新发感染性疾病的未知病毒性病原体检测及鉴定技术的建立
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摘要
本研究利用病毒仅具有“核酸—蛋白质复合体”大分子以及几乎所有医学病毒都具有耐受DNase处理的衣壳蛋白的特征,采用非序列依赖的单引物扩增方法,对DNA病毒经Klenow Fragment补平、RNA病毒反转录成cDNA后再合成双链cDNA,经四碱基限制性内切酶酶切,加接头分子,以此为模板、以接头分子为引物进行单引物PCR扩增；构建PCR产物克隆、测序,在GenBank上BLAST序列比对分析及核酸进化树分析,判断所检测病毒的种属。在此基础上将标本的预处理改在生物安全柜内进行,减少无关核酸的影响。同时,选定乙型肝炎病毒(DNA病毒)和艾滋病毒(RNA病毒)为研究对象,分别收集乙型肝炎和艾滋病病人各3例血清标本,验证上述检测新发未知病毒性感染疾病病原体技术的特异性和灵敏性。结果显示,DNaseⅠ处理血清中内源DNA未观察病毒DNA降解；非序列依赖的单引物扩增血清中病毒DNA核酸可得到多个DNA片段；将所有PCR产物克隆、测序,序列经GenBank BLAST软件比对分析显示,105GE/ml以上拷贝数的病毒被检出,与HBV DNA同源性达98%-100%。与HIV基因序列同源性达91%～97%；以引起手足口病的肠道病毒EV71验证所建立的病毒性病原体的检测技术,非序列依赖的单引物可以扩增粪便标本中病毒核酸得到多个DNA片段：将所有PCR产物克隆、测序,BLAST分析,所得序列中,有的序列与GenBank中登录的EV71基因序列同源性达98%-99%；有的序列与GenBank中登录的单核细胞增生李斯特(Listeria monocytogenes)菌CDC54007同源性达81%。上述研究结果证实,应用所建立的改良的非序列依赖的单引物扩增方法可以检测血清中105GE/ml以上拷贝数的DNA及RNA病毒性病原；上述检测技术不仅适用于血清标本,也适用于粪便标本的检测；检测技术不依赖于被检病毒的核酸序列等信息；可以检测已知病毒,也可以鉴定未知病毒；检测过程不依赖于病毒的分离培养,因此缩短了检测时间,可以快速检测和鉴定病毒性病原体。
The virus-induced infectious disease, such as acute respiratory infection, hemorrhagic fever, diarrhea, AIDS, mad cow disease, bird flu, et al. not only caused a great panic of human life and health, but also resulted in huge losses to the world regional economy. According to statistics, infectious diseases show a kind of tendency that classic diseases reduce but new infectious diseases increase in the past two decades.70% of new infectious diseases are caused by viral pathogens, the new viral disease often shows the characteristics that are a high mortality rate, spread rapidly and controlled difficultly. The present study found that the study sphere of viral disease are expanded to infectious diseases from communicable disease. The experiences and lessons that are got from major viral diseases such as bird flu, AIDS, SARS et al, tell people that there is great significance for the prevention and treatment by early diagnosis or exclusion of viral pathogens. However, a wide range of viruses, now found more than 4000 kinds, among 400 kinds medical virus, the new virus is growing at the rate of one kind one year in the natural world. Specific detected and analysed methods can not analyzed the new virus, and electron microscopy is not suitable for routine use, severe situation of human infectious diseases appeal new technology or union with a variety of methods in order to detect or exclude fast, primitively and effectively unknown viruses pathogens, and it provides the evidence for disease control and drug development.
