H1和H3亚型流感病毒荧光RT-PCR及装甲RNA标准物质的研究和应用
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摘要
A型流感病毒是引起人和动物流感流行的主要病原,H1和H3亚型流感病毒是A型流感病毒中感染宿主范围最广、流行面积最大和危害最为严重的两种病毒。实时荧光RT-PCR是A型流感病毒检测和亚型鉴定的一种主要技术,相继被世界卫生组织(WHO)推荐用于2009年甲型H1N1流感和2013年人感染H7N9禽流感病毒的检测。在我国,实时荧光RT-PCR也被应用于H5、H7、H9等亚型以及A型流感病毒通用检测中,并制定了相应的国家标准。本研究立足于我国尚缺乏针对H1和H3亚型流感病毒荧光RT-PCR检测方法的国家标准,开展了相关研究。同时,采用噬菌体装甲RNA技术,研制了流感病毒的新型核酸标准物质,为流感病毒核酸检测技术的质量控制奠定了物质基础。
1.H1和H3亚型流感病毒荧光RT-PCR检测方法的建立
在对A型流感病毒属病毒株序列比对的基础上,分别针对H1和H3亚型HA基因设计引物和LNA或MGB修饰的TaqMan短探针。在对引物、探针进行筛选和反应条件优化的基础上,分别建立了H1和H3亚型流感病毒实时荧光RT-PCR检测方法。特异性试验表明,H1亚型流感病毒实时荧光RT-PCR可有效用于不同宿主来源和基因型毒株的检测,包括H1亚型人季节性流感病毒、古典H1N1猪流感病毒、类禽H1N1猪流感病毒和甲型H1N1流感病毒(2009)；H3亚型流感病毒实时荧光RT-PCR可有效用于不同宿主来源毒株的检测,包括H3N8马流感病毒、H3亚型禽流感病毒和猪流感病毒；且两种方法与其他亚型流感病毒和动物病毒均无交叉反应。灵敏性试验结果表明,H1和H3亚型流感病毒实时荧光RT-PCR对体外转录cRNA标准品检测极限分别为46和69拷贝/反应；重复性试验中,2种方法对含不同病毒核酸拷贝数样品重复检测3次,其Ct值的变异系数均小于5%。将2种方法应用于612份临床送检组织和拭子样品的检测,并与国家标准规定的普通RT-PCR进行比对,结果符合率均在95%以上,且本研究建立的方法比普通RT-PCR方法灵敏度高10倍和100倍。
2.H1和H3亚型流感病毒荧光RT-PCR检测方法国家标准的起草和验证
在2种实时荧光RT-PCR技术建立的基础上,申请获得了国家标准立项,完成操作过程的标准化研究,起草了《动物流感检测H1亚型流感病毒荧光RT-PCR检测方法》和《动物流感检测H3亚型流感病毒荧光RT-PCR检测方法》2项国家标准草案,明确规定了样品采集与前处理程序、样品核酸提取程序、荧光RT-PCR反应体系配制与加样程序、反应参数设置、结果判定原则、引物序列和探针序列等技术内容。2项标准草案在国内13家实验室对合计1519份临床样品验证的基础上,征求13家科研单位的意见形成送审稿。
3.内含A型流感病毒M基因装甲RNA病毒样颗粒的构建和表达
为构建流感病毒新型装甲RNA核酸质控品,将MS2噬菌体成熟酶蛋白基因、衣壳蛋白基因、包装位点以及流感病毒M基因克隆于表达载体pET32a中,经原核表达后,利用Cellufine sulfate层析柱纯化,经RT-PCR和实时荧光RT-PCR鉴定后研制了包装有流感病毒M基因的装甲RNA(Armored-M, AR-M)病毒样颗粒。稳定性研究表明,AR-M可耐受RNase的降解,且在-20℃和4℃至少可保存3个月以上。AR-M用作流感病毒核酸检测质控样品,具有稳定、无生物传染性、可真实模拟病毒粒子结构、耐RNase等特性,将其用作动物A型流感病毒核酸检测质控品可实现对检测全过程的质量控制。
4.A型流感病毒核酸检测装甲RNA标准物质的研制
将包装有流感病毒M基因RNA的病毒样颗粒,经A型流感病毒通用荧光RT-PCR定值后,制备了流感病毒核酸检测的标准物质。均匀性试验表明病毒样颗粒分布均匀,随机抽取的10管标准物质变异系数小于5%；稳定性试验证实装甲RNA标准物质非常稳定,在室温、2-8℃、-20℃分别可保存14天、3个月和6个月以上；经协作标定及不确定度分析,表明该标准物质的不确定度为0.34×106Copies/μL,含量定值为(3.54±0.34)x106Copies/μL,可实现待检样品的定量分析。流感病毒装甲RNA标准物质的推广应用,将为流感病毒核酸检测工作提供统一的、具有可比性的标准物质。
综上所述,本研究建立了H1和H3亚型流感病毒实时荧光RT-PCR检测方法,进而形成了相应的国家标准送审稿。同时,利用噬菌体病毒包装技术,构建了内含流感病毒核酸的装甲RNA标准物质。本研究制定的国家标准和研制的标准物质,将为流感病毒核酸检测技术的规范化使用提供重要的指导和保障。
