Biomedical Science Letters (대한의생명과학회지)

The Korean Society for Biomedical Laboratory Sciences (대한의생명과학회)
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Application of Loop-Mediated Isothermal Amplification (LAMP) Assay to Rapid Detection of Methicillin-Resistant Staphylococcus aureus from Blood Cultures

  • Baek, Yun-Hee (Department of Microbiology, Chungbuk National University College of Medicine) ;
  • Jo, Mi-Young (Department of Laboratory Medicine, Chungbuk National University College of Medicine) ;
  • Song, Min-Suk (Department of Microbiology, Chungbuk National University College of Medicine) ;
  • Hong, Seung-Bok (Department of Clinical Laboratory Science, Chungbuk Health & Science Unviersity) ;
  • Shin, Kyeong-Seob (Department of Laboratory Medicine, Chungbuk National University College of Medicine)
  • Received : 2019.02.08
  • Accepted : 2019.03.19
  • Published : 2019.03.31
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Abstract

We developed the multiplex LAMP assay using 16S rRNA, femA and mecA genes for direct detection of the methicillin resistance in Staphylococci from positive blood culture. To simultaneously recognize Staphylococci genus, S. aureus and methicillin resistance, three sets of six primers for 16S rRNA, femA and mecA were designed, respectively. The performance of LAMP assay was affirmed using VITEK system for the phenotypic methods of identification and for oxacillin and cefoxitin antimicrobial susceptibility. The optimal condition for LAMP assay was obtained under $64^{\circ}C$ for 50 min. The detection limit was determined to be of 20 copies and CFU/reaction ($10^4CFU/mL$). For clinical application of comparison with phenotypic methods, the sensitivity and specificity of the LAMP with femA gene for detecting S. aureus was 95.31% and 100%, respectively. The sensitivity and specificity of the LAMP with mecA gene for detecting methicillin resistance was 98.46% and 100%, respectively. The multiplex LAMP assay with femA and mecA gene successfully detected all of MRSA (38 isolates) isolates from 103 Staphylococci in blood cultures. The LAMP assay developed in this study is sensitive, specific, and of excellent agreement with the phenotypic methods.

