狂犬病病毒荧光定量RT-PCR检测试剂盒的研制与应用
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摘要
本研究通过对GenBank公布的31株基因型1型狂犬病病毒(RV)N基因全序列进行同源性比较分析,选取序列中保守的区域设计了一对引物和一条Taqman探针,优化了反应体系和条件,建立了可特异针对RV的一步法荧光定量RT-PCR检测方法(qRT-PCR).在此基础上,设计了狂犬病病毒荧光定量RT-PCR检测试剂盒生产工艺,组装并成功研制了狂犬病病毒荧光定量RT-PCR检测试剂盒。该检测方法能够特异检测RV,尤其能够特异检测我国狂犬病流行毒株,所采用的引物和探针经BLAST发现与GenBank公布的我国狂犬病流行毒株相应序列完全匹配,只与少数毒株有个别碱基错配,不会影响检测结果。特异性试验证明该检测方法能特异检测基因Ⅰ型狂犬病病毒,而对狂犬病病毒属其他6个基因型病毒和5种导致动物脑炎疾病常见的病原体(犬瘟热病毒(CDV)、犬细小病毒(CPV)、犬腺病病毒(CAV)、水泡性口炎病毒(VSV)、日本乙型脑炎病毒(JEV))不会产生检测信号。稳定性试验表明,5个重复样品Ct值的变异系数均小于5%,证明该方法在细胞毒和脑组织毒检测方面稳定性较好。敏感性试验证明了该检测方法可检测出处于不同进化群的3株疫苗毒株以及分布于我国不同地域处于不同进化支的7株流行街毒株,由此表明该方法可检测我国广大地区的狂犬病病毒。用已知病毒滴度的狂犬病病毒BHK-21 SRV9细胞毒提取总RNA,10倍稀释后制备了标准品,该标准品可以用于准确定量临床样品中狂犬病病毒滴度,以该标准品进行荧光定量RT-PCR检测方法的灵敏度试验,表明该方法可稳定地检测出4.68TCID50的病毒含量,与国家农业行业狂犬病标准检测技术套式RT-PCR灵敏度相当,建立的Ct值与模板起始浓度对数之间的线性关系良好,相关系数为r=0.999,斜率为-3.495,表达式：Ct=-3.495 log 10TCID50+33.29。在qRT-PCR方法建立之后,通过对34份临床狂犬病发病犬脑组织样品进行荧光抗体实验(FAT)、鼠脑内接种实验(MIT)、套式RT-PCR、qRT-PCR检测,结果表明所建立的qRT-PCR方法与套式RT-PCR灵敏度相当,比FAT和MIT敏感,而且适合于腐败样品检测。
按照所设计的狂犬病病毒荧光定量RT-PCR检测试剂盒生产工艺,分别采用分三次合成、经相应检验合格的引物和探针及其他试剂按优化后反应体系的要求配制了三批狂犬病病毒荧光定量RT-PCR检测试剂,并进行试剂盒的组装,编号为2009001、2009002、2009003,进行可重复性试验和为期12个月的保存期试验,综合评价了该试剂盒的灵敏度和特异性等指标,成功研制了特异、灵敏、快速的狂犬病病毒荧光定量RT-PCR检测试剂盒。
为了将所研制的试剂盒应用于疫苗毒株全程生产质量监控以及街毒株细胞适应性研究,采用该技术对实验室候选疫苗毒株BHK-21 SRV9细胞毒以及狂犬病组织培养分离实验(RTCIT) N2A细胞分离的毒株貉、WS、FJ02细胞毒F1-F8代进行定量试验,并与半数细胞感染量(TCID50)试验测定的病毒滴度进行比较。结果显示各细胞毒定量结果与TCID50测定试验结果相差在10个TCID50/100μl以内。由此表明,试剂盒的定量试验可替代TCID50测定方法对各种疫苗进行质量监控以及对街毒株的细胞适应性进行研究。
采用研制的试剂盒进行人工腐败性脑组织样品的检测,结果表明试剂盒与套式RT-PCR均能灵敏地检测到腐败17天后的脑组织,灵敏度比FAT、MIT要高：试剂盒对1994份临床犬脑组织样品检测也同样发现,在新鲜样品检测方面,试剂盒与FAT和MIT符合率为100%,而在检测过程中FAT未检出的1份样品和MIT未检出的2份样品均为送检时已腐败的狂犬病临床发病犬脑组织。由此表明,无论是人工模拟腐败的样品检测还是临床送检腐败的样品检测,试剂盒检测的准确性均达到了FAT和MIT的水平,而且灵敏度更高,更适合于腐败样品的检测。试剂盒对人工感染狂犬病病毒株小鼠的检测试验表明,试剂盒能在小鼠出现临床症状前更早地检测到狂犬病病毒,在狂犬病早期检测中比FAT和MIT也都要灵敏；对2950份流行病学监测犬唾液样品、8份狂犬病病毒感染的动物唾液样品进行检测,结果2950份流行病学监测犬唾液样品中4份唾液呈检测阳性,阳性率为0.14%,试剂盒与套式RT-PCR符合率达100%,8份狂犬病病毒感染的动物唾液样品中3份羊唾液呈检测阳性,而套式RT-PCR只检出2份阳性唾液。由此证明了试剂盒灵敏度与套式RT-PCR相当,达到了动物狂犬病唾液流行病学监测和检测的要求。
此外,在国际实验室比对样品检测中,该试剂盒所检测出的基因1型狂犬病病毒样品与本室建立的基因分型DNA芯片、套式RT-PCR、SYBR Green通用型荧光定量RT-PCR及FAT检测结果完全相符。由此进一步证明我们研制的狂犬病病毒荧光定量RT-PCR检测试剂盒具有良好的敏感性、特异性和可重复性。
通过对狂犬病病毒实时荧光定量RT-PCR检测试剂盒各指标性能的评价以及对大量临床样品的检测,获得了大量令人满意的试剂盒临床前及临床试验数据,表明该试剂盒在我国动物狂犬病监测和检测中具有巨大的应用价值,为试剂盒商品化及其在基层的推广使用提供可靠的数据支持。另外,随着全自动化技术的不断发展及其推广应用,试剂盒已结合全自动样品研磨设备以及全自动核酸提取系统用于高通量样品的筛查,可实现日检测千份样品的能力。该试剂盒不仅适合单个样品的狂犬病确诊,而且还适合动物狂犬病大规模的流行病学监测和检测,将为我国动物狂犬病的消除和控制提供强有力的技术保障。