Influenza A virus (IAV) is an important causative agent responsible for devastating influenza occurring in both human and animal populations. The subtypes H1and H3are the most prevalent subtypes of IAV, infecting a wide range of host species. Real-time RT-PCR (rRT-PCR) has been widely used in IAV detection and subtyping worldwide, and has been recommended by World Health Organization (WHO) for detection of2009H1N1and2013H7N9influenza viruses. In China, universal rRT-PCR assay has successfully been implemented for molecular subtyping of H5, H7, and H9of IAV, serving as a national standard (GB). Nevertheless, so far there has no rRT-PCR assay available as GB for subtyping of H1and H3IAVs in China. In this study, we sought to develop rRT-PCR assays for detection of H1and H3IAVs. Additionally, we intended to prepare recombinant bacteriophage MS2particles containing armored RNA, which were used as a novel material standard for quality control in testing of IAV nucleic acid.
1. Development of two rRT-PCR assays for detection of H1-and H3-subtype IAVs
Primers and TaqMan probes (LNA or MGB) were designed, respectively, based on the nucleotide sequences of HA gene of H1-and H3-subtype IAVs. rRT-PCR reaction systems have been successfully optimized. The specificities of two rRT-PCR assays were evaluated in this study. Our results indicated that all HI subtype IAVs, including human seasonal H1IAV, classical swine H1N1IAV, avian-like swine H1N1IAV and pandemic A (HIN1)2009virus could be accurately detected by the rRT-PCR assay for H1IAV we developed. As for rRT-PCR for H3IAV, all H3IAVs originated from different species could be detected, including H3N8equine IAVs, H3avian IAVs and swine IAVs. All other viruses as well as all other subtype IAVs, excepting H1and H3, were detected negative employing these two methods. The sensitivity of the two assays were evaluated and the results showed that46copies of H1cRNA and69copies of H3cRNA in each reaction could be detected respectively, indicating high sensitivity of the approach. Reproducibility test showed that coefficient variability of both assays was lower than5%. To evaluate the applicability of the methods,612clinical specimens were tested using the two assays, and the results were compared with conventional RT-PCR from GB/T27521-2011. The consistency is still greater than95%between rRT-PCR and RT-PCR, and rRT-PCR is10-fold and100-fold more sensitive than conventional RT-PCR. Our results suggested that rRT-PCR were suitable for detection of IAVs in practice.
