Browse > Article
http://dx.doi.org/10.15616/BSL.2019.25.1.75

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)
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.
Keywords
Blood culture; Methicillin-resistant Staphylococcus aureus; 16S rRNA; femA; mecA; Loop-mediated isothermal amplification;
Citations & Related Records
연도 인용수 순위
  • Reference
1 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.   DOI
2 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.   DOI
3 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.   DOI
4 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.   DOI
5 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.   DOI
6 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.   DOI
7 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.   DOI
8 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.   DOI
9 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.   DOI
10 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.   DOI
11 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.   DOI
12 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.   DOI
13 Hartman BJ, Tomasz A. Low-affinity penicillin-binding protein associated with beta-lactam resistance in Staphylococcus aureus. J Bacteriol. 1984. 158: 513-516.   DOI
14 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.   DOI
15 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.   DOI
16 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.   DOI
17 Lowy FD. Staphylococcus aureus infections. N Engl J Med. 1998. 339: 520-532.   DOI
18 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.   DOI
19 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.   DOI
20 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.   DOI
21 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.   DOI
22 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.   DOI