内含HCV RNA部分核酸序列的病毒样颗粒的构建及实时荧光RT-PCR分型检测方法的系列研究
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摘要
目的
构建无生物传染危险性且耐RNase的HCV-RNA 1b、2a和6型病毒样颗粒。将病毒样颗粒用于丙型肝炎病毒核酸标准物质和室间质量评价样本的制备,研究其适用性。建立HCV-RNA实时荧光RT-PCR分型(1b、2a和6型)检测方法。
方法
一、内含HCV RNA部分核酸序列的病毒样颗粒的构建
将定量检测试剂盒所检测的区域HCV 5'UTR区和分型检测NS5b区进行overlap连接,并且引入酶切位点。PCR产物进行T载体克隆后用限制性内切酶酶切获得所需要的目的片段,与用酶切后的表达载体相连,构建一新的表达载体。在IPTG诱导下,组装成病毒样颗粒,并验证包装的核酸为HCV reRNA。
二、病毒样颗粒在标准物质的研究及室间质量评价中的应用
靶值的确定:与国家标准物质同时检测,多点比对定值。
稳定性的研究方法:将上述三种不同倍比稀释的质控物置于4℃(对照)、-20℃、室温、37℃、-70℃及-70℃/室温反复冻融3次的条件下保存不同时间观察其稳定性。
室间质量评价方法:向全国开展HCV RNA检测的实验室发放5份样本。所有发放的阳性样本的定量结果根据一级定量标准系列确定,对检测结果进行统计分析。
三、实时荧光RT-PCR分型检测方法
检索已知HCV 1b、2a和6型序列,采用BioEdit软件进行比对,获取HCV 1b、2a和6型序列的保守区域序列。再利用软件Primer Express 3.0进行实时荧光PCR引物和探针设计,初步建立实时荧光PCR检测体系。
结果
一、内含HCV RNA部分核酸序列的病毒样颗粒的构建
成功构建了含有HCV 5'UTR区和NS5b区的三种基因型重组原核表达载体,并转化到BL21-DE3感受态细菌中,得到了可模拟临床样本的HCV RNA病毒样颗粒。
二、质控物的稳定性研究以及应用于临床实验室的PCR检测室间质量评价
采用三种方法分别对VLPs定值,根据系列国家标准品含量,得到待测样本的浓度。在稳定性实验中将检测组与对照组(4℃)数据分别作t检验,结果表明检验组与对照组差异无统计学意义(P＞0.05)。从室间质量评价结果可以看出,整体定量结果与靶值接近,但是不同基因型样本的符合率有所不同。此外,不同试剂和不同仪器检测样本的结果也有差异。
三、实时荧光RT-PCR分型检测方法
通过序列比对设计了三种HCV基因型特异的探针和引物,建立的基因型特异探针和引物基本能检测出已构建的HCV三种基因型的病毒样颗粒。
结论
建立了三种不同基因型的病毒样颗粒,在不同条件下放置依然保持良好的稳定性。与目前冻干血清的标准物质相比,同样具有良好的临床适用性。因此可有效地用于临床实验室HCV-RNA RT-PCR检测的室内质量控制、室间质量评价以及分型检测方法评价。
初步建立了HCV三种基因型特异探针和引物。今后,对于HCV临床治疗有指导意义,具有良好的应用前景。
Objective
To develop the Non-infectious and RNase-resistant 1b, 2a and 6 HCV virus-like particles (VLPs), which were used for positive controls, external quality assessment (EQA) and genotype detections for clinical labs. Meanwhile, we established a new real-time PCR assay for determination of HCV genotypes, and used the VLPs as an internal control.
Methods
1. The VLPs containing HCV genotype 1b, 2a and 6 RNA sequences.
According to the 5' UTR and NS5b sequences of HCV genotypes 1b,2a and 6,which are the prevalent genotypes in China, we designed primers to amplify HCV 5'UTR and NS5b regions by RT-PCR. Restricted sequences were incorporated into the primers. The PCR product was purified and then cloned into T-easy vector. The digested fragments were purified from Gel after digesting the T-easy vector with restriction enzyme. The pET-MS2 previously digested with restriction enzyme was combined with the digested fragment to create a new expression plasmid. Under the induction of IPTG, it assembled with HCV reRNA into VLPs. Then, we examine whether HCV reRNA was packaged into the VLPs.
2. The study in the stability of VLPs and the EQA for HCV RNA test in clinical labs
The VLPs with different concentration were quantified three times with commercialkits for PCR detection. Meanwhile, the stability data of VLPs in different conditions were summarized and evaluated. The EQA involved in HCV RNA test was developed as well. Serum panels were delivered to the clinical laboratories which performed HCV RNA detection in China. Each panel made up of 5 coded samples. All laboratories were requested to carry out the detection within the required time period and report on testing results. All the positive samples were calibrated against the 1st National Standard for HCV RNA.
