus |
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WAN Chunhe,CHEN Cuiteng,FU Qiuling,et al
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| Abstract: |
| 【Objective】This study developed a TaqMan-based real-time PCR method for avian Tembusu virus (ATmV).【Method】A TaqMan-based real-time PCR method was developed for detection of ATmV with specific primers and probe targeting to the nonstructural 1(NS1) gene.The specificity and repeatability of the method were also detected.Fifteen suspected ATmV infection samples were tested by the established method and the coincidence rate was calculated by comparing with conventional RT-PCR method.【Result】The successfully established real-time PCR method had good linear correlation when NS1 gene content was (2.67×102)-(2.67×107) DNA copies/μL with correlation coefficient (R2) of 0.998 and efficiency of 99.9%.The lower detection limit was 2.67×102 copies/μL.No amplification was detected from common waterfowl origin viruses,such as duck hepatitis virus type 1,novel duck reovirus,avian influenza virus,and avian paramyxovirusⅠand Muscovy duck parvovirus.Reproducibility test showed that the intra and inter-assay were 0.39%-1.17% and 0.51%-1.82%,respectively.Fifteen clinical samples were tested by the established TaqMan-based real-time PCR method and the RT-PCR method and their positive rates were 73.33% (11/15) and 60.0% (9/15),respectively.RT-PCR positive samples were tested positive by the real time PCR method,with the coincidence rate was 100.0%.【Conclusion】The established TaqMan-based real-time PCR method provided a useful method for early diagnosis of avian tembusu virus. |
| Key words: avian Tembusu virus NS1 gene TaqMan real time PCR method |
