人偏肺病毒间接免疫荧光方法的建立及各种检测方法的对比研究
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摘要
第一部分人偏肺病毒间接免疫荧光方法的建立及应用
目的人偏肺病毒(Human Metapneumovirus, hMPV)是2001年新发现的呼吸道病毒病原,主要导致婴幼儿上下呼吸道感染。本研究拟建立检测hMPV抗原的间接免疫荧光方法(immunofluorescence assay, IFA)。
方法用hMPV感染非洲绿猴肾细胞株(Vero-E6),待出现明显细胞病变(cytopathogenic effect,CPE)后刮下细胞,制备抗原片。建立以针对hMPV融合蛋白(fusion protein, F )单克隆抗体为一抗的IFA方法并优化,验证hMPV IFA的特异性和灵敏度。选取重庆医科大学儿童医院因急性呼吸道感染新入院的患儿,无菌采集鼻咽分泌物。经离心吹打处理标本,获取细胞悬液制备抗原片。采用IFA方法进行临床适用性评估。
结果建立的IFA可特异性识别hMPV抗原,与其它常见呼吸道病毒病原抗原无交叉反应,其最低检测下限为102.2 TCID50/ml,实验重复性良好。采用该方法检测呼吸道感染患儿气道标本523份,检出hMPV阳性标本48份,阳性率为9.18%。
结论成功建立了具有高特异性和重复性的hMPV IFA检测方法,可用于hMPV感染气道标本的快速诊断,为减少抗生素滥用,hMPV的流行病学和发病机制研究奠定了基础。
第二部分几种人偏肺病毒检测方法的对比研究
目的对逆转录酶联聚合反应(Reverse transcription polymerase chain reaction,RT-PCR )、实时荧光定量酶联聚合反应(Real-time reverse transcriptase PCR,Real-time PCR )、间接免疫荧光(IFA)三种hMPV检测方法灵敏度进行对比研究。评估Real-time PCR及IFA的临床使用价值,为临床选择适当的检测方法提供理论依据。
方法(1)将滴度为105.2TCID50/ml的hMPV按十倍梯度稀释(101～107倍七个稀释度),分别感染培养于六孔板中的Vero-E6细胞。病毒接种六天后,105倍稀释孔中细胞出现CPE。分别收集上清及细胞悬液,采用RT-PCR、Real-time PCR、IFA三种方法进行检测,比较病毒分离及以上三种检测方法灵敏度及重复性。(2)十倍梯度稀释hMPV重组病毒,分别进行RT-PCR及Real-time PCR检测,定量比较二者灵敏度。(3) Real-time PCR、IFA分别检测523份重庆医科大学附属儿童医院因急性呼吸道感染新入院患儿的鼻咽分泌物,分析其临床适用性。
结果(1)IFA可检测到104倍稀释孔中病毒抗原,RT-PCR可检测到105倍稀释孔中病毒核酸,Real-time PCR可检测到107倍稀释孔中病毒核酸。(2)以病毒滴度为定量参考标准,RT-PCR和Real-time PCR检测下限分别为103TCID50/ml、1TCID50/ml。(3)收集鼻咽分泌物标本609份,其中86份无足够细胞进行IFA检测。其余523份标本中,IFA检测到阳性标本48份。以500拷贝/微升为阳性标准时,Real-time PCR检测到阳性标本165份。两种方法均阳性的标本共33份。
结论三种方法比较,Real-time PCR灵敏度最高,RT-PCR其次,IFA灵敏度最低。用病毒滴度进行灵敏度的定量比较,RT-PCR比Real-time PCR低1000倍。在对临床标本的检测中,Real-time PCR较IFA灵敏,应为开展hMPV的流行监测及临床早期诊断的最佳技术手段。
PARTⅠ:Development and application of an indirect immunofluorescence assay for detection of Human Metapneumovirus
Objective Human metapneumovirus (hMPV) is a newly recognized viral pathogen causing upper and lower respiratory infection in all age groups in particular young children. This study was to develop an indirect immunofluorescence assay(IFA) for detection of human Metapneumovirus.
Method Vero-E6 cells were infected by recombinant hMPV strain NL/1/00 and NL/1/99. When maximum CPE was observed, cells were used to prepare cell antigen smear for IFA detection. The assay was validated by using serially diluted human Metapneumovirus infected cells and common respiratory virus (RSV, parainfluenza, adenovirus and influenza viruses) positive anigens. The hMPV IFA was then performed to detect hMPV in clinical specimens.
