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禽流感病毒一步法RT-PCR检测方法的建立
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包红梅1，王秀荣1，刘丽玲1，王昱1，李一经2，陈化兰1
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（1.中国农业科学院 哈尔滨兽医研究所 农业部动物流感重点开放实验室 兽医生物技术国家重点实验室，
黑龙江 哈尔滨150001； 2.东北农业大学 动物医学院，黑龙江 哈尔滨150030）
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摘要：
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为了能快速准确地对禽流感进行病原学诊断，根据流感病毒的M基因序列，在保守区内用Oligo4.0软件设计了1对引物，建立了一步法RT-PCR诊断方法，其目的片段大小为229bp。通过对不同稀释倍数的H5亚型禽流感病毒尿囊液和棉拭子浸出液进行检测，结果显示，病毒尿囊液的最低检出稀释度为1∶104；阳性棉拭子的最低检出稀释度为1∶23。用病毒分离法和一步法RT-PCR同时检测人工感染鸡不同脏器、口咽及泄殖腔棉拭子样品，二者符合率为100%，但前者的检测灵敏度比后者高10～100倍。用该方法检测H1～H15亚型禽流感病毒和鸡新城疫病毒等其他14种禽病病原，所有禽流感病毒均有229bp的目的条带出现，而其他14种禽病病原均无目的条带出现，表明该方法的特异性好。 |
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关键词：
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禽流感病毒； 一步法RT-PCR；快速诊断 |
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中图分类号：S 852.659.5：Q 503 文献标识码：
A 文章编号：1673-4696(2006)09-0701-06 |
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Development of a one-step RT-PCR assay for detection
of A subtype avian influenza virus |
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BAO Hong-mei1, WANG Xiu-rong1，LIU
Li-ling1，WANG Yu1, LI Yi-jing2, CHEN
Hua-lan1 |
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（1.Key Laboratory of Animal Influenza, Ministry of Agriculture/Nation Key Laboratory of Veterinary Biotechnology/
Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences, Harbin 150001, China;
2.College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China） |
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Abstract: A one-step RT-PCR assay was developed to detect avian influenza virus (AIV). A pair of specific oligonucleotide primers was designed for the detection of AIV according to M gene sequences. Sensitivity for detection of allantoic fluid by the one-step RT-PCR reached 10 thousand dilutions and detection of swab samples reached 8 dilutions. The tissue and swab samples infected with AIV were simultaneity detected by the one-step RT-PCR and the virus isolation method. The sensitivity of the PCR assay was 10 to 100-fold lower than that of the virus isolation method but the PCR assay shared an excellent (100%) correlation with the conventional virus isolation method. H1-H15 subtypes of avian influenza virus and other avian diseases etiological agents were detected by the one-step RT-PCR assay. The results showed that the PCR assay was high specific, and displayed no cross reaction to other subtype or other avian diseases. Development of the one-step RT-PCR provides an effective technical support for the rapid diagnosis and surveillance of molecular epidemiology of avian influenza. |
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Key words: avian influenza virus; one-step RT-PCR; diagnoses expeditiousness |
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