尼帕病毒荧光RT-PCR检测方法的建立
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摘要
尼帕病毒病(Nipah Virus,NiV)是1999年新发现的一种烈性人畜共患病,可引起猪的呼吸道症状和人的急性致死性脑炎,首次发现于马来西亚。NiV与1994年在澳大利亚首次发现的人畜共患病病毒——亨德拉病毒(Hendra Virus,HeV)极为相似。NiV与HeV均为副黏病毒科亨尼病毒属成员,虽然只在局部地区暴发,但是它具有很宽的宿主范围,对人的致死率达到40-70%,已成为全球关注的公共卫生问题。虽然我国尚未发现尼帕病,但该病在我国邻近国家,如孟加拉国、印度,频频发生,并且我国西南和华南一带存在大量的此病毒储存宿主——果蝠,因此此病毒对我国构成了巨大威胁。为防范该病传入我国的风险,本研究建立了此病毒荧光RT-PCR检测技术,以期快速检测NiV,并用该方法检测了一些临床样品。
根据GenBank中发表的马来西亚、孟加拉国和印度等国报道的NiV的N基因序列,通过分析比较在其保守区域设计了两对引物和探针,分别对应两套检测体系,筛选出灵敏性和特异性都比较好的一套检测体系。
针对类似检测方法中RNA阳性对照易降解等问题,本研究设计并合成了选定体系的阳性对照序列。该阳性对照序列含有相应引物的结合位点,但两引物之间的阳性对照的一些碱基被去除掉,人工合成此DNA序列后克隆到pBSK质粒,构成重组质粒pBSK-NiV-P,用第一套引物扩增,目的片段纯化后克隆入pMD-18T载体,然后用上下游均含有T7启动子的PUC引物扩增,扩增产物纯化后用T7转录酶系统进行体外转录。体外转录产物本质上是dsRNA,它经适当倍数的稀释后,用作上述荧光RT-PCR的阳性对照。用NanoDrop ND-1000分光光度计对体外转录的dsRNA进行浓度和纯度测定,并分别用DNase、RNase I消化等方法检验其稳定性,结果该阳性对照OD260/OD280值在1.90～2.03之间,纯度较高,经37℃温育24h和RNase I作用1h后仅有微量降解,说明其性质稳定。对反应条件进一步优化后,该方法还可以用于SYBR Green I模式和普通RT-PCR检测。对梯度稀释的RNA和cDNA的检测可知建立的荧光RT-PCR对RNA的检测达到140拷贝/体系,对cDNA的检测达到17拷贝/体系,敏感性比普通RT-PCR高100倍。
从大肠杆菌、Vero细胞及猪各脏器中提取总RNA,用该荧光RT-PCR方法及阳性对照进行检测,结果全为阴性,表明该方法特异性很好。其中有36份猪的脏器来自2005年四川链球菌疫区的病猪,为当时疫病的准确诊断提供了重要参考。从海南采集31只果蝠的血清、咽拭、尿液及各脏器的组织样品。用Vero细胞进行病毒分离。用本研究建立的RT-PCR方法检测组织样品和细胞培养物中是否含有NiV;血清样品用本实验室建立的ELISA方法检测尼帕病毒特异性抗体。ELISA结果与RT-PCR检测结果均为阴性,表明样品来源的果蝠携带或感染过NiV的可能性很小。
Nipah virus (NiV) is a newly recognized zoonotic virus. The virus was discovered in 1999. It has caused severe diseases in animals and in humans through contact with infectious animals. The virus is named after the location where it was first detected in Malaysia. NiV is closely related to another newly recognized zoonotic virus, Hendra virus, named after the town where it first appeared in Australia in 1994. Both Nipah and Hendra viruses are members of the virus family Paramyxoviridae. Although members of this group of viruses have only caused a few local outbreaks, the biologic property of these viruses can infect a wide range of hosts and to produce a disease, causing significant mortality (40%-70%) in humans,which has made this emerging viral infection a public heath concern. NiV outbreak in Malaysia in 1998-1999 claimed 105 human lives and resulted in the culling of about 1.1 million pigs, bringing tremendous economic and social impact to the nation. The presence of NiV in bats has also been demonstrated in Cambodia, India and Bangladesh. Because the natural reservoir of NiV, fruit bats distribute widely in South and West China, and China is near to Bangladesh and India, it is of priority for China to develop some methods including RT-PCR to detect NiV for clinic diagnosis or epidemiological surveillance.