2. Validation of the rRT-PCR assays as GB for detection of animal IAV H1and H3subtypes
Based on the validations of the two assays as described above, two drafts, titled "Real-time RT-PCR Assay for the Detection of H1Subtype Influenza Virus" and "Real-time RT-PCR Assay for the Detection of H3Subtype Influenza Virus" have been completed for standardization of H1and H3AIV detection. Detailed and specific information regarding these two assays were indicated in the drafts, including sampling, sample pretreatments, extraction of nucleic acids, formulating of reaction system, sample loading, setting of reaction parameters, result analysis, sequences of primers and probes, etc. A total of1519clinical samples were analyzed in13independent domestic laboratories using the two assays to evaluate these two GB methods. In addition, we consulted a number of experts from13scientific institutions before we submitted the drafts to the official authorities.
3. Expression of virus like particles (VLPs) of bacteriophage MS2containing cRNA of influenza virus M (Armored RNA)
To develop a novel reference material for detection of IAV RNA, the cDNA fragment of maturation protein (A-protein), coat protein, packaging sites of MS2, and the matrix (M) gene of IAV was cloned into vector pET32a to prokaryotically express fusion proteins. The armored RNA (AR-M), encapsidated with M gene in the presence of bacteriophage coat proteins, was purified by cellufine sulfate mini-column and subsequently identified by RT-PCR and rRT-PCR. The stability test indicated that AR-M particles were resistant to RNase degradation and could be reproducibly used in rRT-PCR for90days when stored at-20℃or4℃. AR-M resembled authentic IAV, noninfectious, stable, RNase-resistant. Accordingly, AR-M was an idea reference control for testing of IAV nucleic acids and monitoring the entire detection process.
4. Armored RNA served as standard material for nucleic acid testing of IAV
Based on the quantitation of AR-M using rRT-PCR for IAV, a new RNA standard for influenza virus was prepared using AR-M. The homogeneity test indicates that the prepared recombinant RNA materials were homogenously distributed, confirmed by a vial-viral variation which was lower than5%when10randomly selected vials were examined. Our data demonstrated that AR-M particles were stable, evidenced by the results that AR-M could be stored for at least14days at room temperature,3months at2-8℃or6months at-20℃without any degradation. The quantitation standard of AR-M was (3.54±0.34)×106copies/μL and the averaged uncertainty value was0.34×106copies/μL, which were verified by our collaborative laboratories. This suggested that the AR-M particles could be used for quantitative assay of positive samples.
