猪流感病毒NASBA-ELISA检测方法的建立
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摘要
猪流感是由猪流感病毒引起的一种猪的急性的呼吸道传染病，病毒可以在猪体内发生重组后可以感染人，不仅给畜禽养殖造成重大经济损失，而且还对人类健康造成严重威胁。因此一种快速、敏感、准确的检测手段的建立显得尤为重要，核酸序列依赖性扩增（NASBA）具有操作简单、敏感性高、特异性好的优点，结合酶联寡核苷酸的高敏感性能够准确的检测到猪流感病毒。
本研究根据GENBANK中已发表的猪流感病毒NP基因的高度保守序列设计引物和探针，通过对核酸序列依赖性扩增（NASBA）方法中酶浓度，离子浓度，反应温度等条件的摸索以及优化，建立了检测猪流感病毒NASBA扩增体系。将NASBA方法扩增产物进行琼脂糖凝胶电泳，结果扩增出了391bp的特异性条带。将H1N1、H3N2和H9N2等亚型猪流感病毒的扩增产物进行测序，与GENEBANK中已经发表的猪流感病毒NP基因进行同源性比较分析，结果同源性在80.9%～98.7%。
将NASBA扩增反应与酶联免疫吸附试验（ELISA）相结合，应用ELISA方法来检测NASBA扩增产物，并对其反应条件进行了优化，最终建立了猪流感病毒NASBA-ELISA检测方法。采用已知猪流感病毒A/Swine/TJ4/2006(H1N1)株（H1N1）鸡胚尿囊液（10-7.5ELD50/.2ml），经过10倍系列连续稀释，使用建立的NASAB-ELISA方法检测，能够检测到10-5稀释倍数的病毒液，而PCR方法只检测到了10-2稀释倍数的病毒液，表明该方法有很强的敏感性。特异性试验结果表明，该方法具有很好的特异性，与猪瘟病毒、蓝耳病病毒、伪狂犬病毒、细小病毒、牛病毒性腹泻病毒等均无交叉反应。
采集可疑猪流感临床病例猪鼻拭子样本14份、气管拭子和肺脏组织样本各8份，应用建立的NASBA-ELISA方法进行检测，同时用鸡胚法进行病毒分离培养，结果二者的符合率为96.7%。
Swine influenza is an acute respiratory disease infected by swine influenza virus（SIV）.The virus can infect people after recombined in the blood of swine. It not only takeheavey losses to livestock and poultry, but also lead to serious threat to human healthy. So it’simportant to establish a fast, sensibility and accurate detection way. Nucleic acid sequence-based amplification(NASBA) has some advantages as convenience, highly sensibility andspecificity. It can detect SIV combined with enzyme-linked immunosorbent assay（ELISA）exactly.
An nucleic acid sequence based amplification (NASBA) method was developed fordetecting swine influenza virus (SIV). Primers and probes used in the NASBA assay wasdesigned according to conserved sequence of NP of SIV by Primerpremier5.0and Oligo6.0.The NASBA amplification system was developed by exploratiing and optimizatingconcentration of the enzymes and ions used in the system, and the temperature of reaction. AnRNA gel e1ectrophoresis of product the NASBA revealed that a specific band was amplifiedand size of the RNA is391bp．The NASBA products of four isolates of SIV were sequencedand blasted with the published genes of NP in GENEBANK, the nucleotide homologies ofthem were from80.9%to98.7%. The four SIV isolates belong to H1N1, H3N2and H9N2subtype separately.
For detecting the SIV specificity and sensitively, an enzyme-linked immunosorbentassay (ELISA) based on probes hybridization was used to detect the products of NASBA. AnNASBA-ELISA mothed was developed for detecting SIV by optimizating the concentrationof reagents and reaction conditions. The A/Swine/TJ4/2006(H1N1) of SIV liquid（ELD5010-7.5/0.2ml）was diluted from10-1to10-7continuouely and tested by theNASBA-ELISA and RT-PCR. The NASBA-ELISA results showed SIV diluton10-1to10-5were positive and others dilution were negative, but results of the RT-PCR only can detect10-2dulution of SIV.This results indicates the NASBA more sensitive than RT-PCR. Theresult of the specificity indicates the NASBA method could detect SIV specifici as well as nointeractions with HCV、PRRSV、PRV、PPV and BVDV.
Fourteen nasal swab samples, eight tracheal swabs and eight lung tissue samples werecollected from suspected SI case in Tianjin area，and all of the samples was detected using theNASBA-ELISA established in this study and isolated SIV by inocluted SPF chicken embryos. The result indicates that the coincidence rate was96.7%between the NASBA-ELISA andvirus isolation and identification.
