逆转录重组酶介导扩增技术快速检测基孔肯雅热病毒
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摘要
本研究通过使用逆转录酶,将基孔肯雅热病毒RNA首先逆转录为cDNA。选取基孔肯雅热病毒保守序列设计引物及探针,建立基孔肯雅热病毒重组酶介导一步法等温核酸扩增RT-RAA(Reverse transcription recombinase aided amplification,RT-RAA)快速检测法,并分析其灵敏度及特异性。结果显示,该方法整个过程在39℃进行,检测时间短(<20 min),检测下限可达100 copy,并与黄热病毒、日本乙型脑炎病毒、西尼罗病毒、登革病毒I型等蚊媒病毒不存在交叉反应。本文建立的基孔肯雅热病毒RT-RAA检测法,灵敏度高、特异性强、操作简便,适应于口岸基层基孔肯雅热病毒的快速检测。
The Chikungunya fever virus( CHIKV) RNA was first transcribed into the cDNA by a reverse-transcriptase reaction,and the resulting product was then served as template for RT-RAA amplification. Universal primers and probe were designed according to the conserved genome sequence of the CHIKV to evaluate the specificity and sensitivity of the methods and develop a rapid one-step reverse transcription recombinase aided amplification( RT-RAA) assay for( CHIKV)detection. The reaction was performed at a constant temperature of 39℃ with a short detection time( < 20 minutes). Our results indicated that the amplification is very sensitive which can detect 100 copies. The approach is also shown very specific as it had no cross reaction with other related viruses,such as Dengue virus,West Nile virus,Japanese encephalitis virus or Yellow fever virus. This RT-RAA assay was rapid,specific and sensitive,and might be utilized in rapid surveillance for CHIKV on ports.
The Chikungunya fever virus( CHIKV) RNA was first transcribed into the cDNA by a reverse-transcriptase reaction,and the resulting product was then served as template for RT-RAA amplification. Universal primers and probe were designed according to the conserved genome sequence of the CHIKV to evaluate the specificity and sensitivity of the methods and develop a rapid one-step reverse transcription recombinase aided amplification( RT-RAA) assay for( CHIKV)detection. The reaction was performed at a constant temperature of 39℃ with a short detection time( < 20 minutes). Our results indicated that the amplification is very sensitive which can detect 100 copies. The approach is also shown very specific as it had no cross reaction with other related viruses,such as Dengue virus,West Nile virus,Japanese encephalitis virus or Yellow fever virus. This RT-RAA assay was rapid,specific and sensitive,and might be utilized in rapid surveillance for CHIKV on ports.
引文
Burt FJ,Rolph MS,Rulli NE,Mahalingam S,Heise MT.Chikungunya:a reemerging virus.Lancet.,2012,379(9816):662-671.
Borgherini G,Poubeau P,Staikowsky F,Lory M,Le Moullec N,Becquart JP,Wengling C,Michault A,Paganin F.Outbreak of chikungunya on Reunion Island:early clinical and laboratory features in 157 adult patients.Clin Infect Dis.,2007,44(11):1 401-1 407.
Huang L,Zhang XG,Zhang W.Analysis of imported chikungunya fever cases detected at frontier ports of Guangdong Entry-Exit Inspection and Quarantine Bureau.Chin J Vectro Biol&Control,2013,24(4):357-360.[黄鹂,张显光,张文.广东出入境检验检疫局口岸输入性基孔肯雅热监测分析.中国媒介生物学及控制杂志,2013,24(4):357-360.]
Laoprasopwattana K,Suntharasaj T,Petmanee P,Suddeaugrai O,Geater A.Chikungunya and dengue virus infections during pregnancy:seroprevalence,seroincidence and maternal-fetal transmission,southern Thailand,2009-2010.Epidemiol Infect.,2016,144(2):381-388.
Li XB,Lu X,Xing LQ.Establishment and application of LAMP for detecting Chikungunya virus.Chinese Journal of Health Laboratory Technology,2012,22(3):506-508.[李小波,鲁曦,幸芦琴.基孔肯雅病毒LAMP检测方法的建立及其应用研究.中国卫生检验杂志,2012,22(3):506-508.]
