猪瘟兔化弱毒一步法荧光定量PCR检测方法的建立及初步应用
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摘要
猪瘟(Classical swine fever, CSF)是由猪瘟病毒(Classical swine fever virus, CSFV)引起的一种急性、发热性、高度接触性的病毒性传染病。其在世界范围内流行并引起巨大的经济损失,发病率高,死亡率高,严重威胁着养猪业。我国研制的猪瘟兔化弱毒疫苗是国际公认的最安全有效的弱毒疫苗,在我国乃至全球得到广泛应用,为猪瘟的防控做出了巨大贡献。猪瘟兔化弱毒疫苗包括组织苗和细胞苗,然而不同厂家之间生产的疫苗的免疫效价以及产生的免疫效果均不一样。为了对猪瘟兔化病毒含量进行准确测定,本研究通过RT-PCR方法和体外转录方法,构建了体外转录RNA作为标准品;并通过对各种反应条件和反应体系进行优化,建立了一种敏感性高、特异性强、重复性好的猪瘟兔化弱毒一步法荧光定量PCR检测方法。同时,利用该方法对3个不同厂家生产的猪瘟兔化弱毒疫苗进行了猪瘟兔化弱毒含量的测定,以及猪瘟兔化弱毒在猪脐静脉血管内皮细胞和牛睾丸细胞中不同增殖情况的测定。本研究获得以下结果:
(1)参照GenBank中公布的猪瘟兔化弱毒全长序列(登录号为AF091507),在其5′端非翻译区设计一对引物,通过RT-PCR成功得到与预期大小相符且包含有T7启动子序列的DNA片段,回收纯化后在T7 RNA聚合酶作用下通过体外转录成功构建了猪瘟兔化弱毒荧光定量PCR检测方法的RNA标准品。其纯度高,稳定性好,浓度高,一次体外转录得到的RNA标准品保存在-70℃下可供长期使用。
(2)通过对退火温度、引物和探针浓度等反应条件和反应体系的优化,成功建立了能准确定量猪瘟兔化弱毒的一步法荧光定量PCR检测方法。该方法对猪瘟病毒有很好的特异性,检测灵敏度可达10拷贝/μL RNA,比RT-nPCR方法高出一个数量级,在较广的范围内(1.0×10~8～1.0×10~2拷贝/μL)具有很好的相关性(R2=0.999)。对高、中、低3种浓度的标准品RNA进行重复性试验,批内变异系数分别为0.54%、0.52%和0.39%;批间变异系数分别为1.47%、1.85%和1.01%,具有良好的可重复性。
(3)对不同厂家生产的不同类型疫苗进行猪瘟兔化弱毒含量测定表明:同一厂家中每一头份脾淋苗中包含的病毒含量比细胞苗中要高出约1～2个数量级,而从不同厂家对比中可以看出,其生产的脾淋苗和细胞苗之间病毒含量有所差异,其中脾淋苗最高相差20.8倍,细胞苗最高相差30.3倍,但所检测疫苗都达到兔体定型热反应要求。
(4)对猪瘟兔化病毒在猪脐静脉血管内皮细胞和牛睾丸细胞中增殖情况进行了初步测定,结果显示猪瘟兔化病毒在前者中含量高出在后者中7.1倍,为进一步探索猪瘟病毒在细胞中的增殖提供了有效的检测手段。
Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is an acute, highly contagious and devastating viral disease with high morbidity and mortality. CSF is endemic in many countries in the world, causing serious losses in the pig industry worldwide. The well-known lapinized Chinese strain of CSFV, also known as C-strain, was developed in China in the mid-1950s and has been proved to be the safest and most effective vaccine in the past half a century. The C-strain vaccine is widespread used in the world and makes huge contribution to the prevention and control of the CSF. The C-strain vaccines manufactured in our country mainly include the tissue vaccine and the cultured cell vaccine, however, they have different virus titrations and immunological efficacy.
In this study, in order to quantify the virus loads of the C-strain precisely, we constructed a RNA Standard through RT-PCR and in vitro transcription and developed a one step real-time PCR assay. Then, we applied the assay to quantify the virus loads of different types of vaccines from 3 different producers, and evaluated the proliferation of the C-strain in swine umbilical vein endothelial cells (SUVECs) and bovine testicular cells (BTCs). We obtained the following results:
(1)A pair of primes were designed with a T7 promotor’s sequence in the 5′end of the forward one, according to the 5′UTR of the CSFV genome whose accession number is AF091507 on Genbank. The target PCR product was purified and then in vitro transcribed to a single-stranded RNA with the T7 RNA Polymerase. The RNA Standard was obtained after purification and identification of the single-stranded RNA, which had high concentration and purity, as well as good stability. The RNA Standard obtained at a time could be used for a long time when stored at -70℃.
(2)The one step real-time PCR assay for detecting and quantifying the lapinized Chinese strain of CSFV was developed succesfully after the optimization of a variety of conditions, such as the annealing temperature, primer’s and probe’s concentration, etc. The assay was highly specific for detecting CSFV and had a minimum detection limit of 10 copies/μL RNA, 10 times higher than the conventional RT-nPCR. The linear range was from 1.0×10~8 copies/μL to 1.0×10~2 copies/μL, and the correlation coefficient was 0.999. Data of the repetitive tests showed good reproducibilities and the coefficient variation of high, medium, low concentration within and between batches were 0.54%, 0.52%, 0.39% and 1.47%, 1.85%, 1.01%, respectively.
