H5亞型禽流感病毒HA序列差異越來越大。為更準(zhǔn)確地檢測H5亞型禽流感病毒，對GenBank中發(fā)表的H5亞型禽流感病毒HA序列以及本實驗室保存病毒序列進(jìn)行比對，同時設(shè)計1對引物和1條探針，建立了H5亞型禽流感病毒實時RT-PCR方法，并對該方法的反應(yīng)體系和反應(yīng)參數(shù)進(jìn)行了優(yōu)化。以本實驗室分離測序確定的30份H5亞型禽流感病毒RNA為模板，將該方法與兩種商品化H5亞型禽流感病毒檢測試劑盒進(jìn)行比對，發(fā)現(xiàn)該方法與兩種商品化試劑盒的檢出率分別為100%、98%、98%。敏感性試驗顯示，該方法可以檢測出0.1 fg的RNA模板，靈敏度比兩種商品化試劑盒均提高了10倍。結(jié)果表明，該方法具有更高的特異性和敏感性，可用于H5亞型高致病性禽流感的早期診斷。
Establishment and Application of Real-time RT-PCR for Detection
of H5 Subtype Avian Influenza Virus
In recent years，thedifference in HA sequence of H5 subtype avian influenza virus（AIV）was increasing. In order to detect H5subtype AIV more accurately，by comparing the HAsequence of H5 subtype AIV published in GenBank with the one conserved in thelaboratory and designing a pair of primers and one TaqMan probe，a real-time RT-PCR method was developed，andits reaction system and parameters were optimized. Taking 30 RNA samples of H5subtype AIV strain isolated and sequenced by the laboratory as the template，the established method was compared with two commercial H5 subtypeAIV detection kits，and it was found that the detectionrates were 100%，98% and 98% respectively. Sensitivitytest showed that RNA template of 0.1 fg could be detected by the establishedmethod whose sensitivity was 10 times higher than that of the two commercialkits. In conclusion，the RT-PCR method was more specificand sensitive，and it could be used for early diagnosisof H5 subtype highly pathogenic avian influenza.
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