Rabies is a deadly enzootic disease caused by rabies virus that attacks the central nervous system and causes acute encephalitis.It is reported that worldwide about 40,000-70,000 people die from rabies each year and most of them occurred in the developing country,of which approximately 98% occur in Asia. In China, which now has the second highest rates of illness and death from human rabies cases in the world after India.The reprots of epidemic diseases issued by China CDC showed that the human death toll is more than 2000 every year and has been increasing since 2002; in the year 2007 alone total case numbers were 3302 and a peak of the current epidemic is yet to be reached. These facts indicate that rabies, the important enzootic disease, bears big threat for the public health.
Classical rabies virus, belonging to the Lyssavirus genus within the rhabdoviridae family, and its genome consists of a non-segmented, single-stranded negative-sense RNA of about 11928-11932 nt, comprising five genes that encode the nucleoprotein (N), phosphoprotein (P),matrix protein (M), glycoprotein (G), and RNA-dependent RNA polymerase (large protein, L).The N gene.because of its most conserved among the Lyssaviruses and high copies in replication of virus.is a ideal target for genotyping and diagnosis.Sequence comparison of the N gene, has provided evidence for division of the Lyssavirus genus into seven established genotypes and other four additional genotypes. Based on the phylogentic analysis of the current rabies virus strains in China, it showed that all RVs in our country belong to Lyssavirus genotype 1 and other genotypes has not been described.
It is currently recommended by the WHO Expert Committee that the fluorescent antibody test is the gold criteria for diagnosis of rabies,with the mouse inoculation test (MIT) being used as confirmatory backup procedures and a method to isolate virus, and the detection of viral nucleic acids by reverse transcription polymerase chain reaction (RT-PCR) are increasingly being used for diagnosis and genotyping and, in combination with nucleotide sequencing, for epidemiological investigations.Because of intuitionistic detection result and being easily identified, These techniques have been applied widely in detecting rabies.However,FAT is not fit for detection of decomposing samples and liquid samples such as salivary glands and cerebrospinal fluid (CSF).And MIT needs to take a long experiment period and sacrifice animal welfare. The disadvantages of RT-PCR are the potential for cross-contamination and test failing. In conclusion,these methods are not recommended to detect the massive clinical samples. Nevertheless,the real time PCR offers a sensitive、specific、rapid and high-throughput assay for virus detection.And with the development of this technology at home and abroad, the real time PCR on rabies virus showed good application prospect.