Morey SH,Kashyap RS,Purohit HJ.An approach towards peptidebased antibody detection for diagnosis of Chikungunya infection.Biomarkers.,2010,15(6):546-552.
Parida M,Santhosh S,Dash P.Rapid and real-time detection of Chikungunya virus by reverse transcription loop-mediated isothermal amplification assay.J Clin Microbiol.,2007,45(2):351-357.
Queyriaux B,Simon F,Grandadam M,Michel R,Tolou H,Boutin JP.Clinical burden of chikungunya virus infection.Lancet Infect Dis.,2008,8(1):2-3.
Schuffenecker I,Iteman I,Michault A.Genome microevolution of chikungunya viruses causing the Indian Ocean outbreak.PLo SMed.,2006,3(7):e263.
Schwartz O,Albert ML.Biology and pathogenesis of chikungunya virus.Nat Rev Microbiol.,2010,8(7):491-500.
Shukla J,Khan M,Tiwari M,Sannarangaiah S,Sharma S.Development and evaluation of antigen capture ELISA for early clinical diagnosis of chikungunya.Diagn Microbiol Infect Dis.,2009,65(2):142-149.
Smalley C,Erasmus JH,Chesson CB,Beasley DW.Status of research and development of vaccines for chikungunya.Vaccine.,2016,34(26):2 976-2 981.
Sudeep A,Hundekar S,Jacob P,Balasubramanian R,Arankalle VA,Mishra AC.Investigation of a Chikungunya-like illness in Tirunelveli district,Tamil Nadu,India 2009-2010.Trop Med Int Health,2011,16(5):585-588.
Telles JN,Le Roux K,Grivard P,Vernet G,Michault A.Evaluation of real-time nucleic acid sequence-based amplification for detection of Chikungunya virus in clinical samples.J Med Microbiol.,2009,58(Pt 9):1 168-1 172.
Tian DQ,Chen W.Chikungunya virus and chikungunya fever.Journal of Microbes and Infections.,2016,11(4):194-206.[田德桥,陈薇.基孔肯雅病毒与基孔肯雅热.微生物与感染,2016,11(4):194-206.]
Tsetsarkin KA,Vanlandingham DL,Mc Gee CE.A single mutation in chikungunya virus affects vector specificity and epidemic potential.PLo S Pathog.,2007,3(12):e201.
Weaver SC,Lecuit M.Chikungunya virus infections.N Engl JMed.,2015,373(1):94-95.
Xu J,Xie Y,Guan DW.Development of a multiplex real-time RT-PCR assay with an internal control for detection of dengue and Chikungunya viruses.Journal of Pathogen Biology,2016,11(4):301-304.[徐静,谢翊,管大伟.含内参的登革病毒与基孔肯雅病毒3重荧光定量PCR检测方法的建立与评估.中国病原生物学杂志,2016,11(4):301-304.]
Yu BB,Lu YY,Xie XY.Simultaneous detection of West Nile virus and Chikungunya virus by duplex real-time quantitative PCR.Chin J Clin Lab Sci.,2013,31(6):412-414.[余蓓蓓,卢亦愚,谢鑫友.双重荧光定量PCR检测西尼罗病毒和基孔肯雅病毒.临床检验杂志,2013,31(6):412-414.]
Borgherini G,Poubeau P,Staikowsky F,Lory M,Le Moullec N,Becquart JP,Wengling C,Michault A,Paganin F.Outbreak of chikungunya on Reunion Island:early clinical and laboratory features in 157 adult patients.Clin Infect Dis.,2007,44(11):1 401-1 407.
Huang L,Zhang XG,Zhang W.Analysis of imported chikungunya fever cases detected at frontier ports of Guangdong Entry-Exit Inspection and Quarantine Bureau.Chin J Vectro Biol&Control,2013,24(4):357-360.[黄鹂,张显光,张文.广东出入境检验检疫局口岸输入性基孔肯雅热监测分析.中国媒介生物学及控制杂志,2013,24(4):357-360.]