3. The research on determination of HCV three mainly genotypes in China by real-time reverse transcriptase-PCR
We searched HCV 1b, 2a and 6 sequences from NCBI website. After the alignment of each genotype sequences of HCV by BioEdit software, we determined the consensus sequences of HCV genome. With regard to the consensus sequences, oligonucleotides were designed with Primer Express 3.0 software. Then the real-time reverse transcriptase-PCR for HCV genotyping was primary developed.
Results
1. The construction of virus-like particles containing HCV genotype 1b, 2a and 6 RNA sequences.
The recombinant plasmids for 3 genotypes VLPs containing HCV RNA 5'UTR and NS5b regions were successfully constructed. The experiment has proved that the VLPs are surely packaging RNA.
2. The study in the stability of VLPs and the EQA for HCV RNA test in clinical labs.
The VLPs concentration were quantified against the first National Standard. The stability testing data indicated the quality control materials were stable at least for one month when stored at4℃,-20℃, room temperate, 37℃, -70℃and -70℃freeze-thaw cycles been repeated 3 times. Frome the result of EQA, we can conclude that the whole results are close to the target value, but different genotypes have different percent of pass. Meanwhile, we compared different reagents and different instruments.
3. The research on determination of HCV three mainly genotypes in China by real-time reverse transcriptase-PCR
The specific probes and primers of HCV 3 genotypes were designed according to the alignment of HCV sequences.The VLPs which we have constructed could be tested by the specific probes and primers.
Conclusions
The internal control of HCV 1b, 2a and 6 genotypes which is Non-infectious and RNase-resistance performed good stability, and it would be help to the quality control, EQA and genotyping detection related to HCV RNA test.
We primary established HCV genotyping assay, and it could be widely used for the clinical diagnose and therapy in the future.
构建无生物传染危险性且耐RNase的HCV-RNA 1b、2a和6型病毒样颗粒。将病毒样颗粒用于丙型肝炎病毒核酸标准物质和室间质量评价样本的制备,研究其适用性。建立HCV-RNA实时荧光RT-PCR分型(1b、2a和6型)检测方法。
方法
一、内含HCV RNA部分核酸序列的病毒样颗粒的构建
将定量检测试剂盒所检测的区域HCV 5'UTR区和分型检测NS5b区进行overlap连接,并且引入酶切位点。PCR产物进行T载体克隆后用限制性内切酶酶切获得所需要的目的片段,与用酶切后的表达载体相连,构建一新的表达载体。在IPTG诱导下,组装成病毒样颗粒,并验证包装的核酸为HCV reRNA。
二、病毒样颗粒在标准物质的研究及室间质量评价中的应用
靶值的确定:与国家标准物质同时检测,多点比对定值。
稳定性的研究方法:将上述三种不同倍比稀释的质控物置于4℃(对照)、-20℃、室温、37℃、-70℃及-70℃/室温反复冻融3次的条件下保存不同时间观察其稳定性。
室间质量评价方法:向全国开展HCV RNA检测的实验室发放5份样本。所有发放的阳性样本的定量结果根据一级定量标准系列确定,对检测结果进行统计分析。
三、实时荧光RT-PCR分型检测方法
检索已知HCV 1b、2a和6型序列,采用BioEdit软件进行比对,获取HCV 1b、2a和6型序列的保守区域序列。再利用软件Primer Express 3.0进行实时荧光PCR引物和探针设计,初步建立实时荧光PCR检测体系。
结果
一、内含HCV RNA部分核酸序列的病毒样颗粒的构建
成功构建了含有HCV 5'UTR区和NS5b区的三种基因型重组原核表达载体,并转化到BL21-DE3感受态细菌中,得到了可模拟临床样本的HCV RNA病毒样颗粒。
二、质控物的稳定性研究以及应用于临床实验室的PCR检测室间质量评价
采用三种方法分别对VLPs定值,根据系列国家标准品含量,得到待测样本的浓度。在稳定性实验中将检测组与对照组(4℃)数据分别作t检验,结果表明检验组与对照组差异无统计学意义(P＞0.05)。从室间质量评价结果可以看出,整体定量结果与靶值接近,但是不同基因型样本的符合率有所不同。此外,不同试剂和不同仪器检测样本的结果也有差异。
三、实时荧光RT-PCR分型检测方法
通过序列比对设计了三种HCV基因型特异的探针和引物,建立的基因型特异探针和引物基本能检测出已构建的HCV三种基因型的病毒样颗粒。
结论
建立了三种不同基因型的病毒样颗粒,在不同条件下放置依然保持良好的稳定性。与目前冻干血清的标准物质相比,同样具有良好的临床适用性。因此可有效地用于临床实验室HCV-RNA RT-PCR检测的室内质量控制、室间质量评价以及分型检测方法评价。
初步建立了HCV三种基因型特异探针和引物。今后,对于HCV临床治疗有指导意义,具有良好的应用前景。
Objective
To develop the Non-infectious and RNase-resistant 1b, 2a and 6 HCV virus-like particles (VLPs), which were used for positive controls, external quality assessment (EQA) and genotype detections for clinical labs. Meanwhile, we established a new real-time PCR assay for determination of HCV genotypes, and used the VLPs as an internal control.