According to the sequences of N gene of NiV reported in Malaysia, Bangladesh and Indonisia in GenBank,we design twosets priers and probes,which according to two sets of detection system,and then choose a better one with superior sensitivity and specificity.
Most RNA positive control currently used for monitoring the quality of RT-PCR assays have some disadvantages, such as instability, inability to monitor the quality of the relevant primers and/or causing indifferentable false positives. To avoid these disadvantages, a simple method to prepare stable and differentiable RNA positive controls is now demonstrated with a real-time RT-PCR assay for the detection of NiV. A DNA sequence which was shorter than its counterpart in the NiV genome and contained the binding sites of the primers of the RT-PCR assay was designed, synthesized and inserted into a vector, and then amplified by PCR with two vector-specific primers both of which contained a T7 promoter at the 5-terminal. The RNA positive control was the dsRNA in vitro transcribed from the PCR amplicons flanked by two T7 promoters. The RNA positive control was stable and able to monitor the quality of the whole concerned RT-PCR assay. False positives caused by contaminations of the RNA positive control or its amplicons could be easily identified because the amplicons of the RNA positive control were obviously shorter than those of real positive samples. Thus, the RNA positive control reported in this study avoided some common disadvantages of current RNA positive controls.
The real-time RT-PCR assay established in this study could detect 140 copies of RNA or 17 copies of cDNA in each reaction. Its sensitivity was approximately 100 times of the conventional RT-PCR. Samples (n=127) from E. coli (n=20), Vero cells (n=7) and swine organs (100) were detected using the RT-PCR assay, and all the results were negative, which demonstrated the high specificity of the assay. Among the 100 swine samples, 36 were collected from the regions in Sichuan in the July of 2005, and the detection facilitated the diagnosis of the notorious Streptococcus suis outbreak.
Samples including serum, nasopharyngeal swabs, urine and organs from 31 bats were collected in Hainan Province for NiV isolation using Vero cells and NiV antibody detection using an in-house ELSA assay. The samples and the cell culture were detected using the RT-PCR assay established in this study. The results of the virus isolation, RT-PCR and ELISA were all negative, which suggested the little possibility for the bat groups in Hainan from which the samples were collected having been infected with NiV.
根据GenBank中发表的马来西亚、孟加拉国和印度等国报道的NiV的N基因序列,通过分析比较在其保守区域设计了两对引物和探针,分别对应两套检测体系,筛选出灵敏性和特异性都比较好的一套检测体系。
针对类似检测方法中RNA阳性对照易降解等问题,本研究设计并合成了选定体系的阳性对照序列。该阳性对照序列含有相应引物的结合位点,但两引物之间的阳性对照的一些碱基被去除掉,人工合成此DNA序列后克隆到pBSK质粒,构成重组质粒pBSK-NiV-P,用第一套引物扩增,目的片段纯化后克隆入pMD-18T载体,然后用上下游均含有T7启动子的PUC引物扩增,扩增产物纯化后用T7转录酶系统进行体外转录。体外转录产物本质上是dsRNA,它经适当倍数的稀释后,用作上述荧光RT-PCR的阳性对照。用NanoDrop ND-1000分光光度计对体外转录的dsRNA进行浓度和纯度测定,并分别用DNase、RNase I消化等方法检验其稳定性,结果该阳性对照OD260/OD280值在1.90～2.03之间,纯度较高,经37℃温育24h和RNase I作用1h后仅有微量降解,说明其性质稳定。对反应条件进一步优化后,该方法还可以用于SYBR Green I模式和普通RT-PCR检测。对梯度稀释的RNA和cDNA的检测可知建立的荧光RT-PCR对RNA的检测达到140拷贝/体系,对cDNA的检测达到17拷贝/体系,敏感性比普通RT-PCR高100倍。
从大肠杆菌、Vero细胞及猪各脏器中提取总RNA,用该荧光RT-PCR方法及阳性对照进行检测,结果全为阴性,表明该方法特异性很好。其中有36份猪的脏器来自2005年四川链球菌疫区的病猪,为当时疫病的准确诊断提供了重要参考。从海南采集31只果蝠的血清、咽拭、尿液及各脏器的组织样品。用Vero细胞进行病毒分离。用本研究建立的RT-PCR方法检测组织样品和细胞培养物中是否含有NiV;血清样品用本实验室建立的ELISA方法检测尼帕病毒特异性抗体。ELISA结果与RT-PCR检测结果均为阴性,表明样品来源的果蝠携带或感染过NiV的可能性很小。
Nipah virus (NiV) is a newly recognized zoonotic virus. The virus was discovered in 1999. It has caused severe diseases in animals and in humans through contact with infectious animals. The virus is named after the location where it was first detected in Malaysia. NiV is closely related to another newly recognized zoonotic virus, Hendra virus, named after the town where it first appeared in Australia in 1994. Both Nipah and Hendra viruses are members of the virus family Paramyxoviridae. Although members of this group of viruses have only caused a few local outbreaks, the biologic property of these viruses can infect a wide range of hosts and to produce a disease, causing significant mortality (40%-70%) in humans,which has made this emerging viral infection a public heath concern. NiV outbreak in Malaysia in 1998-1999 claimed 105 human lives and resulted in the culling of about 1.1 million pigs, bringing tremendous economic and social impact to the nation. The presence of NiV in bats has also been demonstrated in Cambodia, India and Bangladesh. Because the natural reservoir of NiV, fruit bats distribute widely in South and West China, and China is near to Bangladesh and India, it is of priority for China to develop some methods including RT-PCR to detect NiV for clinic diagnosis or epidemiological surveillance.