The unique study on new viral pathogens lies in that the virus is a macromolecules which have "nucleic acid-protein complex", so the earlier research is base on epidemiology and depending on the culture and electron microscopy. The modern molecular biology technology greatly promoted the study of viral pathogens technology, and utilizing adequately macromolecular characteristics of the virus and isolating and identifying with the corresponding immunology and molecular biology methods, a series of new methods developed led to the discovery of most of the new virus. The key problem of molecular virus technology is that the virus content is lower and host nucleic acid and protein content is higher of the disease samples, so the general technological principles are divided into two categories:amplifying (non-specific-PCR) and screening (immunization, nucleic acid hybridization). Non-specific PCR and nucleic acid hybridization techniques based on nucleic acid are relatively shortcut, so the together non-specific PCR with representational difference analysis (RDA) technology of subtractive hybridization found in a series of transfusion-transmitted hepatitis B virokine (1997). However, RDA workload is enormous, with almost all the medical virus have tolerance characteristics for DNase treated capsid protein, Tobias A treated host acid nuclear with DNase and plus joint molecules then amplify enzyme cut spectrum of the virus by single non-specific PCR, and found two new parvovirus in commodity bovine serum by the technology (SISPA). SISPA technology uses commendably the structural characteristics of the virus and very fast and convenient, the defect is that it only detected unknown viruses above 106GE/ml copy. It restricts the application of molecular virus discovery technology because both rapid and sensitive can not take into account, so the early detection of SARS-CoV is still dependent on the electron microscope, that presented a great challenge for molecular virus discovery technology.
Objective To establish a kind of new detected methods for viral pathogen of new infectious diseases by taking hepatitis B virus (DNA virus) and HIV (RNA virus) as the research object, in order to detect or exclude quickly, primitively and effectively viral pathogens and provide the basis for disease control and drug development.
Methods Applying DNase I sequence-independent single primer amplification technology, the HBV serum which were selected by clinical diagnosis and laboratory real-time fluorescence quantitative PCR detection, then were filtered and treated to remove endogenous DNA with DNase I. The complete DNA is digested with four base restriction enzyme digestion Csp6.I after applying Klenow Fragment to filling-in, the pathogenic gene were amplified non-specific with plused joint molecule as the single-primer, then sequenced and analysed, then judged detected pathogen nucleic acid.
Similarly, the HIV serum which were also selected by clinical diagnosis and laboratory real-time fluorescence quantitative PCR and ELIS A detection, then were filtered and treated to remove endogenous DNA with DNase I. then reverse transcripted and synthesized double-stranded cDNA of the RNA virus, The double-stranded cDNA is digested by four base restriction enzyme digestion Taq I, the pathogenic gene were amplified non-specific with plused joint molecule as the single-primer, then sequenced and analysed, then judged detected pathogen nucleic acid.
To verify the detection technology of viral pathogen by enterovirus EV71 which cause hand-foot-mouth disease. According to hand-foot-mouth disease laboratory detect program (for trial implementation), that is, the appendix of " the Guide of Hand-Foot-Mouth Disease Prevention and Control " (2008 edition) that Ministry of Public Health issued, to collect the faeces of infants that had been diagnosed hand-foot-mouth disease in the clinical and were positive by reverse transcription-polymerase chain reaction (RT-PCR) in the laboratory, the specimens were filtered, digested and processed by DNase, then reverse transcripted and synthesized double-stranded cDNA of the RNA virus, the double-stranded cDNA is digested with four base restriction enzyme digestion Csp6. I, the pathogenic gene were amplified non-specific with plused joint molecule as the single-primer, then sequenced and analysed, then judged detected pathogen nucleic acid.
Results
1 The viral DNA can not be degraded when DNase I treated endogenous DNA of the serum. Getting a number of DNA fragments with using non-sequence-dependent single primer to amplify exogenous DNA (viral DNA) from the serum. All PCR products were cloned, sequenced and analysed, the sequences is consistent with HBV DNA sequences of known pathogens, it can detect the virus with 105GE/ml copy number.
2 It does not affect the amount of viral nucleic acid that DNase I treated endogenous DNA of the serum. Getting a number of DNA fragments with using non-sequence-dependent single primer to amplify exogenous DNA (viral DNA) from the serum. All PCR products were cloned, sequenced and BLAST analysis, the homology is up to 94:8% with HIV-sequence in the Gene Bank, so we can judge that the detected pathogens is HIV.