Result The hMPV IFA did not recognize common respiratory viral antigens but hMPV antigen from both subgroup, indicating good specificity and reproducibility. The analytical detection limit of this IFA was 102.2TCID50/ml. A total of 523 nasopharygeal secretions specimens were screened for the presence of hMPV by IFA and 48 specimens were positive.
Conclusion An indirect immunofluorescence assay for detection of hMPV has been successfully developed, which could be utilized in clinical diagnosis and epidemiological studies for hMPV。
PARTⅡ:Comparison of different methods used in detection of human Metapneumovirus
Objective Compared the sensitivity and specificity of conventional RT-PCR、Real-time PCR and IFA in detecting hMPV.And compared the virus detection rate in nasopharyngeal secretions by Real-time PCR with IFA .
Method (1)Tenfold serial dilutions of recombinate hMPV whose titer was 105.2TCID50/ml were inoculated onto Vero-E6 cells in six-well plate.The CPE was observed in cells inoculated with 105 times dilutions virus appeared at 6 post inoculation.Subsequenfly,experiments were undertaken to assess diagnostic criteria such as specificity、sensitivity and reproducibility of RT-PCR、Real-time PCR and IFA respectively. (2)Tenfold serial dilutions of the recombinate hMPV were amplified by Real-time PCR and RT-PCR respectively. (3) The hMPV IFA and Real-time PCR were then performed to detected hMPV in clinical specimens.
Result (1)The hMPV IFA recognized the antigen of 104 times dilution of hMPV , RT-PCR detected the nucleic acid of 105 times dilution of hMPV ,The Real-time PCR detected the nucleic acid of 107 times dilution of hMPV . (2)The analytical detection limit of RT-PCR and Real-time PCR were 103TCID50/ml and 1TCID50/ml respectively .(3) A total of 609 clinical specimens from patients with acute respiratory tract infections were collected.86 of them had no enough cells for IFA. A total of 523 nasopharygeal secretions specimens were screened for the presence of hMPV by IFA and 48 specimens were positive.When 500copies/ul was the reference standard, both of Real-time PCR and IFA were positive for 33 of the 165 Real-time PCR-positive children.
Conclusion The RT-PCR was more sensitive than IFA but less than Real-time PCR . The quantitate analytical detection limit of RT-PCR was lower than Real-time PCR. The Real-time PCR was proved to be more sensitive than IFA when they were used to detect clinical specimens . The results taken together indicate that Real-time PCR is an efficient method for detecting hMPV .
目的人偏肺病毒(Human Metapneumovirus, hMPV)是2001年新发现的呼吸道病毒病原,主要导致婴幼儿上下呼吸道感染。本研究拟建立检测hMPV抗原的间接免疫荧光方法(immunofluorescence assay, IFA)。
方法用hMPV感染非洲绿猴肾细胞株(Vero-E6),待出现明显细胞病变(cytopathogenic effect,CPE)后刮下细胞,制备抗原片。建立以针对hMPV融合蛋白(fusion protein, F )单克隆抗体为一抗的IFA方法并优化,验证hMPV IFA的特异性和灵敏度。选取重庆医科大学儿童医院因急性呼吸道感染新入院的患儿,无菌采集鼻咽分泌物。经离心吹打处理标本,获取细胞悬液制备抗原片。采用IFA方法进行临床适用性评估。
结果建立的IFA可特异性识别hMPV抗原,与其它常见呼吸道病毒病原抗原无交叉反应,其最低检测下限为102.2 TCID50/ml,实验重复性良好。采用该方法检测呼吸道感染患儿气道标本523份,检出hMPV阳性标本48份,阳性率为9.18%。
结论成功建立了具有高特异性和重复性的hMPV IFA检测方法,可用于hMPV感染气道标本的快速诊断,为减少抗生素滥用,hMPV的流行病学和发病机制研究奠定了基础。
第二部分几种人偏肺病毒检测方法的对比研究
目的对逆转录酶联聚合反应(Reverse transcription polymerase chain reaction,RT-PCR )、实时荧光定量酶联聚合反应(Real-time reverse transcriptase PCR,Real-time PCR )、间接免疫荧光(IFA)三种hMPV检测方法灵敏度进行对比研究。评估Real-time PCR及IFA的临床使用价值,为临床选择适当的检测方法提供理论依据。
方法(1)将滴度为105.2TCID50/ml的hMPV按十倍梯度稀释(101～107倍七个稀释度),分别感染培养于六孔板中的Vero-E6细胞。病毒接种六天后,105倍稀释孔中细胞出现CPE。分别收集上清及细胞悬液,采用RT-PCR、Real-time PCR、IFA三种方法进行检测,比较病毒分离及以上三种检测方法灵敏度及重复性。(2)十倍梯度稀释hMPV重组病毒,分别进行RT-PCR及Real-time PCR检测,定量比较二者灵敏度。(3) Real-time PCR、IFA分别检测523份重庆医科大学附属儿童医院因急性呼吸道感染新入院患儿的鼻咽分泌物,分析其临床适用性。