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References

  1. Becker K, Pagnier I, Schuhen B, Wenzelburger F, Friedrich AW, Kipp F, et al. Does nasal cocolonization by methicillinresistant coagulase-negative staphylococci and methicillinsusceptible Staphylococcus aureus strains occur frequently enough to represent a risk of false-positive methicillin-esistant S. aureus determinations by molecular methods? J Clin Microbiol. 2006. 44: 229-231. https://doi.org/10.1128/JCM.44.1.229-231.2006
  2. Chen C, Zhao Q, Guo J, Li Y, Chen Q. Identification of methicillinresistant Staphylococcus aureus (MRSA) using simultaneous detection of mecA, nuc and femB by loop-mediated isothermal amplification (LAMP). Curr Microbiol. 2017. 74: 965-971. https://doi.org/10.1007/s00284-017-1274-2
  3. Clerc O, Prod'hom G, Senn L, et al. Matrix-assisted laser desorption ionization time-of-flight mass spectrophotometry and PCRbased rapid diagnosis of Staphylococcus aureus bacteremia. Clin Microbial Infect. 2014. 20: 355-360. https://doi.org/10.1111/1469-0691.12329
  4. Cosgrove SE, Sakaoulas G, Perencevich EN, Schwaber MJ, Karchmer AW, Carmeli Y. Comparison of mortality associated with methicillin-resistant and methicillin susceptible Staphylococcus aureus bacteremia: a meta-analysis. Clin Infect Dis. 2002. 36: 53-59. https://doi.org/10.1086/345476
  5. Curtis KA, Rudolph DL, Owen SM. Rapid detection of HIV-1 by reverse-transcription, lood-mediated isothermal amplification (RT-LAMP). J Virol Methods. 2008. 151: 264-270. https://doi.org/10.1016/j.jviromet.2008.04.011
  6. Hara-Kudo Y, Konishi N, Ohtsuka K, Hiramatsu R, Tanaka H, Konuma H, Takatori K. Detection of verotoxigenic Escherichia coli O157 and O26 in food by plating methods and LAP method: a collaborative study. Int J Food Microbiol. 2008. 122: 156-161. https://doi.org/10.1016/j.ijfoodmicro.2007.11.078
  7. Hara-Kudo Y, Yoshino M, Kojima T, Ikedo M. Loop-mediated isothermal amplification for the rapid detection of salmonella. FEMS Microbiol Lett. 2005. 253: 155-161. https://doi.org/10.1016/j.femsle.2005.09.032
  8. Hartman BJ, Tomasz A. Low-affinity penicillin-binding protein associated with beta-lactam resistance in Staphylococcus aureus. J Bacteriol. 1984. 158: 513-516. https://doi.org/10.1128/JB.158.2.513-516.1984
  9. Ito M, Watanabe M, Nakagawa N, Ihara T, Okuno Y. Rapid detection and typing of influenza A and B by loop-mediated isothermal amplification: comparison with immunochromatography and virus isolation. J Virol Methods. 2006. 135: 272-275. https://doi.org/10.1016/j.jviromet.2006.03.003
  10. Iwamoto T, Sonobe T, Hayashi K. Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M. avium, and M. intracellulare in sputum samples. J Clin Microbiol. 2003. 41: 2616-2622. https://doi.org/10.1128/JCM.41.6.2616-2622.2003
  11. Li Y, Fan P, Zhou S, Zhang L. Loop-mediated isothermal amplification (LAMP): a novel rapid detection platform for pathogens. Microb Pathog. 2017. 107: 54-61. https://doi.org/10.1016/j.micpath.2017.03.016
  12. Lowy FD. Staphylococcus aureus infections. N Engl J Med. 1998. 339: 520-532. https://doi.org/10.1056/NEJM199808203390806
  13. Misawa Y, Yoshida A, Saito R, Yoshida H, Okuzumi K, Ito N, et al. Application of loop-mediated isothermal amplification technique to rapid and direct detection of methicillin-resistant Staphylococcus aureus (MRSA) in blood cultures. J Infect Chemother. 2007. 13: 134-140. https://doi.org/10.1007/s10156-007-0508-9
  14. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acid Res. 2000. 28: e63. https://doi.org/10.1093/nar/28.12.e63
  15. Opota O, Croxatto A, Prod'hom G, Greub G. Blood culture-based diagnosis of bacteremia: state of the art. Clin Microbiol Infect. 2015. 21: 313-322. https://doi.org/10.1016/j.cmi.2015.01.003
  16. Shands KN, Schmid G, Dan BB, Blum D, Guidotti RJ, Hargrett T, et al. Toxic-shock syndrome in menstruating womenassociation with Tampon use and Staphylococcus aureus and clinical features in 52 cases. N Engl J Med. 1980. 303: 1436-1442. https://doi.org/10.1056/NEJM198012183032502
  17. Stamper PD, Cai M, Howard T, Speser S, Carroll KC. Clinical validation of the molecular BD geneohm staphsr assay for direct detection of Staphylococcus aureus and methicillinresistant Staphylococcus aureus in positive blood cultures. J Clin Microbiol. 2007. 45: 2191-2196. https://doi.org/10.1128/JCM.00552-07
  18. Su J, Liu X, Li Y, Chen D, Li Y, Yu G. Rapid and simple detection of methicillin-resistance Staphylococcus aureus by orfX loopmediated isothermal amplification assay. BMC Biotechnology. 2014. 14: 18. https://doi.org/10.1186/1472-6750-14-18
  19. Verroken A, Defourny L, Lechgar L, Magnette A, Delmee M, Glupczynski Y. Reducing time to identification of positive blood cultures with MALDI-TOF MS analysis after a 5-h subcultlure. Eur J Clin Microbiol Infect Dis. 2015. 34: 405-413. https://doi.org/10.1007/s10096-014-2242-4
  20. Wu SW, Lencastre H, Tomaz A. Recruitment of the mecA gene homologue of Staphylococcus sciuri into a resistance determinant and expression of the resistance phenotype in Staphylococcus aureus. J Bacteriol. 2001. 183: 2417-2424. https://doi.org/10.1128/JB.183.8.2417-2424.2001
  21. Xihong Z, Li Y, Park M, Wang J, Zhang Y, He X, et al. Loopmediated isothermal amplification assay targeting the femA gene for rapid detection of Staphylococcus aureus from clinical and food samples. J Microbiol Biotechnol. 2013. 23: 246-250. https://doi.org/10.4014/jmb.1207.07022
  22. Yamazaki W, Seto K, Taguchi M, Ishibashi M, Inoue K. Sensitive and rapid detection of cholera toxin-producing Vibrio cholerae using a loop-mediated isothermal amplification. BMC Microbiol. 2008. 8: 94. https://doi.org/10.1186/1471-2180-8-94