Real-time polymerase chain reaction in detection of viral pathogen
GAO Xin, ZHU Wu-yang, LU Xue-xin
Rabies Department, National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention, Beijing 102206, China
Abstract:Real-time polymerase chain reaction, a sensitive and fast technology, is a quantitative PCR technology which can monitor the number of gene amplification products by capturing the fluorescence signal. This paper introduces the related principle of real-time PCR and research progress of viral pathogens detection by applying real-time PCR. This technology has been widely applied in quick viral pathogen detection such as HBV, dengue virus, influenza virus and VSV. Real-time PCR is also applied in quick genotyping of viruses, differentiating of similar viruses, detecting of drug-resistant virus mutants and other clinical and public health fields. Rabies is an incurable zoonosis and its mortality rate is almost 100%. This technology is applied in detection of rabies and lyssavirus which overcome the limitation of golden standard-DFA. It has equal sensitivity and specificity with DFA, and it is faster than DFA with no need for extracting brain tissues. It has good detection result of non-neural samples such as saliva, cerebrospinal fluid. Real-time PCR has potential to diagnose rabies. The sensitivity of real-time PCR in rabies detection is 200 times than conventional RT-PCR. Real-time PCR reduces the risk of cross contamination. It also has been applied in detection of rabies-related virus. Multiple real-time PCR can be applied in quick genotyping of many kinds of rabies-related virus in single reaction tube. Applying real-time PCR in detection of rabies and lyssavirus has important significance in public health.
[1] Navarro E, Serrano-Heras G, Castaño M J, et al.Real-time PCR detection chemistry[J]. Clin Chim Acta, 2015, 439(439):231-250. DOI: 10.1016/j.cca.2014.10.017 [2] 钟江华, 张光萍, 柳小英. 实时荧光定量PCR技术的研究进展与应用[J]. 氨基酸和生物资源, 2011, 33(2):68-72. DOI: 10.3969/j.issn.1006-8376.2011.02.019 [3] 陈旭, 齐凤坤, 康立功,等. 实时荧光定量PCR技术研究进展及其应用[J]. 东北农业大学学报, 2010, 41(8):148-155. DOI: 10.3969/j.issn.1005-9369.2010.08.029 [4] Monis PT, Giglio S, Saint CP.Comparison of SYTO9 and SYBR Green I for real-time polymerase chain reaction and investigation of the effect of dye concentration on amplification and DNA melting curve analysis.[J]. Anal Biochem, 2005, 340(1):24-34. DOI: 10.1016/j.ab.2005.01.046 [5] Mao F, Leung WY, Xin X.Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications.[J]. BMC Biotechnol, 2007, 7(1):76. DOI: 10.1186/1472-6750-7-76 [6] 阳成波, 印遇龙, 黄瑞林,等. 实时定量RT-PCR的原理及方法[J]. 免疫学杂志, 2003,19(1):150-155. [7] Hughes GJ, Smith JS, Hanlon CA, et al.Evaluation of a TaqMan PCR assay to detect rabies virus RNA: influence of sequence variation and application to quantification of viral loads[J]. J Clin Microbiol, 2004, 42(1):299-306. DOI: 10.1128/jcm.42.1.299-306.2004 [8] 赵焕英, 包金风. 实时荧光定量PCR 技术的原理及其应用研究进展[J]. 中国组织化学与细胞化学杂志, 2007, 16(4):492-497. DOI: 10.3870/j.issn.1004-1850.2007.04.023 [9] Wu B, Xiao F, Li P, et al.Ultrasensitive detection of serum hepatitis B virus by coupling ultrafiltration DNA extraction with real-time PCR.[J]. PLoS One, 2017, 12(2):e0170290. DOI: 10.1371/journal.pone.0170290 [10] Chen R W, Piiparinen H, Seppänen M, et al.Real-time PCR for detection and quantitation of hepatitis B virus DNA.[J]. J Med Virol, 2001, 65(2):250. DOI: 10.1002/jmv.2027 [11] Welzel TM, Miley WJ, Parks TL, et al.Real-time PCR assay for detection and quantification of hepatitis B virus genotypes A to G.[J]. J Clin Microbiol, 2006, 44(9):3325. DOI: 10.1128/jcm.00024-06 [12] Wang Q, Wang X, Zhang J, et al.LNA real-time PCR probe quantification of hepatitis B virus DNA[J]. J Behav Ther Exp Psychiatry, 2012, 3(3):503-508. DOI: 10.3892/etm.2011.442 [13] Cane PA, Cook P, Ratcliffe D, et al.Use of Real-time PCR and fluorimetry to detect lamivudine resistance-associated mutations in hepatitis B virus[J]. Antimicrob Agents Chemother, 1999, 43(7):1600-1608. [14] Zhao JR, Bai YJ, Zhang QH, et al.Detection of hepatitis B virus DNA by real-time PCR using TaqMan-MGB probe technology.