3 To verify the detection technology of viral pathogen by enterovirus EV71 which cause hand-foot-mouth disease, getting a number of DNA fragments with using non-sequence-dependent single primer to amplify exogenous DNA of the faeces specimen. All PCR products were cloned, sequenced and BLAST analysis, we found homology is up to 99% between some sequences of them and known pathogens EV71 sequence in the Gene Bank,, and is 81% between some sequences of others and Listeria monocytogenes strain CDC54007 in the Gene Bank,.
Conclusion
1 The method can detect DNA viral pathogens of the serum applying the DNase I treatment and sequence-independent single primer amplification technology.
2 The method can detect RNA viral pathogens of the serum applying the DNase I treatment and sequence-independent single primer amplification technology.
3 The method can detect viral pathogens of the faeces specimen applying the DNase I treatment and sequence-independent single primer amplification technology.
4 The improved sequence-independent single primer amplification technology not only can detect serum samples, but also can detect fecal samples, that will expand the sphere of detected samples.
5 The established method is not dependent on sequence-specific.
6 The established method can detect the virus with 105GE/ml copy number.
The virus-induced infectious disease, such as acute respiratory infection, hemorrhagic fever, diarrhea, AIDS, mad cow disease, bird flu, et al. not only caused a great panic of human life and health, but also resulted in huge losses to the world regional economy. According to statistics, infectious diseases show a kind of tendency that classic diseases reduce but new infectious diseases increase in the past two decades.70% of new infectious diseases are caused by viral pathogens, the new viral disease often shows the characteristics that are a high mortality rate, spread rapidly and controlled difficultly. The present study found that the study sphere of viral disease are expanded to infectious diseases from communicable disease. The experiences and lessons that are got from major viral diseases such as bird flu, AIDS, SARS et al, tell people that there is great significance for the prevention and treatment by early diagnosis or exclusion of viral pathogens. However, a wide range of viruses, now found more than 4000 kinds, among 400 kinds medical virus, the new virus is growing at the rate of one kind one year in the natural world. Specific detected and analysed methods can not analyzed the new virus, and electron microscopy is not suitable for routine use, severe situation of human infectious diseases appeal new technology or union with a variety of methods in order to detect or exclude fast, primitively and effectively unknown viruses pathogens, and it provides the evidence for disease control and drug development.
The unique study on new viral pathogens lies in that the virus is a macromolecules which have "nucleic acid-protein complex", so the earlier research is base on epidemiology and depending on the culture and electron microscopy. The modern molecular biology technology greatly promoted the study of viral pathogens technology, and utilizing adequately macromolecular characteristics of the virus and isolating and identifying with the corresponding immunology and molecular biology methods, a series of new methods developed led to the discovery of most of the new virus. The key problem of molecular virus technology is that the virus content is lower and host nucleic acid and protein content is higher of the disease samples, so the general technological principles are divided into two categories:amplifying (non-specific-PCR) and screening (immunization, nucleic acid hybridization). Non-specific PCR and nucleic acid hybridization techniques based on nucleic acid are relatively shortcut, so the together non-specific PCR with representational difference analysis (RDA) technology of subtractive hybridization found in a series of transfusion-transmitted hepatitis B virokine (1997). However, RDA workload is enormous, with almost all the medical virus have tolerance characteristics for DNase treated capsid protein, Tobias A treated host acid nuclear with DNase and plus joint molecules then amplify enzyme cut spectrum of the virus by single non-specific PCR, and found two new parvovirus in commodity bovine serum by the technology (SISPA). SISPA technology uses commendably the structural characteristics of the virus and very fast and convenient, the defect is that it only detected unknown viruses above 106GE/ml copy. It restricts the application of molecular virus discovery technology because both rapid and sensitive can not take into account, so the early detection of SARS-CoV is still dependent on the electron microscope, that presented a great challenge for molecular virus discovery technology.