Given the wide variety of rabies susceptible animal species and diverse characteristics of distribution of the current rabies virus strains in our country, one Pair of specific Taqman probe and primers are designed according to integrated arrangement and comparison of partial N gene squences of 31 RV downloaded from the Genbank. And then a one-step real-time RT-PCR(qRT-PCR) was established to detect the rabies virus,through the optimizaion of reaction system and conditions. On this basis, the technical work-flow of detection kit was designed,and eventually Rabies virus Real Time Fluorogenetic Quantitative RT-PCR Detection kit was successfully developed. This test method can detect RV specifically.especially the rabies epidemic strains in China.According to the analysis of BLAST between the probe/primers and corresponding sequences of RV downloaded from the Genbank,only a few sequences mismatches exists,However,mismatches on primer and/or probe binding sites did not affect amplification or detection.The specificity assay showed that the method could only detect the RV, not other six lyssaviruses from genotype 2 to 7, and other viruses such as CDV、CPV、CAV、VSV、JEV.The stability assay showed that the Ct value of five repeated samples are less than 5% of variation coefficient, which proves this method to be a good stability in detection of Intracellular and brain tissue rabies virus.3 vaccine strains and 7 street strains from different clusters/clades and different areas were tested positive by qRT-PCR,which indicated that the method was able to detect a wide range of rabies virus strains in our country.For determination of the relative sensitivity of the qRT-PCR assay compared to nested RT-PCR,a 10-fold serial dilution of total RNA extracted from BHK-21 infected with rabies virus SRV9 was prepared in nuclease-free water and used as a standard samples to quatify viral titers in the clinical samples.The sensitivity assay showed that The detection limit of the established method was 4.68 TCID50, which is equally sensitive with our lab-developed nested RT-PCR.And there is a good line relationship between Ct value and the start density of RNA template, with the correlation coefficent r 0.999, slope-3.412. standard equation: Ct=-3.495 log 10TCID50+33.29.To validate the method 29 fresh and 5 decomposed dog brain specimens submitted to our laboratory for rabies confirmation were detected in comparison with OIE golden diagnostic standard methods FAT and MIT and routinely-used nested RT-PCR. In the result, the qRT-PCR was equally sensitive with nested RT-PCR and more sensitive than FAT and MIT,which is particularly useful for decomposed brain tissue.
According to the production process of the reagent kit,the reproducibility trial and stability trial for 12 monthes duration have synthesized to evaluate several indexes of the reagent kit that has been built up using three batches of qualified qRT-PCR reagent,three batches are 2009001,2009002, and 2009003.
In order to apply the developed kit to monitor production quality of the rabies virus vaccines and study the cellular adaptability of street strains, viral loads was quantified by this kit in Intracellular rabies virus (SRV9,hao,WS,FJ02)from F1 to F8,in comparison with 50% tissue culture infective dose (TCID50) determination test.Comparison showed that the result discrepancy between the two method within 1OTCID50/100μl,indicating that the kit could replace the TCID50 determination method to monitor vaccines production quality and study the virus cellular adaptability.
In this study,we used the developed kit for the rabies virus detection from incubated decomposed brain tissues, and the results showed that the kit and nested RT-PCR cound detect the virus nucleic acid after 17d,which were more sensitive than FAT and MIT;To validate the kit,1994 clinical dog brain specimens submitted to our laboratory for rabies confirmation were detected in comparison with FAT and MIT. And the results by the three methods showed 100% agreement in fresh samples,while FAT/MIT was negative 1/2 samples in decomposed samples. These two studies showed that the kit was more sensitive than FAT and MIT and particularly useful for decomposed brain tissue.The test results of artificially infected mice brain tissues highlighted the utility of the developed kit in ante mortem diagnosis of rabies with higher sensitivity than FAT and MIT;Amongst the 2950 epidemiological monitoring saliva samples and 8 rabies saliva samples tested, both this kit and nested RT-PCR yielded 4/2950(0.14%) samples positive detection,showing 100% similarity,while this kit was positive in 3/8 samples, and nested RT-PCR positive in 2/8 samples According to the results above,these two studies proves that this kit can be used for early diagnosis of animals rabies and meet the demand of epidemiological investigation and diagnosis of animals rabies.
