甲型流感病毒的逆转录重组酶介导核酸扩增快速检测方法研究
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摘要
目的利用逆转录重组酶介导核酸扩增技术(RT-RAA)建立检测甲型流感病毒的快速方法。方法通过使用逆转录酶,将甲流病毒RNA逆转录为cDNA,根据NCBI基因库中甲型流感病毒保守序列设计引物及探针,用cDNA作模板,进行RT-RAA检测甲型流感病毒;构建携带甲型流感病毒的质粒,检测已知样本,分析该方法的灵敏度和特异性。结果采用甲型流感病毒通用型引物和探针,能有效扩增出相应病毒的核酸,而对其他非甲流病毒无交叉反应;RT-RAA反应过程均在39℃恒温条件下完成,用时30min即可得到扩增结果,反应体系中最低扩增拷贝数为100copies。结论建立的RT-RAA检测甲型流感病毒方法灵敏度和特异性高,反应快速、操作简单,适用于甲型流感病毒通用型的快速检测。
OBJECTIVE To quickly detect the influenza A virus by using reverse transcription recombinase-mediated nucleic acid amplification(RT-RAA).METHODS The influenza A virus RNA was transcribed into cDNA by using reverse transcriptase,the primer and probe were designed based on the conserved sequences of the influenza A virus in NCBI gene pool,the cDNA was used as template,and the influenza A virus was detected by using RTRAA.The plasmids that carried with the influenza A virus were constructed so as to detect the known samples,and the sensitivity and specificity of the RT-RAA were observed.RESULTS The nucleic acids of their matching viruses could be effectively amplified by using the universal primer and probe,which had no cross reaction with noninfluenza A viruses.The whole process of RT-RAA was completed under the constant temperature of 39℃,the amplification results could be acquired within 30 min,and the minimum amplification copy number was 100 copies in the reaction system.CONCLUSION The sensitivity and specificity of the RT-RAA are high in the detection of the influenza A virus,with the reaction rapid,the operation simple,and it is suitable for the rapid detection of the universal influenza A virus.
OBJECTIVE To quickly detect the influenza A virus by using reverse transcription recombinase-mediated nucleic acid amplification(RT-RAA).METHODS The influenza A virus RNA was transcribed into cDNA by using reverse transcriptase,the primer and probe were designed based on the conserved sequences of the influenza A virus in NCBI gene pool,the cDNA was used as template,and the influenza A virus was detected by using RTRAA.The plasmids that carried with the influenza A virus were constructed so as to detect the known samples,and the sensitivity and specificity of the RT-RAA were observed.RESULTS The nucleic acids of their matching viruses could be effectively amplified by using the universal primer and probe,which had no cross reaction with noninfluenza A viruses.The whole process of RT-RAA was completed under the constant temperature of 39℃,the amplification results could be acquired within 30 min,and the minimum amplification copy number was 100 copies in the reaction system.CONCLUSION The sensitivity and specificity of the RT-RAA are high in the detection of the influenza A virus,with the reaction rapid,the operation simple,and it is suitable for the rapid detection of the universal influenza A virus.
引文
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[12]庞耀珊,谢芝勋,许显文,等.多重RT-PCR快速检测鉴别H7亚型禽流感病毒方法的建立[J].中国人畜共患病杂志,2005,21(7):595-597.
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[14]Townsend MB,Dawson ED,Mehlmann M,et al.Experimental evaluation of the FluChip diagnostic microarray for influenza virus surveillance[J].J Clin Microbiol,2006,44(8):2863-2871.
[15]Moore C,Telles JN,Corden S,et al.Development and validation of a commercial real-time NASBA assay for the rapid confirmation of influenza A H5N1virus in clinical samples[J].J Virol Methods,2010,170(1-2):173-176.
[2]胡云光,徐兴丽,王晶晶,等.2005-2012国内报道流感病毒发病情况的Meta分析[J].中华微生物学和免疫学杂志,2015,35(4):265-270.
[3]Yang SG,Wo JE,Li MW,et al.Expression of H5N1influenza virus hemagglutinin protein fused with protein transduction domain in analpha virus replicon system[J].J Virol Methods,2010,163(1):31-39.
[4]徐琦,何蕾,罗鹏,等.流行性感冒病毒监测及检测技术的研究进展[J].现代生物医学进展,2014,14(33):6597-6600.
[5]Leonardi GP,Mitrache I,Pigal A,et al.Public hospital-based laboratory experience during an outbreak of pandemic influenza A(H1N1)virus infections[J].J Clin Microbiol,2010,48(4):1189-1194.
[6]Cheng PK,Wong KK,Mak GC,et al.Performance of laboratory diagnostics for the detection of influenza A(H1N1)virus as correlated with the time after symptom onset and viral load[J].J Clin Virol,2010,47(2):182-185.
[7]朱冰,钟家禹,周荣,等.2010年与2011年流感病毒广州分离株全基因组序列分析[J].中华医院感染学杂志,2013,23(9):2012-2014.
[8]Cappuccio J,Dibarbora M,Lozada I,et al.Two years of surveillance of influenza a virus infection in a swine herd.Results of virological,serological and pathological studies[J].Comp Immunol Microbiol Infect Dis,2017,50:110-115.
[9]李瑾,申红卫,秦萌,等.新型多重PCR方法及其在呼吸道病毒诊断上的应用[J].病毒学报,2013,29(6):638-644.
[10]王国政,崔大伟,谢国良,等.四重荧光定量RT-PCR检测人感染新型甲型流感H7N9病毒[J].临床检验杂志,2014,32(11):801-804.
[11]林方,熊伟,康晓平,等.人类流感病毒多重PCR分型方法的建立[J].军事医学科学院院刊,2010,3(4):331-344.
[12]庞耀珊,谢芝勋,许显文,等.多重RT-PCR快速检测鉴别H7亚型禽流感病毒方法的建立[J].中国人畜共患病杂志,2005,21(7):595-597.
[13]戴玉柱,崔大伟,杨先知,等.五重荧光定量RT-PCR法检测甲型流感病毒[J].临床检验杂志,2015,33(9):641-644.
[14]Townsend MB,Dawson ED,Mehlmann M,et al.Experimental evaluation of the FluChip diagnostic microarray for influenza virus surveillance[J].J Clin Microbiol,2006,44(8):2863-2871.
[15]Moore C,Telles JN,Corden S,et al.Development and validation of a commercial real-time NASBA assay for the rapid confirmation of influenza A H5N1virus in clinical samples[J].J Virol Methods,2010,170(1-2):173-176.