结果(1)IFA可检测到104倍稀释孔中病毒抗原,RT-PCR可检测到105倍稀释孔中病毒核酸,Real-time PCR可检测到107倍稀释孔中病毒核酸。(2)以病毒滴度为定量参考标准,RT-PCR和Real-time PCR检测下限分别为103TCID50/ml、1TCID50/ml。(3)收集鼻咽分泌物标本609份,其中86份无足够细胞进行IFA检测。其余523份标本中,IFA检测到阳性标本48份。以500拷贝/微升为阳性标准时,Real-time PCR检测到阳性标本165份。两种方法均阳性的标本共33份。
结论三种方法比较,Real-time PCR灵敏度最高,RT-PCR其次,IFA灵敏度最低。用病毒滴度进行灵敏度的定量比较,RT-PCR比Real-time PCR低1000倍。在对临床标本的检测中,Real-time PCR较IFA灵敏,应为开展hMPV的流行监测及临床早期诊断的最佳技术手段。
PARTⅠ:Development and application of an indirect immunofluorescence assay for detection of Human Metapneumovirus
Objective Human metapneumovirus (hMPV) is a newly recognized viral pathogen causing upper and lower respiratory infection in all age groups in particular young children. This study was to develop an indirect immunofluorescence assay(IFA) for detection of human Metapneumovirus.
Method Vero-E6 cells were infected by recombinant hMPV strain NL/1/00 and NL/1/99. When maximum CPE was observed, cells were used to prepare cell antigen smear for IFA detection. The assay was validated by using serially diluted human Metapneumovirus infected cells and common respiratory virus (RSV, parainfluenza, adenovirus and influenza viruses) positive anigens. The hMPV IFA was then performed to detect hMPV in clinical specimens.
Result The hMPV IFA did not recognize common respiratory viral antigens but hMPV antigen from both subgroup, indicating good specificity and reproducibility. The analytical detection limit of this IFA was 102.2TCID50/ml. A total of 523 nasopharygeal secretions specimens were screened for the presence of hMPV by IFA and 48 specimens were positive.
Conclusion An indirect immunofluorescence assay for detection of hMPV has been successfully developed, which could be utilized in clinical diagnosis and epidemiological studies for hMPV。
PARTⅡ:Comparison of different methods used in detection of human Metapneumovirus
Objective Compared the sensitivity and specificity of conventional RT-PCR、Real-time PCR and IFA in detecting hMPV.And compared the virus detection rate in nasopharyngeal secretions by Real-time PCR with IFA .
Method (1)Tenfold serial dilutions of recombinate hMPV whose titer was 105.2TCID50/ml were inoculated onto Vero-E6 cells in six-well plate.The CPE was observed in cells inoculated with 105 times dilutions virus appeared at 6 post inoculation.Subsequenfly,experiments were undertaken to assess diagnostic criteria such as specificity、sensitivity and reproducibility of RT-PCR、Real-time PCR and IFA respectively. (2)Tenfold serial dilutions of the recombinate hMPV were amplified by Real-time PCR and RT-PCR respectively. (3) The hMPV IFA and Real-time PCR were then performed to detected hMPV in clinical specimens.
Result (1)The hMPV IFA recognized the antigen of 104 times dilution of hMPV , RT-PCR detected the nucleic acid of 105 times dilution of hMPV ,The Real-time PCR detected the nucleic acid of 107 times dilution of hMPV . (2)The analytical detection limit of RT-PCR and Real-time PCR were 103TCID50/ml and 1TCID50/ml respectively .(3) A total of 609 clinical specimens from patients with acute respiratory tract infections were collected.86 of them had no enough cells for IFA. A total of 523 nasopharygeal secretions specimens were screened for the presence of hMPV by IFA and 48 specimens were positive.When 500copies/ul was the reference standard, both of Real-time PCR and IFA were positive for 33 of the 165 Real-time PCR-positive children.
