基于一种新型再测序芯片的呼吸道症候群检测技术的建立及应用
发布时间：2018-07-17 16:10
【摘要】：背景： 呼吸道症候群感染广泛分布于人群中,尤其在婴幼儿和老年人中,具有相当高的发病率和死亡率。由呼吸道症候群感染引起的新发或突发传染病,对社会经济造成重大损失。准确、快速地鉴定出病原体,对控制传染病的流行、降低经济损失有重要意义。现有的培养等传统方法费时费力,灵敏度低；一些已开发的核酸检测技术虽然能同时检测多种病毒,但检测通量不能满足应急的需要；454等高通量测序仪虽然能检测新病原,但检测成本太高、时间长、容易受到背景的干扰。因此,建立一种高灵敏度、高通量、在短时间内准确鉴定呼吸道病原及其突变的技术,对提高我国应对突发传染病的能力和控制传染病的流行有重大意义。 目的： 建立基于一种新型再测序芯片(Resequencing Pathogen Microarray, RPM)的呼吸道症候群检测方法,该方法可以同时检测19种常见呼吸道病毒、11种甲型流感(FluA)和11种鼻病毒(HRV)、28种肠病毒(EV)、18种不常见呼吸道病毒、14种呼吸道细菌、肺炎支原体、3种衣原体和3种生物防御细菌(炭蛆芽孢杆菌、土拉弗朗西斯菌和耶尔森菌)。评价方法的灵敏度和特异性,然后用临床样品评价方法的实际检测能力。最后将建立和优化的方法应用于不明原因呼吸道感染和突发传染病的检测。 方法： 本研究建立的方法采用一种改良的TSP (Temperature switch PCR)技术来优化扩增条件。筛选呼吸道症候群的病原体,针对检测病原体的保守区及部分病毒的分型区设计特异性嵌合引物,并对常见的呼吸道病毒引物进行验证。将178对特异性引物分成A、B、C、D、E五组分别放在5个反应管内,每个反应体系内分别加入一对内参基因的引物和一对相同的通用引物。用已验证的单个病毒感染的阳性样品为模板检测多重体系的特异性,用梯度稀释的体外转录RNA或腺病毒(HAdV）、博卡病毒(HBoV)的重组质粒DNA检测体系的多引物单模板灵敏度,用等量混合的稀释模板检测多引物多模板的灵敏度。用RPM-IVDC1检测110份社区获得性肺炎(Community-acquired pneumonia, CAP)患者的鼻咽抽吸物,16种常见呼吸道病毒的检测结果与基于GeXP毛细管电泳的多重PCR检测方法比较(Li et al.2012, BMC Infectious Diseases,12:189),其余病毒的检测结果与测序结果比较,细菌和支原体的检测结果与实时荧光定量PCR (qPCR)比较,评价该方法检测临床样品的能力。最后用RPM-IVDC1检测常规方法检测为阴性的不明原因呼吸道感染样品和H7N9重症突发感染样品,评价该方法应用于紧急公共卫生事件的潜力。 结果： 1、成功建立了一种基于RPM-IVDC1的呼吸道症候群检测技术,对于16种常见呼吸道病毒的多引物单模板和多引物多模板检测灵敏度均可达到10-1000拷贝/反应,其中一些探针出现不干扰结果判定的非特异性杂交。检测110份临床样品的结果显示,RPM-IVDC1检测HAdV、PIV2、FluA、HRV和CoV-229E的灵敏度高于GeXP法,检测RSVA的灵敏度略低于GeXP法。RPM-IVDC1同时检出了FluC、麻疹病毒、风疹病毒和疱疹病毒(human herpesvirus, HHV)。检测肺炎链球菌(S. pneumoniae)、流感嗜血杆菌(H. influenzae)、金黄色葡萄球菌(S. aureus)的灵敏度高于qPCR,检测肺炎支原体(M. pneumoniae)和结核分枝杆菌(M. tuberculosis)的灵敏度略低于qPCR。这些表明RPM-IVDC1具有和商业化试剂盒相当的检测灵敏度,具备检测复杂临床样品的能力,并能检测常规方法忽略的不常见的呼吸道病毒和细菌。 2、成功将建立的方法应用于不明原因呼吸通感染和重症发感染的检测。用常规的病毒检测方法检测8份发热儿童的咽拭子样品为阴性,但RPM-IVDC1检测出PIV3、HRV和HHV,检测结果与测序以及质谱(PLEX-ID)结果一致。用RPM-IVDC1检测H7N9重症突发感染的咽拭子样品,检测出FluA、HBoV和HHV-4。这些表明RPM-IVDC1有很高的检测灵敏度,并能快速地检测出病原体,可以满足公共卫生应急检测的需要。 结论： 成功建立了基于RPM-IVDCl的呼吸道症候群检测方法,再测序芯片的再测序和高通量优势在公共卫生领域有广阔的应用前景,为提高我国应对突发传染病的能力和控制传染病的流行有重要意义。
[Abstract]:Background:
Respiratory syndrome is widely distributed among the population, especially in infants and old people, with high morbidity and mortality. New hair or sudden infectious diseases caused by respiratory syndrome infection cause significant losses to the social economy. Accurate, rapid identification of the disease mycoplasma, the control of the epidemic of infectious diseases, and the reduction of economic losses It is of great significance. The existing traditional methods such as culture and other traditional methods are time-consuming and low sensitivity; some developed nucleic acid detection techniques can detect a variety of viruses at the same time, but the flux of detection can not meet the needs of emergency. Although the 454 equal high flux sequencer can detect the new pathogen, the detection cost is too high, the time is long, and the interference of the background is easy. Therefore, the establishment of a high sensitivity, high throughput, accurate identification of respiratory pathogens and mutations in a short period of time is of great significance to improve our country's ability to deal with infectious diseases and to control the epidemic of infectious diseases.
