小鹅瘟和雏鹅新型病毒性肠炎二联PCR检测试剂盒组装和初步应用

本试验旨在研制小鹅瘟和雏鹅新型病毒性肠炎二联PCR检测试剂盒,观察临床应用效果。根据小鹅瘟和鹅病毒性肠炎基因序列,设计合成一对特异性引物,对PCR反应条件进行优化,并分别对试剂盒的特异性、敏感性、重复性和保存期进行研究,之后进行临床扩大试验。该二联PCR诊断试剂盒能扩增出预期的小鹅瘟375 bp核酸片段和鹅病毒性肠炎223 bp核酸片段,与鸭瘟病毒、鹅副黏病毒无交叉反应;最低能检测到小鹅瘟病毒10⁴ ELD₅₀/mL,雏鹅新型病毒性肠炎病毒2.5×10^-1ELD₅₀;不同批次的试剂盒对同一样品检测结果一致;常温下至少可保存5个月;临床应用效果显著。该二联PCR检测试剂盒能够对小鹅瘟和雏鹅病毒性肠炎早期诊断、流行病学调查和卫生检疫提供了检测手段。

Development and Preliminary Application of a Duplex PCR Detection Kit for Gosling Plague and Gosling New Type Viral Enteritis

To develop a duplex PCR detection kit for gosling plague(GP) and gosling new type viral enteritis(NGVE),and observe the effects of clinical appliance.The gene sequences of gosling plague virus(GPV) and gosling new type viral enteritis virus(NGVEV) was chosen to design the species-specific primers.The PCR amplification conditions were optimized and series of tests were conducted respectively in the specificity,sensitivity,stability,reproducibility,and stored period of the kit,and expanded clinical trials were also carried out.This kit could specifically amplify the expected 375 bp DNA fragment of GPV virus and 223 bp DNA fragment of NGVEV.There was no cross-reaction with other virus,such as DPV,GPMV,NGVEV and GPV.It was able to detect the presence of GPV as low as 104ELD50/mL and NGVEV as low as 2.5×10-1ELD50.The test results of different batches of kits of the same sample were consistent.The kit worked well after being stored at room temperature for at least six months.Its effect of clinical application was significant.The results turned out that this duplex PCR detection kit could provide a means for early diagnosis,epidemiological investigation and quarantine of GP and NGVE.