Journal of Microbiology and Biotechnology

  • 제30권3호
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  • Pages.459-468
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  • 2020
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  • 1017-7825(pISSN)
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  • 1738-8872(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
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Rapid and Sensitive Detection of Hepatitis C Virus in Clinical Blood Samples Using Reverse Transcriptase Polymerase Spiral Reaction

  • Sun, Wenying (Clinical Laboratory, the Second Affiliated Hospital of Harbin Medical University) ;
  • Du, Ying (Department of Experimental Diagnosis, Heilongjiang Provincial Hospital) ;
  • Li, Xingku (Experimental Research Center, the Second Affiliated Hospital of Harbin Medical University) ;
  • Du, Bo (Experimental Research Center, the Second Affiliated Hospital of Harbin Medical University)
  • 투고 : 2019.10.21
  • 심사 : 2019.12.17
  • 발행 : 2020.03.28
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초록

This study established a new polymerase spiral reaction (PSR) that combines with reverse transcription reactions for HCV detection targeting 5'UTR gene. To avoid cross-contamination of aerosols, an isothermal amplification tube (IAT), as a separate containment control, was used to judge the result. After optimizing the RT-PSR reaction system, its effectiveness and specificity were tested against 15 different virus strains which included 8 that were HCV positive and 7 as non-HCV controls. The results showed that the RT-PSR assay effectively detected all 8 HCV strains, and no false positives were found among the 7 non-HCV strains. The detection limit of our RT-PSR assay is comparable to the real-time RT-PCR, but is more sensitive than the RT-LAMP. The established RT-PSR assay was further evaluated for detection of HCV in clinical blood samples, and the resulting 80.25% detection rate demonstrated better or similar effectiveness compared to the RT-LAMP (79.63%) and real-time RT-PCR (80.25%). Overall, the results showed that the RT-PSR assay offers high specificity and sensitivity for HCV detection with great potential for screening HCV in clinical blood samples.

