基于一種新型再測序芯片的呼吸道癥候群檢測技術(shù)的建立及應(yīng)用
發(fā)布時間：2018-07-17 16:10
【摘要】：背景： 呼吸道癥候群感染廣泛分布于人群中,尤其在嬰幼兒和老年人中,具有相當(dāng)高的發(fā)病率和死亡率。由呼吸道癥候群感染引起的新發(fā)或突發(fā)傳染病,對社會經(jīng)濟(jì)造成重大損失。準(zhǔn)確、快速地鑒定出病原體,對控制傳染病的流行、降低經(jīng)濟(jì)損失有重要意義。現(xiàn)有的培養(yǎng)等傳統(tǒng)方法費時費力,靈敏度低；一些已開發(fā)的核酸檢測技術(shù)雖然能同時檢測多種病毒,但檢測通量不能滿足應(yīng)急的需要；454等高通量測序儀雖然能檢測新病原,但檢測成本太高、時間長、容易受到背景的干擾。因此,建立一種高靈敏度、高通量、在短時間內(nèi)準(zhǔn)確鑒定呼吸道病原及其突變的技術(shù),對提高我國應(yīng)對突發(fā)傳染病的能力和控制傳染病的流行有重大意義。 目的： 建立基于一種新型再測序芯片(Resequencing Pathogen Microarray, RPM)的呼吸道癥候群檢測方法,該方法可以同時檢測19種常見呼吸道病毒、11種甲型流感(FluA)和11種鼻病毒(HRV)、28種腸病毒(EV)、18種不常見呼吸道病毒、14種呼吸道細(xì)菌、肺炎支原體、3種衣原體和3種生物防御細(xì)菌(炭蛆芽孢桿菌、土拉弗朗西斯菌和耶爾森菌)。評價方法的靈敏度和特異性,然后用臨床樣品評價方法的實際檢測能力。最后將建立和優(yōu)化的方法應(yīng)用于不明原因呼吸道感染和突發(fā)傳染病的檢測。 方法： 本研究建立的方法采用一種改良的TSP (Temperature switch PCR)技術(shù)來優(yōu)化擴(kuò)增條件。篩選呼吸道癥候群的病原體,針對檢測病原體的保守區(qū)及部分病毒的分型區(qū)設(shè)計特異性嵌合引物,并對常見的呼吸道病毒引物進(jìn)行驗證。將178對特異性引物分成A、B、C、D、E五組分別放在5個反應(yīng)管內(nèi),每個反應(yīng)體系內(nèi)分別加入一對內(nèi)參基因的引物和一對相同的通用引物。用已驗證的單個病毒感染的陽性樣品為模板檢測多重體系的特異性,用梯度稀釋的體外轉(zhuǎn)錄RNA或腺病毒(HAdV）、博卡病毒(HBoV)的重組質(zhì)粒DNA檢測體系的多引物單模板靈敏度,用等量混合的稀釋模板檢測多引物多模板的靈敏度。用RPM-IVDC1檢測110份社區(qū)獲得性肺炎(Community-acquired pneumonia, CAP)患者的鼻咽抽吸物,16種常見呼吸道病毒的檢測結(jié)果與基于GeXP毛細(xì)管電泳的多重PCR檢測方法比較(Li et al.2012, BMC Infectious Diseases,12:189),其余病毒的檢測結(jié)果與測序結(jié)果比較,細(xì)菌和支原體的檢測結(jié)果與實時熒光定量PCR (qPCR)比較,評價該方法檢測臨床樣品的能力。最后用RPM-IVDC1檢測常規(guī)方法檢測為陰性的不明原因呼吸道感染樣品和H7N9重癥突發(fā)感染樣品,評價該方法應(yīng)用于緊急公共衛(wèi)生事件的潛力。 結(jié)果： 1、成功建立了一種基于RPM-IVDC1的呼吸道癥候群檢測技術(shù),對于16種常見呼吸道病毒的多引物單模板和多引物多模板檢測靈敏度均可達(dá)到10-1000拷貝/反應(yīng),其中一些探針出現(xiàn)不干擾結(jié)果判定的非特異性雜交。檢測110份臨床樣品的結(jié)果顯示,RPM-IVDC1檢測HAdV、PIV2、FluA、HRV和CoV-229E的靈敏度高于GeXP法,檢測RSVA的靈敏度略低于GeXP法。RPM-IVDC1同時檢出了FluC、麻疹病毒、風(fēng)疹病毒和皰疹病毒(human herpesvirus, HHV)。檢測肺炎鏈球菌(S. pneumoniae)、流感嗜血桿菌(H. influenzae)、金黃色葡萄球菌(S. aureus)的靈敏度高于qPCR,檢測肺炎支原體(M. pneumoniae)和結(jié)核分枝桿菌(M. tuberculosis)的靈敏度略低于qPCR。這些表明RPM-IVDC1具有和商業(yè)化試劑盒相當(dāng)?shù)臋z測靈敏度,具備檢測復(fù)雜臨床樣品的能力,并能檢測常規(guī)方法忽略的不常見的呼吸道病毒和細(xì)菌。 2、成功將建立的方法應(yīng)用于不明原因呼吸通感染和重癥發(fā)感染的檢測。用常規(guī)的病毒檢測方法檢測8份發(fā)熱兒童的咽拭子樣品為陰性,但RPM-IVDC1檢測出PIV3、HRV和HHV,檢測結(jié)果與測序以及質(zhì)譜(PLEX-ID)結(jié)果一致。用RPM-IVDC1檢測H7N9重癥突發(fā)感染的咽拭子樣品,檢測出FluA、HBoV和HHV-4。這些表明RPM-IVDC1有很高的檢測靈敏度,并能快速地檢測出病原體,可以滿足公共衛(wèi)生應(yīng)急檢測的需要。 結(jié)論： 成功建立了基于RPM-IVDCl的呼吸道癥候群檢測方法,再測序芯片的再測序和高通量優(yōu)勢在公共衛(wèi)生領(lǐng)域有廣闊的應(yīng)用前景,為提高我國應(yīng)對突發(fā)傳染病的能力和控制傳染病的流行有重要意義。
[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.
【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R56;R440
本文編號：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.
【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R56;R440
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