流感病毒多亚型检测方法的研制和应用
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摘要
本研究利用分子生物学、病毒学和免疫学等技术和手段,旨在探索H3、H5、H7及H9血凝素亚型流感病毒抗原检测方法。
本研究设计了4对针对流感病毒H3、H5、H7及H9血凝素亚型核酸序列的特异性引物,优化多元RT-PCR反应体系,并通过H3、H5、H7及H9亚型血凝素基因在PBV220载体中的原核表达获得同步阳性对照,建立了四种亚型一步法RT-PCR分型检测方法;制备并纯化了流感病毒H3、H5、H7及H9血凝素亚型单克隆抗体,加之阳性标准血清,通过反应条件的优化,建立了抗原捕获ELISA方法,完成了检测试剂盒的组装。
本研究有以下创新点:实现了流感病毒H3、H5、H7及H9血凝素亚型在一个反应体系中、经一次操作进行检测;将检测对象由禽扩大到人和其它哺乳动物;将PBV220原核表达产物中的RNA用作RT-PCR方法中的实时阳性对照;利用亲和层析法获得了四种亚型高纯度的单克隆抗体;首次建立了H3和H7亚型抗原捕获ELISA方法。
研究证明,建立的方法对H3、H5、H7及H9血凝素亚型流感病毒的检测具有高度的特异性、敏感性和安全性,对流感病毒血凝素亚型抗原的检测和区分具有一定的指导意义,为早期发现流感病毒对人、禽的感染提供了实际应用基础。
Avian influenza(AI)is an acute and contiguous zoonosis which originated from avian influenza virus(AIV).According to the difference of pathogenicity,it could classified as highly pathogenic avian influenza(HPAI),low pathogenic avian influenza(LPAI),and naught pathogenic avian influenza(NPAI),HPAI always induce the general infection,and even the morbidity and mortality could reach 100 percent at the natural condition.The outbreak and epidemic of AI resulted in enormous economic casualty and obstruction of international trade all over the world.Moreover, because that AIV could infect to human being,and even causing death,it also threatened the food safe and sanitation seriously.
The mutation of AIV had already broken through the interspecies barrier,which enforcing us to expand the prevention target from AIV to influenza virus(Ⅳ) for accommodating the present situation.Earlier rapid diagnosis is one of the major methods to control the influenza.In recent years,the study and research ofⅣdiagnostic technique developed quickly,but the routine detection methods had demonstrated certain limitations such as hard to operate,time-consuming,poor reproducibility,et al.As a result,multiplex RT-PCR and antigen capture ELISA(AC-ELISA) became the current focus.Although there were already some reports about the two methods forⅣdetection mentioned above,but they were either short of the self quality control standard,or only aimed directly at few definite subtypes,which is very hard to adapt the loemia progressing tendency and prevention characteristic of China.Accordingly,it possessing the practical importance to establish anⅣdetect method with the rapid velocity,well specificity,high sensitivity, and especially aiming at the prevention characteristic of China,as well as taking along the quality control standard.After considering the above matters,and analyzing the history,status quo,progressing tendency of ChineseⅣ,this study optimized and improved 2Ⅳrapid detection methods—multiplex RT-PCR and AC-ELISA.and the practicality and feasibility of the 2 methods were also validated.
Ⅳhas a highly variability,and the mutation is mainly concentrated on hemagglutinin(HA) gene,which has 16 subtypes.This study had taken H3,H5,H7, H9 subtype as the research target,the main according is as follows:Considering the pathogenicity,all the discovered highly pathogenicⅣstrains were all belonged to H5 and H7 subtypes;Considering the prevention characteristic,H9 subtypesⅣis the pandemic strain in China;Considering the sanitation,H3 subtypeⅣhas the widest host range,it could not only infect mammals represented by swine,but also the main subtype of human influenza.Therefore,research on the mentioned 4 subtypes ofⅣcould accord with not only the situation of influenza prevention,but also the progressing tendency of influenza.Meanwhile,it also expanded the detection target to mammals.On account of the above factors,this study established the detection methods focus onⅣH3,H5,H7,H9 subtypes with the multiplex RT-PCR and AC-ELISA technique.