In summary, in the present study two rRT-PCR assays have successfully been developed for detection of H1or H3subtype IAVs. Consequently, the corresponding GB drafts have been completed and submitted. Additionally, a phage packaging systems, containing armored RNA and genome of IAV was established. The GB drafts and nucleic acid standards obtained in this study would enhance capability of quality control for nucleic-acid testing of IAV.
1.H1和H3亚型流感病毒荧光RT-PCR检测方法的建立
在对A型流感病毒属病毒株序列比对的基础上,分别针对H1和H3亚型HA基因设计引物和LNA或MGB修饰的TaqMan短探针。在对引物、探针进行筛选和反应条件优化的基础上,分别建立了H1和H3亚型流感病毒实时荧光RT-PCR检测方法。特异性试验表明,H1亚型流感病毒实时荧光RT-PCR可有效用于不同宿主来源和基因型毒株的检测,包括H1亚型人季节性流感病毒、古典H1N1猪流感病毒、类禽H1N1猪流感病毒和甲型H1N1流感病毒(2009)；H3亚型流感病毒实时荧光RT-PCR可有效用于不同宿主来源毒株的检测,包括H3N8马流感病毒、H3亚型禽流感病毒和猪流感病毒；且两种方法与其他亚型流感病毒和动物病毒均无交叉反应。灵敏性试验结果表明,H1和H3亚型流感病毒实时荧光RT-PCR对体外转录cRNA标准品检测极限分别为46和69拷贝/反应；重复性试验中,2种方法对含不同病毒核酸拷贝数样品重复检测3次,其Ct值的变异系数均小于5%。将2种方法应用于612份临床送检组织和拭子样品的检测,并与国家标准规定的普通RT-PCR进行比对,结果符合率均在95%以上,且本研究建立的方法比普通RT-PCR方法灵敏度高10倍和100倍。
2.H1和H3亚型流感病毒荧光RT-PCR检测方法国家标准的起草和验证
在2种实时荧光RT-PCR技术建立的基础上,申请获得了国家标准立项,完成操作过程的标准化研究,起草了《动物流感检测H1亚型流感病毒荧光RT-PCR检测方法》和《动物流感检测H3亚型流感病毒荧光RT-PCR检测方法》2项国家标准草案,明确规定了样品采集与前处理程序、样品核酸提取程序、荧光RT-PCR反应体系配制与加样程序、反应参数设置、结果判定原则、引物序列和探针序列等技术内容。2项标准草案在国内13家实验室对合计1519份临床样品验证的基础上,征求13家科研单位的意见形成送审稿。
3.内含A型流感病毒M基因装甲RNA病毒样颗粒的构建和表达
为构建流感病毒新型装甲RNA核酸质控品,将MS2噬菌体成熟酶蛋白基因、衣壳蛋白基因、包装位点以及流感病毒M基因克隆于表达载体pET32a中,经原核表达后,利用Cellufine sulfate层析柱纯化,经RT-PCR和实时荧光RT-PCR鉴定后研制了包装有流感病毒M基因的装甲RNA(Armored-M, AR-M)病毒样颗粒。稳定性研究表明,AR-M可耐受RNase的降解,且在-20℃和4℃至少可保存3个月以上。AR-M用作流感病毒核酸检测质控样品,具有稳定、无生物传染性、可真实模拟病毒粒子结构、耐RNase等特性,将其用作动物A型流感病毒核酸检测质控品可实现对检测全过程的质量控制。
4.A型流感病毒核酸检测装甲RNA标准物质的研制
将包装有流感病毒M基因RNA的病毒样颗粒,经A型流感病毒通用荧光RT-PCR定值后,制备了流感病毒核酸检测的标准物质。均匀性试验表明病毒样颗粒分布均匀,随机抽取的10管标准物质变异系数小于5%；稳定性试验证实装甲RNA标准物质非常稳定,在室温、2-8℃、-20℃分别可保存14天、3个月和6个月以上；经协作标定及不确定度分析,表明该标准物质的不确定度为0.34×106Copies/μL,含量定值为(3.54±0.34)x106Copies/μL,可实现待检样品的定量分析。流感病毒装甲RNA标准物质的推广应用,将为流感病毒核酸检测工作提供统一的、具有可比性的标准物质。
综上所述,本研究建立了H1和H3亚型流感病毒实时荧光RT-PCR检测方法,进而形成了相应的国家标准送审稿。同时,利用噬菌体病毒包装技术,构建了内含流感病毒核酸的装甲RNA标准物质。本研究制定的国家标准和研制的标准物质,将为流感病毒核酸检测技术的规范化使用提供重要的指导和保障。
Influenza A virus (IAV) is an important causative agent responsible for devastating influenza occurring in both human and animal populations. The subtypes H1and H3are the most prevalent subtypes of IAV, infecting a wide range of host species. Real-time RT-PCR (rRT-PCR) has been widely used in IAV detection and subtyping worldwide, and has been recommended by World Health Organization (WHO) for detection of2009H1N1and2013H7N9influenza viruses. In China, universal rRT-PCR assay has successfully been implemented for molecular subtyping of H5, H7, and H9of IAV, serving as a national standard (GB). Nevertheless, so far there has no rRT-PCR assay available as GB for subtyping of H1and H3IAVs in China. In this study, we sought to develop rRT-PCR assays for detection of H1and H3IAVs. Additionally, we intended to prepare recombinant bacteriophage MS2particles containing armored RNA, which were used as a novel material standard for quality control in testing of IAV nucleic acid.