Objective To establish a kind of new detected methods for viral pathogen of new infectious diseases by taking hepatitis B virus (DNA virus) and HIV (RNA virus) as the research object, in order to detect or exclude quickly, primitively and effectively viral pathogens and provide the basis for disease control and drug development.
Methods Applying DNase I sequence-independent single primer amplification technology, the HBV serum which were selected by clinical diagnosis and laboratory real-time fluorescence quantitative PCR detection, then were filtered and treated to remove endogenous DNA with DNase I. The complete DNA is digested with four base restriction enzyme digestion Csp6.I after applying Klenow Fragment to filling-in, the pathogenic gene were amplified non-specific with plused joint molecule as the single-primer, then sequenced and analysed, then judged detected pathogen nucleic acid.
Similarly, the HIV serum which were also selected by clinical diagnosis and laboratory real-time fluorescence quantitative PCR and ELIS A detection, then were filtered and treated to remove endogenous DNA with DNase I. then reverse transcripted and synthesized double-stranded cDNA of the RNA virus, The double-stranded cDNA is digested by four base restriction enzyme digestion Taq I, the pathogenic gene were amplified non-specific with plused joint molecule as the single-primer, then sequenced and analysed, then judged detected pathogen nucleic acid.
To verify the detection technology of viral pathogen by enterovirus EV71 which cause hand-foot-mouth disease. According to hand-foot-mouth disease laboratory detect program (for trial implementation), that is, the appendix of " the Guide of Hand-Foot-Mouth Disease Prevention and Control " (2008 edition) that Ministry of Public Health issued, to collect the faeces of infants that had been diagnosed hand-foot-mouth disease in the clinical and were positive by reverse transcription-polymerase chain reaction (RT-PCR) in the laboratory, the specimens were filtered, digested and processed by DNase, then reverse transcripted and synthesized double-stranded cDNA of the RNA virus, the double-stranded cDNA is digested with four base restriction enzyme digestion Csp6. I, the pathogenic gene were amplified non-specific with plused joint molecule as the single-primer, then sequenced and analysed, then judged detected pathogen nucleic acid.
Results
1 The viral DNA can not be degraded when DNase I treated endogenous DNA of the serum. Getting a number of DNA fragments with using non-sequence-dependent single primer to amplify exogenous DNA (viral DNA) from the serum. All PCR products were cloned, sequenced and analysed, the sequences is consistent with HBV DNA sequences of known pathogens, it can detect the virus with 105GE/ml copy number.
2 It does not affect the amount of viral nucleic acid that DNase I treated endogenous DNA of the serum. Getting a number of DNA fragments with using non-sequence-dependent single primer to amplify exogenous DNA (viral DNA) from the serum. All PCR products were cloned, sequenced and BLAST analysis, the homology is up to 94:8% with HIV-sequence in the Gene Bank, so we can judge that the detected pathogens is HIV.
3 To verify the detection technology of viral pathogen by enterovirus EV71 which cause hand-foot-mouth disease, getting a number of DNA fragments with using non-sequence-dependent single primer to amplify exogenous DNA of the faeces specimen. All PCR products were cloned, sequenced and BLAST analysis, we found homology is up to 99% between some sequences of them and known pathogens EV71 sequence in the Gene Bank,, and is 81% between some sequences of others and Listeria monocytogenes strain CDC54007 in the Gene Bank,.
Conclusion
1 The method can detect DNA viral pathogens of the serum applying the DNase I treatment and sequence-independent single primer amplification technology.
2 The method can detect RNA viral pathogens of the serum applying the DNase I treatment and sequence-independent single primer amplification technology.
3 The method can detect viral pathogens of the faeces specimen applying the DNase I treatment and sequence-independent single primer amplification technology.
4 The improved sequence-independent single primer amplification technology not only can detect serum samples, but also can detect fecal samples, that will expand the sphere of detected samples.
5 The established method is not dependent on sequence-specific.
6 The established method can detect the virus with 105GE/ml copy number.
引文
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