In this study,after searching GenBank for the HA nucleotide sequences ofⅣoccurred in the recent 50 years and include all the AI originated in China.We found 34 pieces for H3HA gene,57 pieces for H5HA gene,78 pieces for H7HA gene,and 121 pieces for H9HA gene,which were accordance with the request above,and took 4 pieces with the best representation as reference strains:H3(CY003712). H5(AY770079),H7(AF831668),H9(DQ664671).4 paires of specific primers aimed directly at conservative region of nucleotide sequence were designed at the two sides of the schizolysis site,which could amplify the fragment of 882 bp,290 bp,693 bp, 550 bp for H3,H5,H7,H9 subtypes respectively,and after analyzed the cross-reactivity and target specificity,the specificity of each pair of primers were verified initially.At first,the study groped the stable react condition of single RT-PCR with the designed primers,and then,RT-PCR of bigeminy,trigeminy,and tetragenus were established step by step,the sensitivity and specificity were also detected.The validation showed that tetragenus RT-PCR could amplify the HA gene ofⅣfrom 1 to 4(H3,H5,H7,and H9) randomly.The amplified fragment were recoveried,and after been gene cloned,enzyme cuted,and sequencing,the result was compared through the BLAST.which show the homology above 98 percent.
Considering the local which positive control of present RT-PCR assay are either positive plasmid DNA or deactivated virus—that positive plasmid DNA can not control the process of detection,embodied on the operation failure of reverse transcription of PCR,which could result in the positive samples showing negative result,and false negative occurred;while,the safe of deactivated virus is anxious.Ad hoc,this study established a suit of safe and isochronous positive control.At first,4 expression plasmid of the 4 subtypesⅣHA gene were constructed through PBV220 prokaryotic expression vector which named pBV-H3HA,pBV-H5HA,pBV-H7HA and pBV-H9HA respectively,after been transformed into BL21 engineering bacteria, prokaryotic thermo-controled expression was progressed,and the expression condition was optimized.The expressed products showed that the HA genes were high performance expressed,the interest protein of H3,H5,H7,and H9 were at the percent of 17.673,18.842,22.519 and 17.125 respectively compared with the total protein of thallus,the quantity of interest protein could reach 9.5,10.84,12.3 and 10.2 mg respectively per-liter.On the base of above studies,the analysis of RNA content in the supernatant was progressed,and mRNA of secondary structure were detected, which could be taken as the isochronous positive control of RT-PCR,and the proper quantity of isochronous positive is 50 mL.At the temperature of 4℃,the mRNAs still could be detected for 6 months,also the temperature of -20℃for 18 months and -70℃for 24 months.
In order to reduce the operational procedure,step down the detection error and environmental pollution,save the time and cost of detection,this study not only introduced one-step RT-PCR system,but also combined the tetragenus RT-PCR and reverse transcription together.Moreover,two sets of negative control were designed: one is the allantoic fluid come from 11 days chick embryo allantoic cavity inoculated with cotton swab enchyma of healthy avian;the other is when progressing the one-step tetragenus RT-PCR,add immediately the reaction agent without any template.On the base of the designed positive control,negative control and tetragenus RT-PCR,the one-step typing RT-PCR assay to detect 4 subtypes ofⅣwas established.The assay was used to detect samples from epidemic area of infectious disease,and the same samples were also detected by chick embryo inoculation, hemagglutination test(HA) and hemagglutination inhibition assay(HI) synchronously. The result showed that the established detection system has a high specificity which coincided with the traditional assays,and the sensitivity could reached 10 pfu.At last, the one-step typing RT-PCR kit was packed,and the definite manipulation procedure and reaction condition were formulated.The typing detection kit based on the above study has not been reported.
AC-ELISA is one of the most sensitive serology assays at present,and the monoclonal antibody(MCAB) of highly purified and high potency is significant to AC-ELISA.This study prepared two strains of H3 subtype MCAB through the syzygium of SP2/0 myeloma cell and spleen cell came from H3 subtype standard antigen immune BALB/c mouse,and the two strains of MCAB were named 1C3 and 1D6 respectively.The cultivated supernatant potency of the two strains of MCAB is 1:320 and 1:640 respectively,and the ascites potency is 1:2×10~5 and 1:1×10~6 respectively.The subtype detection result showed that the 1C3 strain belonged to IgG2a subclass,and the 1D6 strain belonged to IgG1 subclass.The specificity and stability of the two strains of MCAB were also analyzed.The MCAB of H3 subtype and earlier prepared H5,H7 and H9 subtypes were then purified by affinity chromatograph technique with the HiTrap protein G Hp prepacked column,and the high potency MCABs of 10~7 were obtained.
After considering and optimizing the best working concentration of MCABs, best working concentration of standard serum,coating condition,sealing time, reaction condition of MCABs,reaction time of enzyme-signed second antibody,and reaction time of substrate and so on,this study established the AC-ELISA system of standard serum,antigen to be detected,MCABs,enzyme-signed second antibody and substrate colorating.The doubtful interval of negative and positive samples was analyzed by statistics with SPSS10.0 software,the interval is 0.274～0.332.At last, the AC-ELISA kit was packed,and the definite manipulation procedure and reaction condition were formulated.The result showed that the established detection system has a high specificity which coincided with the viral isolation assay,and the sensitivity is 4～8 times higher than traditional hemagglutinin titre.