1. Development of two rRT-PCR assays for detection of H1-and H3-subtype IAVs
Primers and TaqMan probes (LNA or MGB) were designed, respectively, based on the nucleotide sequences of HA gene of H1-and H3-subtype IAVs. rRT-PCR reaction systems have been successfully optimized. The specificities of two rRT-PCR assays were evaluated in this study. Our results indicated that all HI subtype IAVs, including human seasonal H1IAV, classical swine H1N1IAV, avian-like swine H1N1IAV and pandemic A (HIN1)2009virus could be accurately detected by the rRT-PCR assay for H1IAV we developed. As for rRT-PCR for H3IAV, all H3IAVs originated from different species could be detected, including H3N8equine IAVs, H3avian IAVs and swine IAVs. All other viruses as well as all other subtype IAVs, excepting H1and H3, were detected negative employing these two methods. The sensitivity of the two assays were evaluated and the results showed that46copies of H1cRNA and69copies of H3cRNA in each reaction could be detected respectively, indicating high sensitivity of the approach. Reproducibility test showed that coefficient variability of both assays was lower than5%. To evaluate the applicability of the methods,612clinical specimens were tested using the two assays, and the results were compared with conventional RT-PCR from GB/T27521-2011. The consistency is still greater than95%between rRT-PCR and RT-PCR, and rRT-PCR is10-fold and100-fold more sensitive than conventional RT-PCR. Our results suggested that rRT-PCR were suitable for detection of IAVs in practice.
2. Validation of the rRT-PCR assays as GB for detection of animal IAV H1and H3subtypes
Based on the validations of the two assays as described above, two drafts, titled "Real-time RT-PCR Assay for the Detection of H1Subtype Influenza Virus" and "Real-time RT-PCR Assay for the Detection of H3Subtype Influenza Virus" have been completed for standardization of H1and H3AIV detection. Detailed and specific information regarding these two assays were indicated in the drafts, including sampling, sample pretreatments, extraction of nucleic acids, formulating of reaction system, sample loading, setting of reaction parameters, result analysis, sequences of primers and probes, etc. A total of1519clinical samples were analyzed in13independent domestic laboratories using the two assays to evaluate these two GB methods. In addition, we consulted a number of experts from13scientific institutions before we submitted the drafts to the official authorities.
3. Expression of virus like particles (VLPs) of bacteriophage MS2containing cRNA of influenza virus M (Armored RNA)
To develop a novel reference material for detection of IAV RNA, the cDNA fragment of maturation protein (A-protein), coat protein, packaging sites of MS2, and the matrix (M) gene of IAV was cloned into vector pET32a to prokaryotically express fusion proteins. The armored RNA (AR-M), encapsidated with M gene in the presence of bacteriophage coat proteins, was purified by cellufine sulfate mini-column and subsequently identified by RT-PCR and rRT-PCR. The stability test indicated that AR-M particles were resistant to RNase degradation and could be reproducibly used in rRT-PCR for90days when stored at-20℃or4℃. AR-M resembled authentic IAV, noninfectious, stable, RNase-resistant. Accordingly, AR-M was an idea reference control for testing of IAV nucleic acids and monitoring the entire detection process.
4. Armored RNA served as standard material for nucleic acid testing of IAV
Based on the quantitation of AR-M using rRT-PCR for IAV, a new RNA standard for influenza virus was prepared using AR-M. The homogeneity test indicates that the prepared recombinant RNA materials were homogenously distributed, confirmed by a vial-viral variation which was lower than5%when10randomly selected vials were examined. Our data demonstrated that AR-M particles were stable, evidenced by the results that AR-M could be stored for at least14days at room temperature,3months at2-8℃or6months at-20℃without any degradation. The quantitation standard of AR-M was (3.54±0.34)×106copies/μL and the averaged uncertainty value was0.34×106copies/μL, which were verified by our collaborative laboratories. This suggested that the AR-M particles could be used for quantitative assay of positive samples.
In summary, in the present study two rRT-PCR assays have successfully been developed for detection of H1or H3subtype IAVs. Consequently, the corresponding GB drafts have been completed and submitted. Additionally, a phage packaging systems, containing armored RNA and genome of IAV was established. The GB drafts and nucleic acid standards obtained in this study would enhance capability of quality control for nucleic-acid testing of IAV.
引文
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