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참고문헌

  1. Brass V, Moradpour D, Blum HE. 2006. Molecular virology of hepatitis C virus (HCV): 2006 update. Int. J. Med. Sci. 3: 29-34. https://doi.org/10.7150/ijms.3.29
  2. Moradpour D, Penin F, Rice CM. 2007. Replication of hepatitis C virus. Nat. Rev. Microbiol. 5: 453-463. https://doi.org/10.1038/nrmicro1645
  3. Messina JP, Humphreys I, Flaxman A, Brown A, Cooke GS, Pybus OG, et al. 2015. Global distribution and prevalence of hepatitis C virus genotypes. Hepatology 61: 77-87. https://doi.org/10.1002/hep.27259
  4. Campo DS, Xia GL, Dimitrova Z, Lin Y, Forbi JC, Ganova-Raeva L, et al. 2016. Accurate genetic detection of Hepatitis C Virus transmissions in outbreak settings. J. Infect. Dis. 213: 957-965. https://doi.org/10.1093/infdis/jiv542
  5. Chen Y, Yu C, Yin X, Guo X, Wu S, Hou J. 2017. Hepatitis C virus genotypes and subtypes circulating in Mainland China. Emerg. Microbes Infect. 6(11): e95. https://doi.org/10.1038/emi.2017.77
  6. Fu Y, Wang Y, Xia W, Pybus OG, Qin W, Lu L, et al. 2011. New trends of HCV infection in China revealed by genetic analysis of viral sequences determined from first-time volunteer blood donors. J. Viral Hepat. 18: 42-52. https://doi.org/10.1111/j.1365-2893.2010.01280.x
  7. Andrade E, Rocha D, Fontana-Maurell M, Costa E, Ribeiro M, de Godoy DT, et al. 2018. One-step real-time PCR assay for detection and quantification of RNA HCV to monitor patients under treatment in Brazil. Braz. J. Infect. Dis. 22: 418-423. https://doi.org/10.1016/j.bjid.2018.08.003
  8. Nyan DC, Swinson KL. 2016. A method for rapid detection and genotype identification of hepatitis C virus 1-6 by one-step reverse transcription loop-mediated isothermal amplification. Int. J. Infect. Dis. 43: 30-36. https://doi.org/10.1016/j.ijid.2015.12.002
  9. Pawlotsky JM. 2002. Use and interpretation of virological tests for Hepatitis C. Hepatology 36: S65-73. https://doi.org/10.1053/jhep.2002.33998
  10. Shahzamani K, Sabahi F, Merat S, Sadeghizadeh M, Lashkarian HE, Rezvan H, et al. 2011. Rapid low-cost detection of hepatitis C virus RNA in HCV-infected patients by real-time RT-PCR using SYBR Green I. Arch. Iran Med. 14: 396-400.
  11. Laperche S, Elghouzzi MH, Morel P, Asso-Bonnet M, Le Marrec N, Girault A, et al. 2005. Is an assay for simultaneous detection of hepatitis C virus core antigen and antibody a valuable alternative to nucleic acid testing? Transfusion 45: 1965-1972. https://doi.org/10.1111/j.1537-2995.2005.00648.x
  12. Yang JF, Lin YY, Hsieh MH, Tsai CH, Liu SF, Yu ML, et al. 2011. Performance characteristics of a combined hepatitis C virus core antigen and anti-hepatitis C virus antibody test in different patient groups. Kaohsiung J. Med. Sci. 27: 258-263. https://doi.org/10.1016/j.kjms.2010.11.007
  13. Kargar M, Askari A, Doosti A, Ghorbani-Dalini S. 2012. Loop-Mediated isothermal amplification assay for rapid detection of Hepatitis C virus. Indian J. Virol. 23: 18-23. https://doi.org/10.1007/s13337-012-0067-2
  14. Liu W, Yuan C, Zhang L, Feng Y. 2019. Development of isothermal amplification methods for rapid and sensitive detection of heat-labile enterotoxin producing Escherichia coli. J. Microbiol. Methods. 161: 47-55. https://doi.org/10.1016/j.mimet.2019.04.010
  15. Iwamoto T, Sonobe T, Hayashi K. 2003. Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M. avium, and M. intracellulare in sputum samples. J. Clin. Microbiol. 41: 2616-2622. https://doi.org/10.1128/JCM.41.6.2616-2622.2003
  16. Parida M, Sannarangaiah S, Dash PK, Rao PV, Morita K. 2008. Loop mediated isothermal amplification (LAMP): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases. Rev. Med. Virol. 18: 407-421. https://doi.org/10.1002/rmv.593
  17. Zhao N, Liu J, Sun D. 2017. A reverse transcription loop-mediated isothermal amplification assay for detection of HCV genotypes 1b and 2a in China. Bing Du Xue Bao. 33: 49-55.
  18. Liu W, Dong D, Yang Z, Zou D, Chen Z, Yuan J, et al. 2015. Polymerase Spiral Reaction (PSR): A novel isothermal nucleic acid amplification method. Sci. Rep. 5: 12723. https://doi.org/10.1038/srep12723
  19. Jiang X, Dong D, Bian L, Zou D, He X, Ao D, et al. 2016. Rapid detection of candida albicans by polymerase spiral reaction assay in clinical blood samples. Front Microbiol. 7: 916.
  20. Gupta V, Chakravarti S, Chander V, Majumder S, Bhat SA, Gupta VK, et al. 2017. Polymerase spiral reaction (PSR): a novel, visual isothermal amplification method for detection of canine parvovirus 2 genomic DNA. Arch. Virol. 162: 1995-2001. https://doi.org/10.1007/s00705-017-3321-5
  21. Das A, Kumar B, Chakravarti S, Prakash C, Singh RP, Gupta V, et al. 2018. Rapid visual isothermal nucleic acidbased detection assay of Brucella species by polymerase spiral reaction. J. Appl. Microbiol. 125: 646-654. https://doi.org/10.1111/jam.13882
  22. Malla JA, Chakravarti S, Gupta V, Chander V, Sharma GK, Qureshi S, et al. 2018. Novel Polymerase Spiral Reaction (PSR) for rapid visual detection of Bovine Herpesvirus 1 genomic DNA from aborted bovine fetus and semen. Gene 644: 107-112. https://doi.org/10.1016/j.gene.2017.11.004
  23. Athar MA, Xu Y, Xie X, Xu Z, Ahmad V, Hayder Z, et al. 2015. Rapid detection of HCV genotyping 1a, 1b, 2a, 3a, 3b and 6a in a single reaction using two-melting temperature codes by a real-time PCR-based assay. J. Virol. Methods 222: 85-90. https://doi.org/10.1016/j.jviromet.2015.05.013
  24. Khalvati Fahlyani B, Behzad-Behbahani A, Taghavi SA, Farhadi A, Salehi S, Adibzadeh S, et al. 2015. Development of an in-house TaqMan real time RT-PCR assay to quantify Hepatitis C Virus RNA in serum and peripheral blood mononuclear cells in patients with chronic Hepatitis C Virus infection. Hepat. Mon. 15: e28895.
  25. Martinez AW, Phillips ST, Whitesides GM, Carrilho E. 2010. Diagnostics for the developing world: microfluidic paper-based analytical devices. Anal. Chem. 82: 3-10. https://doi.org/10.1021/ac9013989