According to the sequences of N gene of NiV reported in Malaysia, Bangladesh and Indonisia in GenBank,we design twosets priers and probes,which according to two sets of detection system,and then choose a better one with superior sensitivity and specificity.
Most RNA positive control currently used for monitoring the quality of RT-PCR assays have some disadvantages, such as instability, inability to monitor the quality of the relevant primers and/or causing indifferentable false positives. To avoid these disadvantages, a simple method to prepare stable and differentiable RNA positive controls is now demonstrated with a real-time RT-PCR assay for the detection of NiV. A DNA sequence which was shorter than its counterpart in the NiV genome and contained the binding sites of the primers of the RT-PCR assay was designed, synthesized and inserted into a vector, and then amplified by PCR with two vector-specific primers both of which contained a T7 promoter at the 5-terminal. The RNA positive control was the dsRNA in vitro transcribed from the PCR amplicons flanked by two T7 promoters. The RNA positive control was stable and able to monitor the quality of the whole concerned RT-PCR assay. False positives caused by contaminations of the RNA positive control or its amplicons could be easily identified because the amplicons of the RNA positive control were obviously shorter than those of real positive samples. Thus, the RNA positive control reported in this study avoided some common disadvantages of current RNA positive controls.
The real-time RT-PCR assay established in this study could detect 140 copies of RNA or 17 copies of cDNA in each reaction. Its sensitivity was approximately 100 times of the conventional RT-PCR. Samples (n=127) from E. coli (n=20), Vero cells (n=7) and swine organs (100) were detected using the RT-PCR assay, and all the results were negative, which demonstrated the high specificity of the assay. Among the 100 swine samples, 36 were collected from the regions in Sichuan in the July of 2005, and the detection facilitated the diagnosis of the notorious Streptococcus suis outbreak.
Samples including serum, nasopharyngeal swabs, urine and organs from 31 bats were collected in Hainan Province for NiV isolation using Vero cells and NiV antibody detection using an in-house ELSA assay. The samples and the cell culture were detected using the RT-PCR assay established in this study. The results of the virus isolation, RT-PCR and ELISA were all negative, which suggested the little possibility for the bat groups in Hainan from which the samples were collected having been infected with NiV.
引文
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2. Ahmad K., Malaysia culls pigs as Nipah virus strikes again. Lancet, 2000. 356(9225): p. 230.
3. Hsu V.P., Hossain M.J., Parashar U.D., et al., Nipah virus encephalitis reemergence, Bangladesh. Emerg Infect Dis, 2004. 10(12): p. 2082-2087.
4. Harit A.K., Ichhpujani R.L., Gupta S., et al., Nipah/Hendra virus outbreak in Siliguri, West Bengal, India in 2001. Indian J Med Res, 2006. 123(4): p. 553-560.
5. Chadha M.S., Comer J.A., Lowe L., et al., Nipah virus-associated encephalitis outbreak, Siliguri, India. Emerg Infect Dis, 2006. 12(2): p. 235-240.
6. Olson J.G., Rupprecht C., Rollin P.E., et al., Antibodies to Nipah-like virus in bats (Pteropus lylei), Cambodia. Emerg Infect Dis, 2002. 8(9): p. 987-988.