Methods
1. The VLPs containing HCV genotype 1b, 2a and 6 RNA sequences.
According to the 5' UTR and NS5b sequences of HCV genotypes 1b,2a and 6,which are the prevalent genotypes in China, we designed primers to amplify HCV 5'UTR and NS5b regions by RT-PCR. Restricted sequences were incorporated into the primers. The PCR product was purified and then cloned into T-easy vector. The digested fragments were purified from Gel after digesting the T-easy vector with restriction enzyme. The pET-MS2 previously digested with restriction enzyme was combined with the digested fragment to create a new expression plasmid. Under the induction of IPTG, it assembled with HCV reRNA into VLPs. Then, we examine whether HCV reRNA was packaged into the VLPs.
2. The study in the stability of VLPs and the EQA for HCV RNA test in clinical labs
The VLPs with different concentration were quantified three times with commercialkits for PCR detection. Meanwhile, the stability data of VLPs in different conditions were summarized and evaluated. The EQA involved in HCV RNA test was developed as well. Serum panels were delivered to the clinical laboratories which performed HCV RNA detection in China. Each panel made up of 5 coded samples. All laboratories were requested to carry out the detection within the required time period and report on testing results. All the positive samples were calibrated against the 1st National Standard for HCV RNA.
3. The research on determination of HCV three mainly genotypes in China by real-time reverse transcriptase-PCR
We searched HCV 1b, 2a and 6 sequences from NCBI website. After the alignment of each genotype sequences of HCV by BioEdit software, we determined the consensus sequences of HCV genome. With regard to the consensus sequences, oligonucleotides were designed with Primer Express 3.0 software. Then the real-time reverse transcriptase-PCR for HCV genotyping was primary developed.
Results
1. The construction of virus-like particles containing HCV genotype 1b, 2a and 6 RNA sequences.
The recombinant plasmids for 3 genotypes VLPs containing HCV RNA 5'UTR and NS5b regions were successfully constructed. The experiment has proved that the VLPs are surely packaging RNA.
2. The study in the stability of VLPs and the EQA for HCV RNA test in clinical labs.
The VLPs concentration were quantified against the first National Standard. The stability testing data indicated the quality control materials were stable at least for one month when stored at4℃,-20℃, room temperate, 37℃, -70℃and -70℃freeze-thaw cycles been repeated 3 times. Frome the result of EQA, we can conclude that the whole results are close to the target value, but different genotypes have different percent of pass. Meanwhile, we compared different reagents and different instruments.
3. The research on determination of HCV three mainly genotypes in China by real-time reverse transcriptase-PCR
The specific probes and primers of HCV 3 genotypes were designed according to the alignment of HCV sequences.The VLPs which we have constructed could be tested by the specific probes and primers.
Conclusions
The internal control of HCV 1b, 2a and 6 genotypes which is Non-infectious and RNase-resistance performed good stability, and it would be help to the quality control, EQA and genotyping detection related to HCV RNA test.
We primary established HCV genotyping assay, and it could be widely used for the clinical diagnose and therapy in the future.
引文
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[1]Simmonds P,Holmes EC,Cha TA,et al.Classification of hepatitis C virus into six major genotypes and a series of subtypes by phylogenetic analysis of the NS-5 region[J].J Gen Virol.1993,74(11):2391-2399.
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[5] Mellor J, Walsh EA, et al. Survey of type 6 group variants of hepatitis C virus in Southeast Asia by using a core-based genotyping assay [J]. J Clin Microbiol. 1996, 34(2):417-423.
[6] Simmonds P, Holmes EC, et al. Classification of hepatitis C virus into six major genotypes and a series of subtypes by phylogenetic analysis of the NS-5 region [J]. J Gen Virol. 1993, 74(11):2391-2399.
[7] Hraber PT, Fischer W, et al. Comparative analysis of hepatitis C virus phytogenies from coding and non-coding regions: the 5' untranslated region (UTR) fails to classify subtypes [J]. Virol J. 2006, 14(3): 103-111.
[8] Laperche S, Lunel F, et al. Comparison of hepatitis C virus NS5b and 5' noncoding gene sequencing methods in a multicenter study [J]. J Clin Microbiol. 2005,43(2):733-739.
[9] Revie D, Alberti MO, et al. Discovery of significant variants containing large deletions in the 5'UTR of human hepatitis C virus(HCV) [J]. Virology Journal 2006,29(3): 82-86.
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[11] Noppornpanth S, Sablon E, et al. Genotyping hepatitis C viruses from Southeast Asia by a novel line probe assay that simultaneously detects core and 5' untranslated regions [J]. J Clin Microbiol. 2006,44(11): 3969-3974.
[12] Saracco G, Olivero A, et al. Therapy of chronic hepatitis C: a critical review [J].Curr Drug Targets Infect Disord. 2003, 3(1):25-32.
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[14] Shiffman ML, Di Bisceglie AM, et al. Hepatitis C Antiviral Long-Term Treatment Against Cirrhosis Trial Group. Peginterferon alfa-2a and ribavirin in patients with chronic hepatitis C who have failed prior treatment [J]. Gastroenterology. 2004, 126(4):1015-1023.
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