The above studies provided technique according to establish and consummate the rapid,accurate,sensitive and specificⅣdetection,and settled a rationale for influenza prevention in China.
本研究设计了4对针对流感病毒H3、H5、H7及H9血凝素亚型核酸序列的特异性引物,优化多元RT-PCR反应体系,并通过H3、H5、H7及H9亚型血凝素基因在PBV220载体中的原核表达获得同步阳性对照,建立了四种亚型一步法RT-PCR分型检测方法;制备并纯化了流感病毒H3、H5、H7及H9血凝素亚型单克隆抗体,加之阳性标准血清,通过反应条件的优化,建立了抗原捕获ELISA方法,完成了检测试剂盒的组装。
本研究有以下创新点:实现了流感病毒H3、H5、H7及H9血凝素亚型在一个反应体系中、经一次操作进行检测;将检测对象由禽扩大到人和其它哺乳动物;将PBV220原核表达产物中的RNA用作RT-PCR方法中的实时阳性对照;利用亲和层析法获得了四种亚型高纯度的单克隆抗体;首次建立了H3和H7亚型抗原捕获ELISA方法。
研究证明,建立的方法对H3、H5、H7及H9血凝素亚型流感病毒的检测具有高度的特异性、敏感性和安全性,对流感病毒血凝素亚型抗原的检测和区分具有一定的指导意义,为早期发现流感病毒对人、禽的感染提供了实际应用基础。
Avian influenza(AI)is an acute and contiguous zoonosis which originated from avian influenza virus(AIV).According to the difference of pathogenicity,it could classified as highly pathogenic avian influenza(HPAI),low pathogenic avian influenza(LPAI),and naught pathogenic avian influenza(NPAI),HPAI always induce the general infection,and even the morbidity and mortality could reach 100 percent at the natural condition.The outbreak and epidemic of AI resulted in enormous economic casualty and obstruction of international trade all over the world.Moreover, because that AIV could infect to human being,and even causing death,it also threatened the food safe and sanitation seriously.
The mutation of AIV had already broken through the interspecies barrier,which enforcing us to expand the prevention target from AIV to influenza virus(Ⅳ) for accommodating the present situation.Earlier rapid diagnosis is one of the major methods to control the influenza.In recent years,the study and research ofⅣdiagnostic technique developed quickly,but the routine detection methods had demonstrated certain limitations such as hard to operate,time-consuming,poor reproducibility,et al.As a result,multiplex RT-PCR and antigen capture ELISA(AC-ELISA) became the current focus.Although there were already some reports about the two methods forⅣdetection mentioned above,but they were either short of the self quality control standard,or only aimed directly at few definite subtypes,which is very hard to adapt the loemia progressing tendency and prevention characteristic of China.Accordingly,it possessing the practical importance to establish anⅣdetect method with the rapid velocity,well specificity,high sensitivity, and especially aiming at the prevention characteristic of China,as well as taking along the quality control standard.After considering the above matters,and analyzing the history,status quo,progressing tendency of ChineseⅣ,this study optimized and improved 2Ⅳrapid detection methods—multiplex RT-PCR and AC-ELISA.and the practicality and feasibility of the 2 methods were also validated.
Ⅳhas a highly variability,and the mutation is mainly concentrated on hemagglutinin(HA) gene,which has 16 subtypes.This study had taken H3,H5,H7, H9 subtype as the research target,the main according is as follows:Considering the pathogenicity,all the discovered highly pathogenicⅣstrains were all belonged to H5 and H7 subtypes;Considering the prevention characteristic,H9 subtypesⅣis the pandemic strain in China;Considering the sanitation,H3 subtypeⅣhas the widest host range,it could not only infect mammals represented by swine,but also the main subtype of human influenza.Therefore,research on the mentioned 4 subtypes ofⅣcould accord with not only the situation of influenza prevention,but also the progressing tendency of influenza.Meanwhile,it also expanded the detection target to mammals.On account of the above factors,this study established the detection methods focus onⅣH3,H5,H7,H9 subtypes with the multiplex RT-PCR and AC-ELISA technique.
In this study,after searching GenBank for the HA nucleotide sequences ofⅣoccurred in the recent 50 years and include all the AI originated in China.We found 34 pieces for H3HA gene,57 pieces for H5HA gene,78 pieces for H7HA gene,and 121 pieces for H9HA gene,which were accordance with the request above,and took 4 pieces with the best representation as reference strains:H3(CY003712). H5(AY770079),H7(AF831668),H9(DQ664671).4 paires of specific primers aimed directly at conservative region of nucleotide sequence were designed at the two sides of the schizolysis site,which could amplify the fragment of 882 bp,290 bp,693 bp, 550 bp for H3,H5,H7,H9 subtypes respectively,and after analyzed the cross-reactivity and target specificity,the specificity of each pair of primers were verified initially.At first,the study groped the stable react condition of single RT-PCR with the designed primers,and then,RT-PCR of bigeminy,trigeminy,and tetragenus were established step by step,the sensitivity and specificity were also detected.The validation showed that tetragenus RT-PCR could amplify the HA gene ofⅣfrom 1 to 4(H3,H5,H7,and H9) randomly.The amplified fragment were recoveried,and after been gene cloned,enzyme cuted,and sequencing,the result was compared through the BLAST.which show the homology above 98 percent.