Objective:
A new method of detection of respiratory syndrome based on a new Resequencing Pathogen Microarray (RPM) was established. This method can simultaneously detect 19 common respiratory viruses, 11 kinds of influenza A (FluA) and 11 rhinoviruses (HRV), 28 intestinal viruses (EV), 18 uncommon respiratory viruses, 14 respiratory bacteria, Mycoplasma pneumoniae, 3. Chlamydia and 3 kinds of biological defense bacteria (Bacillus charcoal, Turafrancisrand and Jerson). The sensitivity and specificity of the evaluation method were evaluated and the actual detection ability of the method was evaluated with clinical samples. Finally, the method of establishing and optimizing the method was applied to the detection of unexplained respiratory tract infection and sudden infectious disease.
Method:
The method established in this study uses an improved TSP (Temperature switch PCR) technique to optimize the conditions of amplification. Screening the pathogens of the respiratory syndrome, designing specific chimeric primers for the detection of the pathogen's conservative areas and some viruses, and verifying the common primers of the respiratory tract virus. 178 pairs of specific primers are used. Five groups of A, B, C, D, and E were placed in 5 reaction tubes. Each reaction system was added to a pair of primers and a pair of common primers respectively. The specificity of the multiple system was detected by the tested positive samples of single virus infection, and the gradient dilute release of RNA or adenovirus (HAdV), Boka virus (HBoV) was used. The sensitivity of the multi primer template sensitivity of the recombinant plasmid DNA detection system and the sensitivity of multiple primers and multiple templates were detected with the same dilution template. RPM-IVDC1 was used to detect nasopharyngeal aspirates in 110 Community-acquired pneumonia (CAP) patients and the results of 16 common respiratory viruses and GeXP based capillary electricity. Comparison of multiple PCR detection methods (Li et al.2012, BMC Infectious Diseases, 12:189), the results of the other viruses were compared with the sequencing results. The detection results of bacteria and Mycoplasma were compared with the real-time fluorescent quantitative PCR (qPCR), and the ability to detect the clinical samples was evaluated. Finally, the test was negative by RPM-IVDC1 detection routine method. Samples of unexplained respiratory tract infection and H7N9 severe acute infection samples were evaluated to assess the potential of this method in emergency public health events.