[J]. World J Gastroenterol, 2005, 11(4):508-510. DOI: 10.3748/wjg.v11.i4.508 [15] Yang ZJ, Tu MZ, Liu J, et al.Comparison of amplicon-sequencing, pyrosequencing and real-time PCR for detection of YMDD mutants in patients with chronic hepatitis B[J]. World J Gastroenterol, 2006, 14(44):7192-7196. DOI: 10.3748/wjg.v12.i44.7192 [16] Shih YH, Yeh SH, Chen PJ, et al.Hepatitis B virus quantification and detection of YMDD mutants in a single reaction by real-time PCR and annealing curve analysis[J]. Antivir Ther, 2008, 13(4):469-480. [17] Ntziora F, Paraskevis D, Haida C, et al.Quantitative detection of the M204V hepatitis B virus minor variants by amplification refractory mutation system real-time PCR combined with molecular beacon technology[J]. J Clin Microbiol, 2009, 47(8):2544-2550. DOI: 10.1128/JCM.00045-09 [18] Takkenberg RB, Zaaijer HL, Molenkamp R, et al.Validation of a sensitive and specific real-time PCR for detection and quantitation of hepatitis B virus covalently closed circular DNA in plasma of chronic hepatitis B patients[J]. J Med Virol, 2009, 81(6):988-95. DOI: 10.1002/jmv.21477 [19] Shu PY, Chang SF, Kuo YC, et al.Development of group- and serotype-specific one-step SYBR green I-based real-time reverse transcription-PCR assay for dengue virus[J]. J Clin Microbiol, 2003, 41(6):2408-2416. DOI: 10.1128/JCM.41.6.2408-2416.2003 [20] Alm E, Lindegren G, Falk KI, et al.One-step real-time RT-PCR assays for serotyping dengue virus in clinical samples[J]. BMC Infect Dis, 2015, 15(1):493. DOI: 10.1186/s12879-015-1226-z [21] Callahan JD, Wu SJ, DionSchultz A, et al. Development and evaluation of serotype- and group-specific fluorogenic reverse transcriptase PCR (TaqMan) assays for dengue virus[J]. J Clin Microbiol, 2001, 39(11):4119. DOI: 10.1128/JCM.39.11.4119-4124.2001 [22] Santiago GA, Vergne E, Quiles Y, et al.Analytical and clinical performance of the CDC real time RT-PCR assay for detection and typing of dengue virus[J]. PLoS Negl Trop Dis, 2013, 7(7):e2311. DOI: 10.1371/journal.pntd.0002311 [23] Ellis J, Iturriza M, Allen R, et al.Evaluation of four real-time PCR assays for detection of influenza A(H1N1)v viruses[J]. Euro Surveill, 2009, 14(22):1891-1898. [24] Song HO, Kim JH, Ryu HS, et al.Polymeric LabChip real-time PCR as a point-of-care-potential diagnostic tool for rapid detection of influenza A/H1N1 virus in human clinical specimens[J]. PLoS One, 2012, 7(12):e53325. DOI: 10.1371/journal.pone.0053325 [25] Stone B, Burrows J, Schepetiuk S, et al.Rapid detection and simultaneous subtype differentiation of influenza A viruses by real time PCR[J]. J Virol Methods, 2004, 117(2):103-112. DOI: 10.1016/j.jviromet.2003.12.005 [26] Wei W, Ren PJ, Mardi S, et al.Design of multiplexed detection assays for identification of avian influenza A virus subtypes pathogenic to humans by SmartCycler real-time reverse transcription-PCR.[J]. J Clin Microbiol, 2009, 47(1):86-92. DOI: 10.1128/JCM.01090-08 [27] Zhang Z, Liu D, Sun W, et al.Multiplex one-step Real-time PCR by Taqman-MGB method for rapid detection of pan and H5 subtype avian influenza viruses:[J]. PLoS One, 2017, 12(6):e0178634. DOI: 10.1371/journal.pone.0178634 [28] Wu C, Cheng X, He J, et al.A multiplex real-time RT-PCR for detection and identification of influenza virus types A and B and subtypes H5 and N1[J]. J Virol Methods, 2008, 148(1/2):81-88. DOI: 10.1016/j.jviromet.2007.10.023 [29] Matsuzaki Y, Ikeda T, Abiko C, et al.Detection and quantification of influenza C virus in pediatric respiratory specimens by real-time PCR and comparison with infectious viral counts[J]. J Clin Virol, 2012, 54(2):130-134. DOI: 10.1016/j.jcv.2012.02.012 [30] Esposito S, Scala A, Tagliabue C, et al.Evaluation of a multiplex real-time polymerase chain reaction assay for the detection of influenza and respiratory syncytial viruses[J]. Diagn Microbiol Infect Dis, 2016, 84(1):40-42. DOI: 10.1016/j.diagmicrobio.2015.09.014 [31] 周航, 李昱, 陈瑞丰,等. 狂犬病预防控制技术指南(2016版)[J]. 