Conclusion The RT-PCR was more sensitive than IFA but less than Real-time PCR . The quantitate analytical detection limit of RT-PCR was lower than Real-time PCR. The Real-time PCR was proved to be more sensitive than IFA when they were used to detect clinical specimens . The results taken together indicate that Real-time PCR is an efficient method for detecting hMPV .
引文
[1] van den Hoogen BG,de Jong JC,Groen J,et al.A newly discovered human pneumovirusisolated from young children with respiratory tract disease[J].Nat Med,2001 ,7(6):719-724.
[2] Mackay IM,Jacob KC,Woolhouse D,et al.Molecular assays for detection of human metapneumovires.[J] Clin Microbiol ,2003 ;41(1):100-5.
[3] Stockton J,Stephenson I,Fleming D,et al.Human metapneumovirus as a cause of community-acquired respiratory illness. [J]Emerg Infect Dis,2002 ;8(9):897-901.
[4]Crowe JE Jr.Human metapneumovirus as a major cause of human respiratory tracr disease. [J]Pediatr Infect Dis J,2004 ;23(11 suppl):S215-S221.
[5] Falsey AR,Erdman D,Anderson LJ,et al.Human metapneumovirus infections in young and elderly adults. [J] Infect Dis,2003 ,187(5):785-790.
[6]Chung JY,Han TH,Kim BE,et al.Human Metapneumovirus infection in hospitalized children with acute respiratory disease in Korea. [J] Korena Med Sci.2006,21(5):838-42.
[7]Peret TC,Boivin G,Li Y,et al.Characterization of human metapneumoviruses isolated from patients in North America. [J] Infect Dis,2002 ,185(11):1660-1663.
[8]Gray GC,Capuano AW,Setterquist SF,et al.Human Metapnemovirus,Peru. [J]Emerg infect Dis,2006,12(2):347-350.
[9]Smuts H,Workman L,Zar HJ.Role of human metapneumovirus,human coronavirus NL63 and human bocavirus in infants and young children with acute wheezing. [J]Med Virol,2008,80(5):906-12.
[10]Mackay IM,Jacob KC,Woolhouse D.et al.Molecular assays for detection of human metapneumovirus. [J] Clin Microbiol,2003 ,41(1):100-5.
[11]Manoha C,Espinosa S,Aho SL,et al.Epidemiological and clinical features of hMPV , RSV and RVs infections in young children. [J]Clin Virol,2007,38(3):221-226.
[12]Mao HW,Yang XQ,Wang LJ.et al.Isolation of human metapneumovirus from children with acute respiratory tract infection in Chongqing, [J]China.Zhonghua Er Ke Za Zhi.2007 Jan;45(1):42-5.
[13]Zhu RN,Qian Y,Deng J.et al.Human metapneumovirus may associate with acute respiratory infections in hospitalized pediatric patients in Beijing,China. [J]Zhonghua Er Ke Za Zhi.2003 Jun;41(6):441-4.
[14]Kikuta H,Sakata C,Gamo R,et al.Comparison of a lateral-flow immunochromatography assay with real-time reverse transcription-PCR for detection of human metapneumovirus. [J] Clin Microbiol.2008 Mar;46(3):928-932.
[1] van den Hoogen BG,de Jong JC,Groen J,et al.A newly discovered human pneumovirusisolated from young children with respiratory tract disease[J].Nat Med,2001 ,7(6):719-724.
[2] Hideaki Kikuta,Chikako Sakata,Reiko Gamo.,et al.Comparison of a Lateral-Flow Immunochromatography Assay with Real-Time Reverse Transcription-PCR for Detection of Human Metapneumovirus.[J]Journal of clinical microbiology.2008, p. 928–932.
[3] Gregory A. Storch. Diagnostic Virology.[M]Clinical Infectious Diseases 2000;31:739–51.
[4] Jane Kuypers, Nancy Wright, James Ferrenberg,et al. Comparison of Real-Time PCR Assays with Fluorescent-Antibody Assays for Diagnosis of Respiratory Virus Infections in Children. [J]Journal of clinical microbiology, July 2006, p. 2382–2388.