Result:
1, a RPM-IVDC1 based respiratory syndrome detection technique was successfully established. The sensitivity of multiple primers and multiple primers to multiple primers and multiple templates for the detection of 16 common respiratory viruses could reach 10-1000 copies / reactions, some of which were nonspecific hybridization that did not interfere with the determination of the results. The results of detection of 110 clinical samples were significant. The sensitivity of RPM-IVDC1 to HAdV, PIV2, FluA, HRV and CoV-229E was higher than the GeXP method. The sensitivity of RSVA was slightly lower than GeXP method.RPM-IVDC1 and FluC, measles virus, rubella virus and herpes virus (human). The sensitivity of (S. aureus) is higher than that of qPCR, and the sensitivity of the detection of Mycoplasma pneumoniae (M. pneumoniae) and Mycobacterium tuberculosis (M. tuberculosis) is slightly lower than that of qPCR., which indicates that RPM-IVDC1 has the equivalent detection sensitivity with the commercial kits, has the ability to detect complex clinical samples, and can detect uncommon breathing that is ignored by conventional methods. Virus and bacteria.
2, the established method was successfully applied to the detection of unexplained respiratory infection and severe infection. Detection of swab samples from 8 febrile children was negative by routine virus detection, but PIV3, HRV and HHV were detected by RPM-IVDC1, and the results were consistent with the results of sequencing and mass spectrometry (PLEX-ID). RPM-IVDC1 was used to detect the severe sudden sense of H7N9. The dyed swab samples were used to detect FluA, HBoV and HHV-4., which showed that RPM-IVDC1 had high detection sensitivity and could quickly detect pathogens, which could meet the needs of public health emergency testing.
Conclusion:
The RPM-IVDCl based respiratory syndrome detection method has been successfully established, and the re sequencing and high throughput advantage of the sequenced chip have broad application prospects in the public health field. It is of great significance to improve our country's ability to deal with the outbreak of infectious diseases and control the epidemic of infectious diseases.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R56;R440
本文编号：2130199
[Abstract]:Background:
Respiratory syndrome is widely distributed among the population, especially in infants and old people, with high morbidity and mortality. New hair or sudden infectious diseases caused by respiratory syndrome infection cause significant losses to the social economy. Accurate, rapid identification of the disease mycoplasma, the control of the epidemic of infectious diseases, and the reduction of economic losses It is of great significance. The existing traditional methods such as culture and other traditional methods are time-consuming and low sensitivity; some developed nucleic acid detection techniques can detect a variety of viruses at the same time, but the flux of detection can not meet the needs of emergency. Although the 454 equal high flux sequencer can detect the new pathogen, the detection cost is too high, the time is long, and the interference of the background is easy. Therefore, the establishment of a high sensitivity, high throughput, accurate identification of respiratory pathogens and mutations in a short period of time is of great significance to improve our country's ability to deal with infectious diseases and to control the epidemic of infectious diseases.
Objective:
A new method of detection of respiratory syndrome based on a new Resequencing Pathogen Microarray (RPM) was established. This method can simultaneously detect 19 common respiratory viruses, 11 kinds of influenza A (FluA) and 11 rhinoviruses (HRV), 28 intestinal viruses (EV), 18 uncommon respiratory viruses, 14 respiratory bacteria, Mycoplasma pneumoniae, 3. Chlamydia and 3 kinds of biological defense bacteria (Bacillus charcoal, Turafrancisrand and Jerson). The sensitivity and specificity of the evaluation method were evaluated and the actual detection ability of the method was evaluated with clinical samples. Finally, the method of establishing and optimizing the method was applied to the detection of unexplained respiratory tract infection and sudden infectious disease.
