鸭甲肝病毒3型、鸭星状病毒3型、鸭腺病毒3型多重PCR检测方法的建立及初步应用OA
Development and Preliminary Application of a Multiplex PCR for Detection of DHAV-3,DAstV-3 and DAdV-3
为建立能同时检测鸭甲肝病毒3型(Duck hepatitis A virus-3,DHAV-3)、鸭星状病毒3型(Duck astrovirus-3,DAstV-3)和鸭腺病毒3型(Duck adenovirus-3,DAdV-3)的多重PCR方法,根据DHAV-3、DAstV-3、DAdV-3基因组保守区域设计3对特异性引物,预期扩增片段大小分别为298、505、668 bp.通过对反应体系、反应条件进行优化,建立了DHAV-3、DAstV-3和DAdV-3的多重PCR检测方法,对其特异性、敏感性和重复性进行验证,并在临床上进行初步应用.结果显示:建立的多重PCR方法可同时扩增DHAV-3、DAstV-3、DAdV-3的目的片段,对其他鸭常见病原的检测均无扩增条带,3种病毒的最低检测限分别是1.0×103、1.0×104和1.0×104 copies/μL,同一模板的3次重复性试验结果一致;应用多重PCR方法对临床样品进行检测,检测结果与常规单一(RT-)PCR符合率为100%.总之,建立的多重PCR方法特异性强、敏感性高、重复性好,可用于DHAV-3、DAstV-3、DAdV-3的临床检测和流行病学调查.
In order to develop a multiplex PCR method for simultaneous detection of Duck hepatitis A virus-3(DHAV-3),Duck astrovirus-3(DAstV-3)and Duck adenovirus-3(DAdV-3),three sets of specific primers were designed based on the conserved regions of their genomes.The resulting amplified products were 298,505 and 668 bp,respectively.The multiplex PCR assay was developed and optimized for testing DHAV-3,DAstV-3 and DAdV-3.The specificity,sensitivity and reproducibility of the method were then validated for clinical application.The results showed that the multiplex PCR method developed here simultaneously amplified the target fragments of DHAV-3,DAstV-3 and DAdV-3 and there were no amplification products of other common duck pathogens.The detection limits of the three viruses were 1.0×103,1.0×104 and 1.0×104 copies/μL,respectively.Additionally,the method showed excellent repeatability with coefficients of three tests using the same templates.The multiplex PCR method was applied to test the clinical samples and the results showed 100%agreement with routine single(RT-)PCR tests.In conclusion,the multiplex PCR method developed here was specific,sensitive and reproducible and could be used for clinical detection and epidemiological investigation of DHAV-3,DAstV-3 and DAdV-3.
钟敏;程龙飞;刘荣昌;万春和;傅光华;黄瑜
赣州市畜牧水产研究所,赣州 341401||福建省禽病防治重点实验室,福州 350013福建省农业科学院畜牧兽医研究所,福州 350013福建省禽病防治重点实验室,福州 350013||福建省农业科学院畜牧兽医研究所,福州 350013福建省禽病防治重点实验室,福州 350013||福建省农业科学院畜牧兽医研究所,福州 350013福建省禽病防治重点实验室,福州 350013||福建省农业科学院畜牧兽医研究所,福州 350013福建省禽病防治重点实验室,福州 350013||福建省农业科学院畜牧兽医研究所,福州 350013
农业科技
鸭甲肝病毒3型鸭星状病毒3型鸭腺病毒3型多重PCR
Duck hepatitis A virus-3Duck astrovirus-3Duck adenovirus-3multiplex PCR
《中国动物传染病学报》 2026 (2)
70-78,9
福建省禽病防治重点实验室开放基金(FKADL-2024-02)福建省农业科学院自由探索科技创新项目(ZYTS2024014)
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