7. Reynes J.M., Counor D., Ong S., et al., Nipah virus in Lyle's flying foxes, Cambodia. Emerg Infect Dis, 2005. 11(7): p. 1042-1047.
8. Wacharapluesadee S., Lumlertdacha B., Boongird K., et al., Bat Nipah virus, Thailand. Emerg Infect Dis, 2005. 11(12): p. 1949-1951.
9. Chua K.B., Nipah virus outbreak in Malaysia. J Clin Virol, 2003. 26(3): p. 265-275.
10. 陈继明,王志亮,赵永刚等, 2004 年孟加拉尼帕病毒病疫情简介. 中华传染病杂志, 2005. 23: p. 144.
11. Harcourt B.H., Lowe L., Tamin A., et al., Genetic characterization of Nipah virus, Bangladesh, 2004. Emerg Infect Dis, 2005. 11(10): p. 1594-1597.
12. 郭丽霞,陈继明,曲光刚等, 尼帕病毒病及其病原的研究进展. 中国兽医科学, 2006. 36(11): p. 940-944.
13. Wang L., Harcourt B.H., Yu M., et al., Molecular biology of Hendra and Nipah viruses. Microbes Infect, 2001. 3(4): p. 279-287.
14. AbuBakar S., Chang L.Y., Ali A.R., et al., Isolation and molecular identification of Nipah virus from pigs. Emerg Infect Dis, 2004. 10(12): p. 2228-2230.
15. Bossart K.N., Wang L.F., Flora M.N., et al., Membrane fusion tropism and heterotypic functional activities of the Nipah virus and Hendra virus envelope glycoproteins. J Virol, 2002. 76(22): p. 11186-11198.
16. Chan Y.P., Koh C.L., Lam S.K., et al., Mapping of domains responsible for nucleocapsidprotein-phosphoprotein interaction of Henipaviruses. J Gen Virol, 2004. 85(Pt 6): p. 1675-1684.
17. Pager C.T., Craft W.W., Jr., Patch J., et al., A mature and fusogenic form of the Nipah virus fusion protein requires proteolytic processing by cathepsin L. Virology, 2006. 346(2): p. 251-257.
18. Moll M., Diederich S., Klenk H.D., et al., Ubiquitous activation of the Nipah virus fusion protein does not require a basic amino acid at the cleavage site. J Virol, 2004. 78(18): p. 9705-9712.
19. Negrete O.A., Levroney E.L., Aguilar H.C., et al., EphrinB2 is the entry receptor for Nipah virus, an emergent deadly paramyxovirus. Nature, 2005. 436(7049): p. 401-405.
20. Bonaparte M.I., Dimitrov A.S., Bossart K.N., et al., Ephrin-B2 ligand is a functional receptor for Hendra virus and Nipah virus. Proc Natl Acad Sci U S A, 2005. 102(30): p. 10652-10657.
21. Negrete O.A., Wolf M.C., Aguilar H.C., et al., Two key residues in ephrinB3 are critical for its use as an alternative receptor for Nipah virus. PLoS Pathog, 2006. 2(2): p. e7.
22. Wong K.T., Grosjean I., Brisson C., et al., A golden hamster model for human acute Nipah virus infection. Am J Pathol, 2003. 163(5): p. 2127-2137.
23. Chua K.B., Koh C.L., Hooi P.S., et al., Isolation of Nipah virus from Malaysian Island flying-foxes. Microbes Infect, 2002. 4(2): p. 145-151.
24. Halpin K., Young P.L., Field H.E., et al., Isolation of Hendra virus from pteropid bats: a natural reservoir of Hendra virus. J Gen Virol, 2000. 81(Pt 8): p. 1927-1932.
25. Field H., Young P., Yob J.M., et al., The natural history of Hendra and Nipah viruses. Microbes Infect, 2001. 3(4): p. 307-314.
26. Chua K.B., A novel approach for collecting samples from fruit bats for isolation of infectious agents. Microbes Infect, 2003. 5(6): p. 487-490.
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38. Chen J.M., Guo L.X., Sun C.Y., et al., A stable and differentiable RNA positive control for reverse transcription-polymerase chain reaction. Biotechnol Lett, 2006. 28(22): p. 1787-1792.
39. 陈继明,王志亮,赵永刚等, 我国尼帕病毒病风险与防控措施初步分析. 中国动物检疫, 2005. 22(1): p. 42-44.