Moreover, the results of test for the international inter-laboratory unknown samples showed that this kit has a complete consistency with DNA microarray for simultaneous detection and genotyping of lyssaviruses and nested RT-PCR and one step SYBR Green I real time RT-PCR for the detection of all lyssavirus genotypes.Thus it is demonstrated that the developed kit has good sensitivity and specificity and repeatability.
The preclinical and clinical results for evaluation of several indexes and detection of clinical samples indicated that the method was very applicable in surveillance and diagnosis of animal rabies,which also provided reliable and supporting datas for its commercialization and widespread use. In addition, with the development of automation technology and its application, this kit has combined with the completely automatic sample grinder and nucleic acid Delivery protocol used for high-throughput screening.with the ability to detect thousands of samples every weekday.So it is not only suitable for diagnosis of single sample, but also suitable for large scale of epidemiological surveillance and rapid detection of animal rabies, which rendering this kit very applicable in elimination and control of rabies.
按照所设计的狂犬病病毒荧光定量RT-PCR检测试剂盒生产工艺,分别采用分三次合成、经相应检验合格的引物和探针及其他试剂按优化后反应体系的要求配制了三批狂犬病病毒荧光定量RT-PCR检测试剂,并进行试剂盒的组装,编号为2009001、2009002、2009003,进行可重复性试验和为期12个月的保存期试验,综合评价了该试剂盒的灵敏度和特异性等指标,成功研制了特异、灵敏、快速的狂犬病病毒荧光定量RT-PCR检测试剂盒。
为了将所研制的试剂盒应用于疫苗毒株全程生产质量监控以及街毒株细胞适应性研究,采用该技术对实验室候选疫苗毒株BHK-21 SRV9细胞毒以及狂犬病组织培养分离实验(RTCIT) N2A细胞分离的毒株貉、WS、FJ02细胞毒F1-F8代进行定量试验,并与半数细胞感染量(TCID50)试验测定的病毒滴度进行比较。结果显示各细胞毒定量结果与TCID50测定试验结果相差在10个TCID50/100μl以内。由此表明,试剂盒的定量试验可替代TCID50测定方法对各种疫苗进行质量监控以及对街毒株的细胞适应性进行研究。
采用研制的试剂盒进行人工腐败性脑组织样品的检测,结果表明试剂盒与套式RT-PCR均能灵敏地检测到腐败17天后的脑组织,灵敏度比FAT、MIT要高：试剂盒对1994份临床犬脑组织样品检测也同样发现,在新鲜样品检测方面,试剂盒与FAT和MIT符合率为100%,而在检测过程中FAT未检出的1份样品和MIT未检出的2份样品均为送检时已腐败的狂犬病临床发病犬脑组织。由此表明,无论是人工模拟腐败的样品检测还是临床送检腐败的样品检测,试剂盒检测的准确性均达到了FAT和MIT的水平,而且灵敏度更高,更适合于腐败样品的检测。试剂盒对人工感染狂犬病病毒株小鼠的检测试验表明,试剂盒能在小鼠出现临床症状前更早地检测到狂犬病病毒,在狂犬病早期检测中比FAT和MIT也都要灵敏；对2950份流行病学监测犬唾液样品、8份狂犬病病毒感染的动物唾液样品进行检测,结果2950份流行病学监测犬唾液样品中4份唾液呈检测阳性,阳性率为0.14%,试剂盒与套式RT-PCR符合率达100%,8份狂犬病病毒感染的动物唾液样品中3份羊唾液呈检测阳性,而套式RT-PCR只检出2份阳性唾液。由此证明了试剂盒灵敏度与套式RT-PCR相当,达到了动物狂犬病唾液流行病学监测和检测的要求。
此外,在国际实验室比对样品检测中,该试剂盒所检测出的基因1型狂犬病病毒样品与本室建立的基因分型DNA芯片、套式RT-PCR、SYBR Green通用型荧光定量RT-PCR及FAT检测结果完全相符。由此进一步证明我们研制的狂犬病病毒荧光定量RT-PCR检测试剂盒具有良好的敏感性、特异性和可重复性。
通过对狂犬病病毒实时荧光定量RT-PCR检测试剂盒各指标性能的评价以及对大量临床样品的检测,获得了大量令人满意的试剂盒临床前及临床试验数据,表明该试剂盒在我国动物狂犬病监测和检测中具有巨大的应用价值,为试剂盒商品化及其在基层的推广使用提供可靠的数据支持。另外,随着全自动化技术的不断发展及其推广应用,试剂盒已结合全自动样品研磨设备以及全自动核酸提取系统用于高通量样品的筛查,可实现日检测千份样品的能力。该试剂盒不仅适合单个样品的狂犬病确诊,而且还适合动物狂犬病大规模的流行病学监测和检测,将为我国动物狂犬病的消除和控制提供强有力的技术保障。