[5] Takashi Ebihara, Rika Endo, Xiaoming Ma,et al. Detection of Human Metapneumovirus Antigens in Nasopharyngeal Secretions by an Immunofluorescent-Antibody Test. [J]Journal of clinical microbiology , Mar. 2005, p. 1138–1141
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[3] Jane Kuypers, Nancy Wright, James Ferrenberg,et al. Comparison of Real-Time PCR Assays with Fluorescent-Antibody Assays for Diagnosis of Respiratory Virus Infections in Children. [J] Clin Microbiol , July 2006, p. 2382–2388
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[7] Percivalle E, Sarasini A, Visai L,et al.Rapid detection of human metapneumovirus strains in nasopharyngeal aspirates and shell vial cultures by monoclonal antibodies. [J] Clin Microbiol 2005;43:3443–6
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[9] Marie L. Landry,Sandra Cohen, David Ferguson. Prospective study of human metapneumovirus detection in clinical samples by use of light diagnostics directimmunofluorescence reagent and Real-time PCR.[J] Clin Microbiol, Mar. 2008, p. 1098–1100
[10] Hideaki Kikuta,Chikako Sakata, Reiko Gamo.Comparison of a Lateral-Flow Immunochromatography Assay with Real-Time Reverse Transcription-PCR for Detection of Human Metapneumovirus. [J] Hideaki Kikuta,Chikako Sakata, Reiko Gamo. , Mar. 2008, p. 928–932
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[15] Elena Percivalle, Antonella Sarasini,Livia Visai,et al. Rapid Detection of Human Metapneumovirus Strains in Nasopharyngeal Aspirates and Shell Vial Cultures by Monoclonal Antibodies. [J] Clin Microbiol , July 2005, p. 3443–3446 Vol. 43, No. 7
[16]Takashi Ebihara, Rika Endo, Xiaoming Ma,et al. Detection of Human Metapneumovirus Antigens in Nasopharyngeal Secretions by an Immunofluorescent-Antibody Test.[J] Clin Microbiol , Mar. 2005, p. 1138–1141
[17]Gregory A. Storch. Diagnostic Virology. [M]Clinical Infectious Diseases 2000;31:739–51.
[18]Kanti Pabbaraju a,, Sallene Wong a, Thomas McMillan,et al. Diagnosis and epidemiological studies of human metapneumovirus using real-time PCR. Journal of Clinical Virology 40 (2007) 186–192.
[19] Hamelin, M. E., Y. Abed, and G. Boivin. 2004. Human metapneumovirus: a new player among respiratory viruses. Clin. Infect. Dis. 38:983–990.
[2] Mackay IM,Jacob KC,Woolhouse D,et al.Molecular assays for detection of human metapneumovires.[J] Clin Microbiol ,2003 ;41(1):100-5.
[3] Stockton J,Stephenson I,Fleming D,et al.Human metapneumovirus as a cause of community-acquired respiratory illness. [J]Emerg Infect Dis,2002 ;8(9):897-901.
[4]Crowe JE Jr.Human metapneumovirus as a major cause of human respiratory tracr disease. [J]Pediatr Infect Dis J,2004 ;23(11 suppl):S215-S221.
[5] Falsey AR,Erdman D,Anderson LJ,et al.Human metapneumovirus infections in young and elderly adults. [J] Infect Dis,2003 ,187(5):785-790.
[6]Chung JY,Han TH,Kim BE,et al.Human Metapneumovirus infection in hospitalized children with acute respiratory disease in Korea. [J] Korena Med Sci.2006,21(5):838-42.
[7]Peret TC,Boivin G,Li Y,et al.Characterization of human metapneumoviruses isolated from patients in North America. [J] Infect Dis,2002 ,185(11):1660-1663.
[8]Gray GC,Capuano AW,Setterquist SF,et al.Human Metapnemovirus,Peru. [J]Emerg infect Dis,2006,12(2):347-350.
[9]Smuts H,Workman L,Zar HJ.Role of human metapneumovirus,human coronavirus NL63 and human bocavirus in infants and young children with acute wheezing. [J]Med Virol,2008,80(5):906-12.
[10]Mackay IM,Jacob KC,Woolhouse D.et al.Molecular assays for detection of human metapneumovirus. [J] Clin Microbiol,2003 ,41(1):100-5.
[11]Manoha C,Espinosa S,Aho SL,et al.Epidemiological and clinical features of hMPV , RSV and RVs infections in young children. [J]Clin Virol,2007,38(3):221-226.
[12]Mao HW,Yang XQ,Wang LJ.et al.Isolation of human metapneumovirus from children with acute respiratory tract infection in Chongqing, [J]China.Zhonghua Er Ke Za Zhi.2007 Jan;45(1):42-5.
[13]Zhu RN,Qian Y,Deng J.et al.Human metapneumovirus may associate with acute respiratory infections in hospitalized pediatric patients in Beijing,China. [J]Zhonghua Er Ke Za Zhi.2003 Jun;41(6):441-4.
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