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2. Ahmad K., Malaysia culls pigs as Nipah virus strikes again. Lancet, 2000. 356(9225): p. 230.
3. Hsu V.P., Hossain M.J., Parashar U.D., et al., Nipah virus encephalitis reemergence, Bangladesh. Emerg Infect Dis, 2004. 10(12): p. 2082-2087.
4. Harit A.K., Ichhpujani R.L., Gupta S., et al., Nipah/Hendra virus outbreak in Siliguri, West Bengal, India in 2001. Indian J Med Res, 2006. 123(4): p. 553-560.
5. Chadha M.S., Comer J.A., Lowe L., et al., Nipah virus-associated encephalitis outbreak, Siliguri, India. Emerg Infect Dis, 2006. 12(2): p. 235-240.
6. Olson J.G., Rupprecht C., Rollin P.E., et al., Antibodies to Nipah-like virus in bats (Pteropus lylei), Cambodia. Emerg Infect Dis, 2002. 8(9): p. 987-988.
7. Reynes J.M., Counor D., Ong S., et al., Nipah virus in Lyle's flying foxes, Cambodia. Emerg Infect Dis, 2005. 11(7): p. 1042-1047.
8. Wacharapluesadee S., Lumlertdacha B., Boongird K., et al., Bat Nipah virus, Thailand. Emerg Infect Dis, 2005. 11(12): p. 1949-1951.
9. Chua K.B., Nipah virus outbreak in Malaysia. J Clin Virol, 2003. 26(3): p. 265-275.
10. 陈继明,王志亮,赵永刚等, 2004 年孟加拉尼帕病毒病疫情简介. 中华传染病杂志, 2005. 23: p. 144.
11. Harcourt B.H., Lowe L., Tamin A., et al., Genetic characterization of Nipah virus, Bangladesh, 2004. Emerg Infect Dis, 2005. 11(10): p. 1594-1597.
12. 郭丽霞,陈继明,曲光刚等, 尼帕病毒病及其病原的研究进展. 中国兽医科学, 2006. 36(11): p. 940-944.
13. Wang L., Harcourt B.H., Yu M., et al., Molecular biology of Hendra and Nipah viruses. Microbes Infect, 2001. 3(4): p. 279-287.
14. AbuBakar S., Chang L.Y., Ali A.R., et al., Isolation and molecular identification of Nipah virus from pigs. Emerg Infect Dis, 2004. 10(12): p. 2228-2230.
15. Bossart K.N., Wang L.F., Flora M.N., et al., Membrane fusion tropism and heterotypic functional activities of the Nipah virus and Hendra virus envelope glycoproteins. J Virol, 2002. 76(22): p. 11186-11198.
16. Chan Y.P., Koh C.L., Lam S.K., et al., Mapping of domains responsible for nucleocapsidprotein-phosphoprotein interaction of Henipaviruses. J Gen Virol, 2004. 85(Pt 6): p. 1675-1684.
17. Pager C.T., Craft W.W., Jr., Patch J., et al., A mature and fusogenic form of the Nipah virus fusion protein requires proteolytic processing by cathepsin L. Virology, 2006. 346(2): p. 251-257.
18. Moll M., Diederich S., Klenk H.D., et al., Ubiquitous activation of the Nipah virus fusion protein does not require a basic amino acid at the cleavage site. J Virol, 2004. 78(18): p. 9705-9712.
19. Negrete O.A., Levroney E.L., Aguilar H.C., et al., EphrinB2 is the entry receptor for Nipah virus, an emergent deadly paramyxovirus. Nature, 2005. 436(7049): p. 401-405.
20. Bonaparte M.I., Dimitrov A.S., Bossart K.N., et al., Ephrin-B2 ligand is a functional receptor for Hendra virus and Nipah virus. Proc Natl Acad Sci U S A, 2005. 102(30): p. 10652-10657.
21. Negrete O.A., Wolf M.C., Aguilar H.C., et al., Two key residues in ephrinB3 are critical for its use as an alternative receptor for Nipah virus. PLoS Pathog, 2006. 2(2): p. e7.
22. Wong K.T., Grosjean I., Brisson C., et al., A golden hamster model for human acute Nipah virus infection. Am J Pathol, 2003. 163(5): p. 2127-2137.
23. Chua K.B., Koh C.L., Hooi P.S., et al., Isolation of Nipah virus from Malaysian Island flying-foxes. Microbes Infect, 2002. 4(2): p. 145-151.
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