Method:
The method established in this study uses an improved TSP (Temperature switch PCR) technique to optimize the conditions of amplification. Screening the pathogens of the respiratory syndrome, designing specific chimeric primers for the detection of the pathogen's conservative areas and some viruses, and verifying the common primers of the respiratory tract virus. 178 pairs of specific primers are used. Five groups of A, B, C, D, and E were placed in 5 reaction tubes. Each reaction system was added to a pair of primers and a pair of common primers respectively. The specificity of the multiple system was detected by the tested positive samples of single virus infection, and the gradient dilute release of RNA or adenovirus (HAdV), Boka virus (HBoV) was used. The sensitivity of the multi primer template sensitivity of the recombinant plasmid DNA detection system and the sensitivity of multiple primers and multiple templates were detected with the same dilution template. RPM-IVDC1 was used to detect nasopharyngeal aspirates in 110 Community-acquired pneumonia (CAP) patients and the results of 16 common respiratory viruses and GeXP based capillary electricity. Comparison of multiple PCR detection methods (Li et al.2012, BMC Infectious Diseases, 12:189), the results of the other viruses were compared with the sequencing results. The detection results of bacteria and Mycoplasma were compared with the real-time fluorescent quantitative PCR (qPCR), and the ability to detect the clinical samples was evaluated. Finally, the test was negative by RPM-IVDC1 detection routine method. Samples of unexplained respiratory tract infection and H7N9 severe acute infection samples were evaluated to assess the potential of this method in emergency public health events.
Result:
1, a RPM-IVDC1 based respiratory syndrome detection technique was successfully established. The sensitivity of multiple primers and multiple primers to multiple primers and multiple templates for the detection of 16 common respiratory viruses could reach 10-1000 copies / reactions, some of which were nonspecific hybridization that did not interfere with the determination of the results. The results of detection of 110 clinical samples were significant. The sensitivity of RPM-IVDC1 to HAdV, PIV2, FluA, HRV and CoV-229E was higher than the GeXP method. The sensitivity of RSVA was slightly lower than GeXP method.RPM-IVDC1 and FluC, measles virus, rubella virus and herpes virus (human). The sensitivity of (S. aureus) is higher than that of qPCR, and the sensitivity of the detection of Mycoplasma pneumoniae (M. pneumoniae) and Mycobacterium tuberculosis (M. tuberculosis) is slightly lower than that of qPCR., which indicates that RPM-IVDC1 has the equivalent detection sensitivity with the commercial kits, has the ability to detect complex clinical samples, and can detect uncommon breathing that is ignored by conventional methods. Virus and bacteria.
2, the established method was successfully applied to the detection of unexplained respiratory infection and severe infection. Detection of swab samples from 8 febrile children was negative by routine virus detection, but PIV3, HRV and HHV were detected by RPM-IVDC1, and the results were consistent with the results of sequencing and mass spectrometry (PLEX-ID). RPM-IVDC1 was used to detect the severe sudden sense of H7N9. The dyed swab samples were used to detect FluA, HBoV and HHV-4., which showed that RPM-IVDC1 had high detection sensitivity and could quickly detect pathogens, which could meet the needs of public health emergency testing.
Conclusion:
The RPM-IVDCl based respiratory syndrome detection method has been successfully established, and the re sequencing and high throughput advantage of the sequenced chip have broad application prospects in the public health field. It is of great significance to improve our country's ability to deal with the outbreak of infectious diseases and control the epidemic of infectious diseases.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R56;R440
【参考文献】
相关期刊论文 前3条
1 胡秀梅;张勇;徐邦牢;杨梦婕;王淼;张晨;李瑾;白如银;周小棉;许文波;马学军;;GeXP多重基因表达遗传分析系统在手足口病病原分型检测中的应用[J];病毒学报;2011年04期
2 李瑾;毛乃颖;秦萌;胡秀梅;杨梦婕;王淼;张晨;许文波;马学军;;GeXP多重PCR技术同时检测12种常见呼吸道病毒[J];病毒学报;2011年06期
3 杨玉霞;乔俊英;程秀永;王新霞;;新生儿下呼吸道感染病原学及耐药性检测[J];中国当代儿科杂志;2008年04期
，本文编号：2130199
本文链接：https://www.wllwen.com/yixuelunwen/huxijib/2130199.html
最近更新
教材专著
