禽流感病毒A型和H5亚型RT-PCR检测试剂盒研究

Preparation of RT-PCR Kits for Identification of Avian Influnza Virus Type A and Subtype H5

目的 检测和鉴定A型、H5亚型禽流感病毒 (AIV) ,研发一种高效实用的检测手段。方法 根据Ming ShiuhLee报道的文献设计、合成引物 ,采用反转录和PCR一步法对A型、H5亚型禽流感病毒cDNA进行扩增和电泳鉴定 ,组装成禽流感病毒RT PCR试剂盒 ,对H1～ 15亚型AIV参考株、38份AIV国内分离株进行检测试验。结果 建立了A型、H5亚型禽流感病毒RT PCR检测方法 ,并在此基础上组装试剂盒 ,用A型试剂盒检测时 ,全部AIV毒株均为阳性 ,能检测 1 10 2 4血凝单位禽流感病毒 ;用H5亚型试剂盒检测时 ,仅有H5亚型AIV参考株和 19株H5亚型AIV分离株呈阳性 ,其余H1～H4、H6～H15参考株和H7、H9分离株以及 1株H5分株均为阴性 ,能检测1 6 4血凝单位禽流感病毒。 2种试剂盒对实验感染鸡病料检出率均为 10 0 %。结论 研制的AIVA型、H5亚型RT PCR试剂盒具有特异性强、敏感性高、稳定性和重复性好的特点。

Objective To develop RT-PCR kits for detection and identification of type A and subtype H5 avien influnza virus (AIV). Methods Two pairs of primes were designed for type A and subtype H5 avian influenza vir us according to the report by Ming-Shiuh Lee. The cDNA of AIV were amplified by one step RT-PCR for AIV type A and subtype H5, and identified by electrophores is analysis. The RT-PCT kits for AIV type A and subtype H5 were assembled. The H1-H15 reference strains and 38 isolated strains were detected using the RT -PCR kits. Results The RT-PCR method for detection of AIV type A and subt ype H5 was established. All stains of AIV were positive using RT-PCR kits for A type. The sensitivity of this kit was 1/1024 hemagglutinin unit. Subtype H5 reference strains and 19 subtype H5 isolated strains were positively detected using the RT-PCR kit for AIV subty pe H5. Subtypes H1-H4 and H6-H15 reference strains, subtypes H7 and H9 isolated strains, and 1 subtype H5 isolated strain showed a negative result. The sensiti vity of this kit was 1/64 hemagglutinin unit for AIV subtype H5. The samples of H5N1 AIV infected chicken were all positively detected using these two types of kits. Conclusion The RT-PCR kits for AIV type A and subtype H5 are excellent in specificity, sensitivity, stability and reproducibility.