应用PCR-RFLP技术检测短喙矮小综合征鹅细小病毒

Application of PCR-RFLP in the detection of short beak and dwarfism syndrome viruses

为准确诊断鸭短喙矮小综合征(SBDS)以及鉴别鹅细小病毒感染鸭群中新发短喙矮小综合征鹅细小病毒(SBDS-GPV),本研究通过对GenBank登录的水禽细小病毒基因组核苷酸序列的比对分析,根据该病毒5'非编码区基因序列特征设计一对特异性引物,建立了SBDS-GPV聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)检测方法。特异性试验和敏感性试验显示,水禽细小病毒特异性引物可以扩增得到约200 bp的目的片段,SBDS-GPV PCR产物能够被SspⅠ酶切为140 bp和60 bp两个片段,番鸭细小病毒与德兹西氏病小鹅瘟病毒则不能被酶切,检出SBDS-GPV DNA的最低浓度为2.28×10~7拷贝/μL。利用该方法对14份疑似SBDS临床样品检测,阳性样本采用SPF鸭胚进行病毒分离,结果显示SBDS-GPV PCR-RFLP检测的阳性样本经病毒分离均为SBDS-GPV。上述研究表明,该SBDS-GPV PCR-RFLP检测方法特异性强、敏感性高,为SBDS的防控提供了有效手段。

To rapidly diagnose short beak and dwarfism syndrome (SBDS)in ducklings and identify SBDS vires (SBDS-GPV) in domestic duck farms,a pair of primers was designed according to the 5'-terminal untranslated region (5'-UTR)sequence of waterfowl parvovirus strains,and a polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP)assay was established for rapid detection of SBDS-GPV infection.The results showed that a fragment of 200bp was amplified from SBDS-GPV which could be digested into two fragments of 140bp and 60bp with Ssp I ,while the amplified fragment from the Derzsy's disease vires (DD-GPV)and Muscovy duck parvovims (MDPV)were unable to be digested with Ssp I .The lowest concentration of SBDS-GPV DNA could be detected with the established assay was 2.28×10^7copies/μL.We evaluated the PCR-RFLP assay by detection of 14clinically suspected duck cases of SBDS,SBDS-GPV could be isolated from all positive tissues by inoculating in SPF duck embryonated eggs,which was confirmed by SBDS-GPV PCR-RFLP.The developed PCR-RFLP assay in this study is a specific and sensitive method for detection and differentiation of SBDS-GPV and classical waterfowl parvovirus.