CAJ | 학술논문

虽然H3、H9亚型禽流感病毒（Avian influenza virus, AIV）是低致病性禽流感病毒，但其具有跨宿主感染哺乳动物的能力，对社会公共卫生安全具有重大的潜在危害，因而对这些亚型病毒的监测极其重要。通过比对GenBank上H3、H9亚型禽流感病毒HA基因序列，设计了分别针对H3 AIV和H9 AIV的特异性探针。特异性试验和标准曲线试验的结果显示，这两个探针仅分别特异性识别H3、H9亚型AIV，且具有较好的扩增效率。通过重组质粒pMD19-T-H3HA和pMD19-T-H9HA 10倍梯度稀释液为模板，进行敏感性实验。结果表明，本研究所建立的荧光定量PCR方法能检测到100个DNA拷贝数H3 AIV和10个DNA拷贝数的H9 AIV，比传统PCR方法敏感性分别提高了100倍和1000倍。此外，本方法还能检测到10 EID50的H3亚型AIV或H9亚型AIV，比传统PCR方法检测敏感性均提高了1000倍。批间和批内试验的变异系数均小于3%，表明本研究建立的方法具有较好的重复性。此外，与传统PCR方法相比，用H3、H9亚型AIV双重荧光定量PCR方法检测人工感染动物的肺组织时，针对H3 AIV的敏感性提高了10~100倍，针对H9 AIV的敏感性提高了100倍。综上所述，本研究所建立的H3、H9亚型禽流感病毒双重荧光定量PCR方法具有较高的特异性和敏感性，对H3、H9亚型禽流感病毒监测具有重要的应用价值。
수연H3、H9아형금류감병독（Avian influenza virus, AIV）시저치병성금류감병독，단기구유과숙주감염포유동물적능력，대사회공공위생안전구유중대적잠재위해，인이대저사아형병독적감측겁기중요。통과비대GenBank상H3、H9아형금류감병독HA기인서렬，설계료분별침대H3 AIV화H9 AIV적특이성탐침。특이성시험화표준곡선시험적결과현시，저량개탐침부분별특이성식별H3、H9아형AIV，차구유교호적확증효솔。통과중조질립pMD19-T-H3HA화pMD19-T-H9HA 10배제도희석액위모판，진행민감성실험。결과표명，본연구소건립적형광정량PCR방법능검측도100개DNA고패수H3 AIV화10개DNA고패수적H9 AIV，비전통PCR방법민감성분별제고료100배화1000배。차외，본방법환능검측도10 EID50적H3아형AIV혹H9아형AIV，비전통PCR방법검측민감성균제고료1000배。비간화비내시험적변이계수균소우3%，표명본연구건립적방법구유교호적중복성。차외，여전통PCR방법상비，용H3、H9아형AIV쌍중형광정량PCR방법검측인공감염동물적폐조직시，침대H3 AIV적민감성제고료10~100배，침대H9 AIV적민감성제고료100배。종상소술，본연구소건립적H3、H9아형금류감병독쌍중형광정량PCR방법구유교고적특이성화민감성，대H3、H9아형금류감병독감측구유중요적응용개치。
Although H3 and H9 subtypes of Avian influenza virus (AIV) are low pathogenic in birds, they have adapted abilities to infect mammalians, which poses potential threat to human beings. Therefore, surveillance of H3 and H9 subtypes of AIV is important for public health. In the present study, TaqMan primers specific for either H3 or H9 subtype were designed to develop fluorescent quantitative PCR (qPCR) by comparing the homology of HA gene sequences submitted on the GenBank. The specificity testing and amplification trials demonstrated that TaqMan primers only recognized their respective H3 or H9 subtype. The sensitivity testing of the assay was performed using 10 fold serial dilutions of pMD19-T-H3HA and pMD19-T-H9HA as templates. The result showed that qPCR was able to detect 100 DNA copies of H3 subtype or 10 DNA copies of H9 subtype, which was 100 fold or 1000 fold higher than conventional PCR. In addition, this assay was also able to detect 10 EID50 of both H3 and H9 subtypes, which was 1000 fold higher than conventional PCR. The inter-and intra-assay trials demonstrated that the variations were less than 3%for both subtypes, indicating its good reproducibility. Moreover, qPCR was used to detect viruses in lung samples from experimentally infected chickens. As compared to conventional PCR, qPCR was 10-100 fold more sensitive for H3 subtype or 100 fold more sensitive for H9 subtype than conventional PCR. Therefore, qPCR method developed in this study showed high specificity and sensitivity and could be used for epidemiological study of H3 and H9 subtypes.