Rabies is a deadly enzootic disease caused by rabies virus that attacks the central nervous system and causes acute encephalitis.It is reported that worldwide about 40,000-70,000 people die from rabies each year and most of them occurred in the developing country,of which approximately 98% occur in Asia. In China, which now has the second highest rates of illness and death from human rabies cases in the world after India.The reprots of epidemic diseases issued by China CDC showed that the human death toll is more than 2000 every year and has been increasing since 2002; in the year 2007 alone total case numbers were 3302 and a peak of the current epidemic is yet to be reached. These facts indicate that rabies, the important enzootic disease, bears big threat for the public health.
Classical rabies virus, belonging to the Lyssavirus genus within the rhabdoviridae family, and its genome consists of a non-segmented, single-stranded negative-sense RNA of about 11928-11932 nt, comprising five genes that encode the nucleoprotein (N), phosphoprotein (P),matrix protein (M), glycoprotein (G), and RNA-dependent RNA polymerase (large protein, L).The N gene.because of its most conserved among the Lyssaviruses and high copies in replication of virus.is a ideal target for genotyping and diagnosis.Sequence comparison of the N gene, has provided evidence for division of the Lyssavirus genus into seven established genotypes and other four additional genotypes. Based on the phylogentic analysis of the current rabies virus strains in China, it showed that all RVs in our country belong to Lyssavirus genotype 1 and other genotypes has not been described.
It is currently recommended by the WHO Expert Committee that the fluorescent antibody test is the gold criteria for diagnosis of rabies,with the mouse inoculation test (MIT) being used as confirmatory backup procedures and a method to isolate virus, and the detection of viral nucleic acids by reverse transcription polymerase chain reaction (RT-PCR) are increasingly being used for diagnosis and genotyping and, in combination with nucleotide sequencing, for epidemiological investigations.Because of intuitionistic detection result and being easily identified, These techniques have been applied widely in detecting rabies.However,FAT is not fit for detection of decomposing samples and liquid samples such as salivary glands and cerebrospinal fluid (CSF).And MIT needs to take a long experiment period and sacrifice animal welfare. The disadvantages of RT-PCR are the potential for cross-contamination and test failing. In conclusion,these methods are not recommended to detect the massive clinical samples. Nevertheless,the real time PCR offers a sensitive、specific、rapid and high-throughput assay for virus detection.And with the development of this technology at home and abroad, the real time PCR on rabies virus showed good application prospect.