Considering the local which positive control of present RT-PCR assay are either positive plasmid DNA or deactivated virus—that positive plasmid DNA can not control the process of detection,embodied on the operation failure of reverse transcription of PCR,which could result in the positive samples showing negative result,and false negative occurred;while,the safe of deactivated virus is anxious.Ad hoc,this study established a suit of safe and isochronous positive control.At first,4 expression plasmid of the 4 subtypesⅣHA gene were constructed through PBV220 prokaryotic expression vector which named pBV-H3HA,pBV-H5HA,pBV-H7HA and pBV-H9HA respectively,after been transformed into BL21 engineering bacteria, prokaryotic thermo-controled expression was progressed,and the expression condition was optimized.The expressed products showed that the HA genes were high performance expressed,the interest protein of H3,H5,H7,and H9 were at the percent of 17.673,18.842,22.519 and 17.125 respectively compared with the total protein of thallus,the quantity of interest protein could reach 9.5,10.84,12.3 and 10.2 mg respectively per-liter.On the base of above studies,the analysis of RNA content in the supernatant was progressed,and mRNA of secondary structure were detected, which could be taken as the isochronous positive control of RT-PCR,and the proper quantity of isochronous positive is 50 mL.At the temperature of 4℃,the mRNAs still could be detected for 6 months,also the temperature of -20℃for 18 months and -70℃for 24 months.
In order to reduce the operational procedure,step down the detection error and environmental pollution,save the time and cost of detection,this study not only introduced one-step RT-PCR system,but also combined the tetragenus RT-PCR and reverse transcription together.Moreover,two sets of negative control were designed: one is the allantoic fluid come from 11 days chick embryo allantoic cavity inoculated with cotton swab enchyma of healthy avian;the other is when progressing the one-step tetragenus RT-PCR,add immediately the reaction agent without any template.On the base of the designed positive control,negative control and tetragenus RT-PCR,the one-step typing RT-PCR assay to detect 4 subtypes ofⅣwas established.The assay was used to detect samples from epidemic area of infectious disease,and the same samples were also detected by chick embryo inoculation, hemagglutination test(HA) and hemagglutination inhibition assay(HI) synchronously. The result showed that the established detection system has a high specificity which coincided with the traditional assays,and the sensitivity could reached 10 pfu.At last, the one-step typing RT-PCR kit was packed,and the definite manipulation procedure and reaction condition were formulated.The typing detection kit based on the above study has not been reported.
AC-ELISA is one of the most sensitive serology assays at present,and the monoclonal antibody(MCAB) of highly purified and high potency is significant to AC-ELISA.This study prepared two strains of H3 subtype MCAB through the syzygium of SP2/0 myeloma cell and spleen cell came from H3 subtype standard antigen immune BALB/c mouse,and the two strains of MCAB were named 1C3 and 1D6 respectively.The cultivated supernatant potency of the two strains of MCAB is 1:320 and 1:640 respectively,and the ascites potency is 1:2×10~5 and 1:1×10~6 respectively.The subtype detection result showed that the 1C3 strain belonged to IgG2a subclass,and the 1D6 strain belonged to IgG1 subclass.The specificity and stability of the two strains of MCAB were also analyzed.The MCAB of H3 subtype and earlier prepared H5,H7 and H9 subtypes were then purified by affinity chromatograph technique with the HiTrap protein G Hp prepacked column,and the high potency MCABs of 10~7 were obtained.
After considering and optimizing the best working concentration of MCABs, best working concentration of standard serum,coating condition,sealing time, reaction condition of MCABs,reaction time of enzyme-signed second antibody,and reaction time of substrate and so on,this study established the AC-ELISA system of standard serum,antigen to be detected,MCABs,enzyme-signed second antibody and substrate colorating.The doubtful interval of negative and positive samples was analyzed by statistics with SPSS10.0 software,the interval is 0.274～0.332.At last, the AC-ELISA kit was packed,and the definite manipulation procedure and reaction condition were formulated.The result showed that the established detection system has a high specificity which coincided with the viral isolation assay,and the sensitivity is 4～8 times higher than traditional hemagglutinin titre.
The above studies provided technique according to establish and consummate the rapid,accurate,sensitive and specificⅣdetection,and settled a rationale for influenza prevention in China.
引文
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