本研究根据GENBANK中已发表的猪流感病毒NP基因的高度保守序列设计引物和探针，通过对核酸序列依赖性扩增（NASBA）方法中酶浓度，离子浓度，反应温度等条件的摸索以及优化，建立了检测猪流感病毒NASBA扩增体系。将NASBA方法扩增产物进行琼脂糖凝胶电泳，结果扩增出了391bp的特异性条带。将H1N1、H3N2和H9N2等亚型猪流感病毒的扩增产物进行测序，与GENEBANK中已经发表的猪流感病毒NP基因进行同源性比较分析，结果同源性在80.9%～98.7%。
将NASBA扩增反应与酶联免疫吸附试验（ELISA）相结合，应用ELISA方法来检测NASBA扩增产物，并对其反应条件进行了优化，最终建立了猪流感病毒NASBA-ELISA检测方法。采用已知猪流感病毒A/Swine/TJ4/2006(H1N1)株（H1N1）鸡胚尿囊液（10-7.5ELD50/.2ml），经过10倍系列连续稀释，使用建立的NASAB-ELISA方法检测，能够检测到10-5稀释倍数的病毒液，而PCR方法只检测到了10-2稀释倍数的病毒液，表明该方法有很强的敏感性。特异性试验结果表明，该方法具有很好的特异性，与猪瘟病毒、蓝耳病病毒、伪狂犬病毒、细小病毒、牛病毒性腹泻病毒等均无交叉反应。
采集可疑猪流感临床病例猪鼻拭子样本14份、气管拭子和肺脏组织样本各8份，应用建立的NASBA-ELISA方法进行检测，同时用鸡胚法进行病毒分离培养，结果二者的符合率为96.7%。
Swine influenza is an acute respiratory disease infected by swine influenza virus（SIV）.The virus can infect people after recombined in the blood of swine. It not only takeheavey losses to livestock and poultry, but also lead to serious threat to human healthy. So it’simportant to establish a fast, sensibility and accurate detection way. Nucleic acid sequence-based amplification(NASBA) has some advantages as convenience, highly sensibility andspecificity. It can detect SIV combined with enzyme-linked immunosorbent assay（ELISA）exactly.
An nucleic acid sequence based amplification (NASBA) method was developed fordetecting swine influenza virus (SIV). Primers and probes used in the NASBA assay wasdesigned according to conserved sequence of NP of SIV by Primerpremier5.0and Oligo6.0.The NASBA amplification system was developed by exploratiing and optimizatingconcentration of the enzymes and ions used in the system, and the temperature of reaction. AnRNA gel e1ectrophoresis of product the NASBA revealed that a specific band was amplifiedand size of the RNA is391bp．The NASBA products of four isolates of SIV were sequencedand blasted with the published genes of NP in GENEBANK, the nucleotide homologies ofthem were from80.9%to98.7%. The four SIV isolates belong to H1N1, H3N2and H9N2subtype separately.
For detecting the SIV specificity and sensitively, an enzyme-linked immunosorbentassay (ELISA) based on probes hybridization was used to detect the products of NASBA. AnNASBA-ELISA mothed was developed for detecting SIV by optimizating the concentrationof reagents and reaction conditions. The A/Swine/TJ4/2006(H1N1) of SIV liquid（ELD5010-7.5/0.2ml）was diluted from10-1to10-7continuouely and tested by theNASBA-ELISA and RT-PCR. The NASBA-ELISA results showed SIV diluton10-1to10-5were positive and others dilution were negative, but results of the RT-PCR only can detect10-2dulution of SIV.This results indicates the NASBA more sensitive than RT-PCR. Theresult of the specificity indicates the NASBA method could detect SIV specifici as well as nointeractions with HCV、PRRSV、PRV、PPV and BVDV.
Fourteen nasal swab samples, eight tracheal swabs and eight lung tissue samples werecollected from suspected SI case in Tianjin area，and all of the samples was detected using theNASBA-ELISA established in this study and isolated SIV by inocluted SPF chicken embryos. The result indicates that the coincidence rate was96.7%between the NASBA-ELISA andvirus isolation and identification.
引文
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[2]丁铲.病毒的进化与传播.中国动物传染病学报,2010,18(4):71-75.
[3]寇晓霞,吴清平,范宏英.轮状病毒NASBA检测研究.微生物学报,2006,46(2):328-330.
[4]高闪电,常惠芸,丛国正等. NASBA(依赖核酸序列的扩增)技术及其在病毒检测中的应用.中国生物工程杂志,2009,29（1）:80-85.
[5]姜晓慧,陈寅,卢亦愚等. H3型流行性感冒病毒环介导逆转录等温扩增检测方法的建立与应用.中国疫苗和免疫,380-382.
[6]孔令青,李勇,高洪等．生物素亲和素标记技术.动物医学进展,2008,29(4):100-102．
[7]李小兰,聂福平,李应国等.猪传染性胃肠炎病毒NASBA检测方法的建立.中国预防兽医学报,2010,32(6):451-454.
[8]李海燕,于康震,杨焕良等.中国猪源H5N1和H9N2亚型流感病毒的分离鉴定.中国预防兽医学报，2004,26(1):1-6.
[9]梁海霞,薛青红,高金源等.高致病性猪生殖与呼吸综合征病毒NASBA-ELISA检测方法的建立.中国兽医科学,2009,39(05):401-404.
[10]骆利敏,黄建生,李明等.应用NASBA定量检测HCV RNA的研究进展. Tropical Medicine,2002,52(1):64-66.
[11]马义才,陈李容, Alison Cox.661份HIV抗体阳性血浆标本HIV-1RNA浓度测定.中国输血杂志.2003,16:312-313.
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