Given the wide variety of rabies susceptible animal species and diverse characteristics of distribution of the current rabies virus strains in our country, one Pair of specific Taqman probe and primers are designed according to integrated arrangement and comparison of partial N gene squences of 31 RV downloaded from the Genbank. And then a one-step real-time RT-PCR(qRT-PCR) was established to detect the rabies virus,through the optimizaion of reaction system and conditions. On this basis, the technical work-flow of detection kit was designed,and eventually Rabies virus Real Time Fluorogenetic Quantitative RT-PCR Detection kit was successfully developed. This test method can detect RV specifically.especially the rabies epidemic strains in China.According to the analysis of BLAST between the probe/primers and corresponding sequences of RV downloaded from the Genbank,only a few sequences mismatches exists,However,mismatches on primer and/or probe binding sites did not affect amplification or detection.The specificity assay showed that the method could only detect the RV, not other six lyssaviruses from genotype 2 to 7, and other viruses such as CDV、CPV、CAV、VSV、JEV.The stability assay showed that the Ct value of five repeated samples are less than 5% of variation coefficient, which proves this method to be a good stability in detection of Intracellular and brain tissue rabies virus.3 vaccine strains and 7 street strains from different clusters/clades and different areas were tested positive by qRT-PCR,which indicated that the method was able to detect a wide range of rabies virus strains in our country.For determination of the relative sensitivity of the qRT-PCR assay compared to nested RT-PCR,a 10-fold serial dilution of total RNA extracted from BHK-21 infected with rabies virus SRV9 was prepared in nuclease-free water and used as a standard samples to quatify viral titers in the clinical samples.The sensitivity assay showed that The detection limit of the established method was 4.68 TCID50, which is equally sensitive with our lab-developed nested RT-PCR.And there is a good line relationship between Ct value and the start density of RNA template, with the correlation coefficent r 0.999, slope-3.412. standard equation: Ct=-3.495 log 10TCID50+33.29.To validate the method 29 fresh and 5 decomposed dog brain specimens submitted to our laboratory for rabies confirmation were detected in comparison with OIE golden diagnostic standard methods FAT and MIT and routinely-used nested RT-PCR. In the result, the qRT-PCR was equally sensitive with nested RT-PCR and more sensitive than FAT and MIT,which is particularly useful for decomposed brain tissue.
According to the production process of the reagent kit,the reproducibility trial and stability trial for 12 monthes duration have synthesized to evaluate several indexes of the reagent kit that has been built up using three batches of qualified qRT-PCR reagent,three batches are 2009001,2009002, and 2009003.
In order to apply the developed kit to monitor production quality of the rabies virus vaccines and study the cellular adaptability of street strains, viral loads was quantified by this kit in Intracellular rabies virus (SRV9,hao,WS,FJ02)from F1 to F8,in comparison with 50% tissue culture infective dose (TCID50) determination test.Comparison showed that the result discrepancy between the two method within 1OTCID50/100μl,indicating that the kit could replace the TCID50 determination method to monitor vaccines production quality and study the virus cellular adaptability.
In this study,we used the developed kit for the rabies virus detection from incubated decomposed brain tissues, and the results showed that the kit and nested RT-PCR cound detect the virus nucleic acid after 17d,which were more sensitive than FAT and MIT;To validate the kit,1994 clinical dog brain specimens submitted to our laboratory for rabies confirmation were detected in comparison with FAT and MIT. And the results by the three methods showed 100% agreement in fresh samples,while FAT/MIT was negative 1/2 samples in decomposed samples. These two studies showed that the kit was more sensitive than FAT and MIT and particularly useful for decomposed brain tissue.The test results of artificially infected mice brain tissues highlighted the utility of the developed kit in ante mortem diagnosis of rabies with higher sensitivity than FAT and MIT;Amongst the 2950 epidemiological monitoring saliva samples and 8 rabies saliva samples tested, both this kit and nested RT-PCR yielded 4/2950(0.14%) samples positive detection,showing 100% similarity,while this kit was positive in 3/8 samples, and nested RT-PCR positive in 2/8 samples According to the results above,these two studies proves that this kit can be used for early diagnosis of animals rabies and meet the demand of epidemiological investigation and diagnosis of animals rabies.
Moreover, the results of test for the international inter-laboratory unknown samples showed that this kit has a complete consistency with DNA microarray for simultaneous detection and genotyping of lyssaviruses and nested RT-PCR and one step SYBR Green I real time RT-PCR for the detection of all lyssavirus genotypes.Thus it is demonstrated that the developed kit has good sensitivity and specificity and repeatability.
The preclinical and clinical results for evaluation of several indexes and detection of clinical samples indicated that the method was very applicable in surveillance and diagnosis of animal rabies,which also provided reliable and supporting datas for its commercialization and widespread use. In addition, with the development of automation technology and its application, this kit has combined with the completely automatic sample grinder and nucleic acid Delivery protocol used for high-throughput screening.with the ability to detect thousands of samples every weekday.So it is not only suitable for diagnosis of single sample, but also suitable for large scale of epidemiological surveillance and rapid detection of animal rabies, which rendering this kit very applicable in elimination and control of rabies.
引文
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