中华流行病学杂志, 2016, 37(2):139-163. DOI: 10.3760/cma.j.issn.0254-6450.2016.02.001 [32] Nagaraj T, Vasanth JP, Desai A, et al.Ante mortem diagnosis of human rabies using saliva samples: comparison of real time and conventional RT-PCR techniques.[J]. J Clin Virol, 2006, 36(1):17. DOI: 10.1016/j.jcv.2006.01.009 [33] Saengseesom W, Mitmoonpitak C, Kasempimolporn S, et al.Real-time PCR analysis of dog cerebrospinal fluid and saliva samples for ante-mortem diagnosis of rabies[J]. Southeast Asian J Trop Med Public Health, 2007, 38(1):53-57. [34] Wacharapluesadee S, Tepsumethanon V, Supavonwong P, et al.Detection of rabies viral RNA by TaqMan real-time RT-PCR using non-neural specimens from dogs infected with rabies virus.[J]. J Virol Methods, 2012, 184(1/2):109-112. DOI: 10.1016/j.jviromet.2012.05.013 [35] Mani RS, Madhusudana SN, Mahadevan A, et al.Utility of real-time Taqman PCR for antemortem and postmortem diagnosis of human rabies[J]. J Med Virol, 2014, 86(10):1804-1812. DOI: 10.1002/jmv.23814 [36] Faye M, Dacheux L, Weidmann M, et al.Development and validation of sensitive real-time RT-PCR assay for broad detection of rabies virus.[J]. J Virol Methods, 2017, 243:120. DOI: 10.1016/j.jviromet.2016.12.019 [37] Black EM, Lowings JP, Smith J, et al.A rapid RT-PCR method to differentiate six established genotypes of rabies and rabies-related viruses using TaqManTM technology[J]. J Virol Methods, 2002, 105(1):25-35. DOI: 10.1016/S0166-0934(02)00062-9 [38] Coertse J, Weyer J, Nel LH, et al.Improved PCR methods for detection of African rabies and rabies-related lyssaviruses.[J]. J Clin Microbiol, 2010, 48(11):3949-3955. DOI: 10.1128/JCM.01256-10 [39] Wadhwa A, Wilkins K, Gao J, et al.A pan-Lyssavirus taqman real-time RT-PCR assay for the detection of highly variable rabies virus and other lyssaviruses[J]. PLoS Negl Trop Dis, 2017, 11(1):e0005258. DOI: 10.1371/journal.pntd.0005258 [40] Wakeley PR, Johnson N, Mcelhinney LM, et al.Development of a real-time, differential RT-PCR TaqMan○R assay for lyssavirus genotypes 1, 5 and 6.[J]. Dev Biol, 2006, 126(126):227. [41] Deubelbeiss A, Zahno ML, Zanoni M, et al.Real-time RT-PCR for the Detection of lyssavirus species[J]. J Vet Med, 2014. DOI: 10.1155/2014/476091 [42] Hayman DTS, Banyard AC, Wakeley PR, et al.A universal real-time assay for the detection of Lyssaviruses[J]. J Virol Methods, 2011, 177(1):87-93. DOI: 10.1016/j.jviromet.2011.07.002 [43] Dacheux L, Larrous F, Lavenir R, et al.Dual combined real-time reverse transcription polymerase chain reaction assay for the diagnosis of lyssavirus infection[J]. PLoS Negl Trop Dis, 2016, 10(7):e0004812. DOI: 10.1371/journal.pntd.0004812 [44] 花群义, 徐自忠, 杨云庆,等. TaqMan(R) RT-PCR对水疱性口炎病毒的鉴定检测[J]. 动物医学进展, 2004, 25(2):64-68. DOI: 10.3969/j.issn.1007-5038.2004.02.022 [45] Tolardo AL, Souza WMD, Romeiro MF, et al.A real-time reverse transcriptase polymerase chain reaction for detectionand quantification of Vesiculovirus[J]. Mem Inst Oswaldo Cruz, 2016, 111(6):385-390. DOI: 10.1590/0074-02760150456 [46] Hole K, Clavijo A, Pineda LA.Detection and serotype-specific differentiation of vesicular stomatitis virus using a multiplex, real-time, reverse transcription-polymerase chain reaction assay.[J]. J Vet Diagn Invest, 2006, 18(2):139-146. DOI: 10.1177/104063870601800201 [47] Chan FW, Yip CY, Tee KM, et al.Improved detection of Zika virus RNA in human and animal specimens by a novel, highly sensitive and specific real-time RT-PCR assay targeting the 5'-untranslated region of Zika virus[J]. Trop Med Int Health, 2017, 22(5):594. DOI: 10.1111/tmi.12857 [48] Pabbaraju K, Wong S, Gill K, et al.Simultaneous detection of Zika, Chikungunya and Dengue viruses by a multiplex real-time RT-PCR assay[J]. J Clin Virol, 2016, 83:66-71. DOI: 10.1016/j.jcv.2016.09.001 [49] Xu MY, Liu SQ, Deng CL, et al.Detection of Zika virus by SYBR green one-step real-time RT-PCR[J]. J Virol Methods, 2016, 236:93-97. DOI:10.1016/j.jviromet.2016.07.014 [50] Burkhalter KL, Savage HM.Detection of Zika virus in desiccated mosquitoes by real-time reverse transcription PCR and plaque assay[J]. Emerg Infect Dis, 2017, 23(4):680. DOI:10.3201/eid2304.161772