Laoprasopwattana K,Suntharasaj T,Petmanee P,Suddeaugrai O,Geater A.Chikungunya and dengue virus infections during pregnancy:seroprevalence,seroincidence and maternal-fetal transmission,southern Thailand,2009-2010.Epidemiol Infect.,2016,144(2):381-388.
Li XB,Lu X,Xing LQ.Establishment and application of LAMP for detecting Chikungunya virus.Chinese Journal of Health Laboratory Technology,2012,22(3):506-508.[李小波,鲁曦,幸芦琴.基孔肯雅病毒LAMP检测方法的建立及其应用研究.中国卫生检验杂志,2012,22(3):506-508.]
Morey SH,Kashyap RS,Purohit HJ.An approach towards peptidebased antibody detection for diagnosis of Chikungunya infection.Biomarkers.,2010,15(6):546-552.
Parida M,Santhosh S,Dash P.Rapid and real-time detection of Chikungunya virus by reverse transcription loop-mediated isothermal amplification assay.J Clin Microbiol.,2007,45(2):351-357.
Queyriaux B,Simon F,Grandadam M,Michel R,Tolou H,Boutin JP.Clinical burden of chikungunya virus infection.Lancet Infect Dis.,2008,8(1):2-3.
Schuffenecker I,Iteman I,Michault A.Genome microevolution of chikungunya viruses causing the Indian Ocean outbreak.PLo SMed.,2006,3(7):e263.
Schwartz O,Albert ML.Biology and pathogenesis of chikungunya virus.Nat Rev Microbiol.,2010,8(7):491-500.
Shukla J,Khan M,Tiwari M,Sannarangaiah S,Sharma S.Development and evaluation of antigen capture ELISA for early clinical diagnosis of chikungunya.Diagn Microbiol Infect Dis.,2009,65(2):142-149.
Smalley C,Erasmus JH,Chesson CB,Beasley DW.Status of research and development of vaccines for chikungunya.Vaccine.,2016,34(26):2 976-2 981.
Sudeep A,Hundekar S,Jacob P,Balasubramanian R,Arankalle VA,Mishra AC.Investigation of a Chikungunya-like illness in Tirunelveli district,Tamil Nadu,India 2009-2010.Trop Med Int Health,2011,16(5):585-588.
Telles JN,Le Roux K,Grivard P,Vernet G,Michault A.Evaluation of real-time nucleic acid sequence-based amplification for detection of Chikungunya virus in clinical samples.J Med Microbiol.,2009,58(Pt 9):1 168-1 172.
Tian DQ,Chen W.Chikungunya virus and chikungunya fever.Journal of Microbes and Infections.,2016,11(4):194-206.[田德桥,陈薇.基孔肯雅病毒与基孔肯雅热.微生物与感染,2016,11(4):194-206.]
Tsetsarkin KA,Vanlandingham DL,Mc Gee CE.A single mutation in chikungunya virus affects vector specificity and epidemic potential.PLo S Pathog.,2007,3(12):e201.
Weaver SC,Lecuit M.Chikungunya virus infections.N Engl JMed.,2015,373(1):94-95.
Xu J,Xie Y,Guan DW.Development of a multiplex real-time RT-PCR assay with an internal control for detection of dengue and Chikungunya viruses.Journal of Pathogen Biology,2016,11(4):301-304.[徐静,谢翊,管大伟.含内参的登革病毒与基孔肯雅病毒3重荧光定量PCR检测方法的建立与评估.中国病原生物学杂志,2016,11(4):301-304.]
Yu BB,Lu YY,Xie XY.Simultaneous detection of West Nile virus and Chikungunya virus by duplex real-time quantitative PCR.Chin J Clin Lab Sci.,2013,31(6):412-414.[余蓓蓓,卢亦愚,谢鑫友.双重荧光定量PCR检测西尼罗病毒和基孔肯雅病毒.临床检验杂志,2013,31(6):412-414.]