H5、H7和H9亚型禽流感分子鉴别诊断方法的建立Establishment of Molecular Method for Differentiating and Detecting H5、H7and H9 Subtype of Avian Influenza Virus
黄淑坚,龚中贵,陈秀玲,梁小英,冼琼珍,顾万军,王林川,辛朝安
摘要(Abstract)：
为快速、敏感、特异地鉴别诊断现较为重要的禽流感病毒亚型,根据H5、H7和H9亚型禽流感病毒HA基因序列的保守区域设计出并合成3对特异性引物,利用这3对引物对H5、H7和H9亚型禽流感病毒的HA基因片断进行多联RT-PCR扩增,并对多联RT-PCR检测方法进行敏感性及特异性实验。结果表明:3对引物能特异性地扩增出H5、H7和H9亚型禽流感病毒HA基因片段,大小分别为490bp,365bp和686bp;该检测方法敏感性高,特异性强;初步建立起快速、敏感、特异地鉴别检测H5、H7和H9亚型禽流感病毒的分子诊断方法。
关键词(KeyWords)： 禽流感病毒;H5亚型;H7亚型;H9亚型;多联RT-PCR
基金项目(Foundation): 广东省自然科学基金项目(04300710);; 广东省科技攻关项目(2005A20901003);; 佛山市专项基金项目(03020051)。
作者(Author): 黄淑坚,龚中贵,陈秀玲,梁小英,冼琼珍,顾万军,王林川,辛朝安
参考文献(References)：
- [1]殷震,刘景华.动物病毒学[M].第2版.北京:科学出社社,1997:731-734.
- [2]甘孟侯.禽流感[M].第2版.北京:中国农业出版社,1995:1.
- [3]Isoda N,Sakoda Y,Kishida N,et al.Pathogenicity of a highly pathogenic avian influenza virus,A/chicken/Yamaguchi/7/04(H5N1)in different species of birds and mammals[J].Arch Virol,2006,151(7):1267-1279.
- [4]Gapua I,Alexander D J.Human health implication of Avian influenza viruses and paramyxoviruses[J].Eur J.Clin Microbiol Infect Dis,2004,23:1-5.
- [5]Capua I,Mutinelli F and Hablovarid M H.Avian embryo susceptibility to Italian H7N1avian influenza viruses belonging to different genetic lineages[J].Arch Virol,2002,147:1611-1621.
- [6]Banks J,Speidel E C,McCauley J W,et al.Phylogenetic analysis of H7haemagglutinin subtype influenza A viruses[J].Arch Virol,2000,145:1047-1058.
- [7]Choi Y K,Goya S M,Kang S W,et al.Detection and subtyping of swine influenza H1N1,H1N2and H3N2viruses in clinical samples using two multiplex RT-PCR assays[J].Journal of Virological Methods,2002,102:53-59.
- [8]Akira Yamada,Lap-yee Lam,John S.Tam.Typing and subtyping of influenza viruses and respiratory syncytial viruses by multiplex RT-PCR[J].International Congress Series,2004,1263:381-385.
- [9]Munch M,Nielsen L P,Handberg K J,et al.Detection and subtyping(H5and H7)of avian type A influenza virus by reverse transcription-PCR and PCR-ELISA[J].Arch Virol,2001,146:87-97.
- [10]Osek J.Development of a multiplex PCR approach for the identification of Shiga toxin-producing Escherichia coli strains and their major virulence factor genes[J].Journal of Applied Microbiology,2003,95:1217-1225.
- [11]Starick E,Romer-oberdo-rffr A and Werner O.Type-and Subtype-Specific RT-PCR Assays for Avian Influenza A Viruses(AIV)[J].Vet Med B,2000,47:295-301.
- [12]Banks J,Speide E S,Moore E,et al.Changes in the haemagglutinin and the neuraminidase genes prior to the emergence of highly pathogenic H7N1avian influenza viruses in Italy[J].Arch Virol,2001,146:963-973.
- [13]Kishida N,Sakoda Y,Isoda N,et al.Pathogenicity of H5influenza viruses for ducks[J].Arch Virol,2005,150(7):1383-1392.