(3)Data of quantifying the virus loads of several lapinized virus vaccines against CSFV showed that the virus loads of the tissue vaccines were 10~100 times higher than that of the cultured cell vaccines comparing each producers’vaccines. However, when comparing with different producers, the virus loads of tissue vaccines and cultured cell vaccines were relatively discrepant. The maximum discrepancy was up to 20.8 times among the tissue vaccines and 30.2 times among the cultured cell vaccines. However, all the vaccines tested in this study achieved the standard made by the conventional rabbit fever reaction.
(4)Data of evaluating the proliferation of the C-strain proliferated in two types of cells showed that the virus loads in SUVECs were approximately 7.1 times higher than that in BTCs, which supplied an effective techniques support for the evaluating the proliferation of the C-strain proliferated in different cell lines.
(1)参照GenBank中公布的猪瘟兔化弱毒全长序列(登录号为AF091507),在其5′端非翻译区设计一对引物,通过RT-PCR成功得到与预期大小相符且包含有T7启动子序列的DNA片段,回收纯化后在T7 RNA聚合酶作用下通过体外转录成功构建了猪瘟兔化弱毒荧光定量PCR检测方法的RNA标准品。其纯度高,稳定性好,浓度高,一次体外转录得到的RNA标准品保存在-70℃下可供长期使用。
(2)通过对退火温度、引物和探针浓度等反应条件和反应体系的优化,成功建立了能准确定量猪瘟兔化弱毒的一步法荧光定量PCR检测方法。该方法对猪瘟病毒有很好的特异性,检测灵敏度可达10拷贝/μL RNA,比RT-nPCR方法高出一个数量级,在较广的范围内(1.0×10~8～1.0×10~2拷贝/μL)具有很好的相关性(R2=0.999)。对高、中、低3种浓度的标准品RNA进行重复性试验,批内变异系数分别为0.54%、0.52%和0.39%;批间变异系数分别为1.47%、1.85%和1.01%,具有良好的可重复性。
(3)对不同厂家生产的不同类型疫苗进行猪瘟兔化弱毒含量测定表明:同一厂家中每一头份脾淋苗中包含的病毒含量比细胞苗中要高出约1～2个数量级,而从不同厂家对比中可以看出,其生产的脾淋苗和细胞苗之间病毒含量有所差异,其中脾淋苗最高相差20.8倍,细胞苗最高相差30.3倍,但所检测疫苗都达到兔体定型热反应要求。
(4)对猪瘟兔化病毒在猪脐静脉血管内皮细胞和牛睾丸细胞中增殖情况进行了初步测定,结果显示猪瘟兔化病毒在前者中含量高出在后者中7.1倍,为进一步探索猪瘟病毒在细胞中的增殖提供了有效的检测手段。
Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is an acute, highly contagious and devastating viral disease with high morbidity and mortality. CSF is endemic in many countries in the world, causing serious losses in the pig industry worldwide. The well-known lapinized Chinese strain of CSFV, also known as C-strain, was developed in China in the mid-1950s and has been proved to be the safest and most effective vaccine in the past half a century. The C-strain vaccine is widespread used in the world and makes huge contribution to the prevention and control of the CSF. The C-strain vaccines manufactured in our country mainly include the tissue vaccine and the cultured cell vaccine, however, they have different virus titrations and immunological efficacy.
In this study, in order to quantify the virus loads of the C-strain precisely, we constructed a RNA Standard through RT-PCR and in vitro transcription and developed a one step real-time PCR assay. Then, we applied the assay to quantify the virus loads of different types of vaccines from 3 different producers, and evaluated the proliferation of the C-strain in swine umbilical vein endothelial cells (SUVECs) and bovine testicular cells (BTCs). We obtained the following results:
(1)A pair of primes were designed with a T7 promotor’s sequence in the 5′end of the forward one, according to the 5′UTR of the CSFV genome whose accession number is AF091507 on Genbank. The target PCR product was purified and then in vitro transcribed to a single-stranded RNA with the T7 RNA Polymerase. The RNA Standard was obtained after purification and identification of the single-stranded RNA, which had high concentration and purity, as well as good stability. The RNA Standard obtained at a time could be used for a long time when stored at -70℃.
(2)The one step real-time PCR assay for detecting and quantifying the lapinized Chinese strain of CSFV was developed succesfully after the optimization of a variety of conditions, such as the annealing temperature, primer’s and probe’s concentration, etc. The assay was highly specific for detecting CSFV and had a minimum detection limit of 10 copies/μL RNA, 10 times higher than the conventional RT-nPCR. The linear range was from 1.0×10~8 copies/μL to 1.0×10~2 copies/μL, and the correlation coefficient was 0.999. Data of the repetitive tests showed good reproducibilities and the coefficient variation of high, medium, low concentration within and between batches were 0.54%, 0.52%, 0.39% and 1.47%, 1.85%, 1.01%, respectively.
(3)Data of quantifying the virus loads of several lapinized virus vaccines against CSFV showed that the virus loads of the tissue vaccines were 10~100 times higher than that of the cultured cell vaccines comparing each producers’vaccines. However, when comparing with different producers, the virus loads of tissue vaccines and cultured cell vaccines were relatively discrepant. The maximum discrepancy was up to 20.8 times among the tissue vaccines and 30.2 times among the cultured cell vaccines. However, all the vaccines tested in this study achieved the standard made by the conventional rabbit fever reaction.
(4)Data of evaluating the proliferation of the C-strain proliferated in two types of cells showed that the virus loads in SUVECs were approximately 7.1 times higher than that in BTCs, which supplied an effective techniques support for the evaluating the proliferation of the C-strain proliferated in different cell lines.
引文
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