Detection of Vancomycin-resistant Enterococci using Multiplex Real-time PCR Assay and Melting Curve Analysis
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Background : We developed and evaluated the utility of a multiplex real-time PCR assay that uses melting curve analysis and allows simultaneous identification of vancomycin-resistant genotypes and clinically relevant enterococci. Methods : The specificity of the assay was tested using 4 reference strains of vancomycin-resistant enterococci (VRE) and 2 reference strains of vancomycin-susceptible enterococci. Ninety-three clinical isolates of enterococci with different glycopeptide-resistant phenotypes were genotyped and identified using a multiplex real-time PCR assay and melting curve analysis. Results : Representative melting curves were obtained for Enterococcus faecium, Enterococcus faecalis, vanA-containing E. faecium, vanB-containing E. faecalis, Enterococcus gallinarum, and Enterococcus casseliflavus. Phenotypic and genotypic analysis of the isolates revealed same results for 82 enterococcal isolates, while in 4 isolates, the glycopeptide-resistant phenotypes were inconsistent with the glycopeptide-resistant genotypes and in the 4 other isolates, species could not be accurately identified. Three isolates with mixed strains, which were detected by the PCR assay, could not be correctly identified using phenotypic methods. Conclusions : VRE genotyping and identification of clinically relevant enterococci were rapidly and correctly performed using multiplex real-time PCR assay and melting curve analysis. (Korean J Lab Med 2010;30:138-46)
Abstract
Background : We developed and evaluated the utility of a multiplex real-time PCR assay that uses melting curve analysis and allows simultaneous identification of vancomycin-resistant genotypes and clinically relevant enterococci. Methods : The specificity of the assay was tested using 4 reference strains of vancomycin-resistant enterococci (VRE) and 2 reference strains of vancomycin-susceptible enterococci. Ninety-three clinical isolates of enterococci with different glycopeptide-resistant phenotypes were genotyped and identified using a multiplex real-time PCR assay and melting curve analysis. Results : Representative melting curves were obtained for Enterococcus faecium, Enterococcus faecalis, vanA-containing E. faecium, vanB-containing E. faecalis, Enterococcus gallinarum, and Enterococcus casseliflavus. Phenotypic and genotypic analysis of the isolates revealed same results for 82 enterococcal isolates, while in 4 isolates, the glycopeptide-resistant phenotypes were inconsistent with the glycopeptide-resistant genotypes and in the 4 other isolates, species could not be accurately identified. Three isolates with mixed strains, which were detected by the PCR assay, could not be correctly identified using phenotypic methods. Conclusions : VRE genotyping and identification of clinically relevant enterococci were rapidly and correctly performed using multiplex real-time PCR assay and melting curve analysis. (Korean J Lab Med 2010;30:138-46)
참고문헌 (37)
- Leclercq R, "Plasmid-mediated resistance to vancomycin and teicoplanin in Enterococcus faecium", N Engl J Med, 319, pp.157~161 (1988)
- Willems RJ, "Global spread of vancomycin-resistant Enterococcus faecium from distinct nosocomial genetic complex", Emerg Infect Dis, 11, pp.821~828 (2005)
- 박지원, "Vancomycin 내성 enterococci에대한감수성검사", 감염, 24, pp.133~137 (1992)
- 이위교, "Vancomycin 내성 장구균의 분리율, 항균제 감수성 및 내성형에 관한 연구", 대한임상병리학회지, 18, pp.51~56 (1998)
- "Konis web-based reports & analysis program"[URL]
- Leven M, "Molecular methods for the detection of antibacterial resistance genes in Antibiotics in laboratory medicine. 5th ed.", pp.509~531 (2005), Lippincott Williams & Wilkins
- Clark NC, "Characterization of glycopeptide-resistant enterococci from U.S. hospitals", Antimicrob Agents Chemother, 37, pp.2311~2317 (1993)
- Dutka-Malen S, "Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR", J Clin Microbiol, 33, pp.24~27 (1995)
- Satake S, "Tenover FC. Detection of vancomycin-resistant enterococci in fecal samples by PCR", J Clin Microbiol, 35, pp.2325~2330 (1997)
- Kariyama R, "Simple and reliable multiplex PCR assay for surveillance isolates of vancomycin-resistant enterococci", J Clin Microbiol, 38, pp.3092~3095 (2000)
- Perez-Hernandez X, "Claverie-Martin F. A PCR assay for rapid detection of vancomycin-resistant enterococci", Diagn Microbiol Infect Dis, 42, pp.273~277 (2002)
- Palladino S, "Real-time PCR for the rapid detection of vanA and vanB genes", Diagn Microbiol Infect Dis, 45, pp.81~84 (2003)
- Palladino S, "Rapid detection of vanA and vanB genes directly from clinical specimens and enrichment broths by real-time multiplex PCR assay", J Clin Microbiol, 41, pp.2483~2486 (2003)
- Clinical and Laboratory Standards Institute, "Performance standards for antimicrobial susceptibility testing: nineteenth informational supplement" (2009), Clinical and Laboratory Standards Institute
- Rozen S, "Primer3 on the WWW for general users and for biologist programmers in Bioinformatics methods and protocols", pp.365~386 (2000), Humana Press
- Shipley GL, "An introduction to real-time PCR in Real-time PCR", pp.1~26 (2006), Taylor & Francis
- Ririe KM, "Product differentiation by analysis of DNA melting curves during the polymerase chain reaction", Anal Biochem, 245, pp.154~160 (1997)
- Wittwer CT, "Highresolution genotyping by amplicon melting analysis using LCGreen", Clin Chem, 49, pp.853~860 (2003)
- Henegariu O, "Multiplex PCR: critical parameters and step-by-step protocol", Biotechniques, 23, pp.504~511 (1997)
- Markoulatos P, "Multiplex polymerase chain reaction: a practical approach", J Clin Lab Anal, 16, pp.47~51 (2002)
- Wittwer CT, "Elenitoba-Johnson KS. Real-time multiplex PCR assays", Methods, 25, pp.430~442 (2001)
- Chang S, "Infection with vancomycin-resistant Staphylococcus aureus containing the vanA resistance gene", N Engl J Med, 348, pp.1342~1347 (2003)
- Boyd DA, "Molecular characterization of Enterococcus faecalis N06-0364 with low-level vancomycin resistance harboring a novel D-Ala-D-Ser gene cluster, vanL", Antimicrob Agents Chemother, 52, pp.2667~2672 (2008)
- 홍기숙, "Vancomycin 내성 Enterococci의 빈도 조사 및 중합효소연쇄반응을 이용한 유전자형의 분석", 대한임상병리학회지, 18, pp.372~378 (1998)
- 이수연, "Glycopeptide 내성장구균의항균제 감수성 검사법 비교: E-test, Vitek, 디스크 확산법 및 한천 희석법", 대한임상병리학회지, 20, pp.301~307 (2000)
- 박진희, "반코마이신 내성 장구균 장내 군집 및 감염의 위험인자 분석", 대한임상병리학회지, 20, pp.308~313 (2000)
- 권오건, "원주지역 3차 병원에서 분리된 vancomycin 내성 장구균의 분리 추이와 내성 유전자형", 대한임상병리학회지, 20, pp.486~493 (2000)
- Navarro F, "Analysis of genes encoding D-alanine-D-alanine ligase-related enzymes in Enterococcus casseliflavus and Enterococcus flavescens", Antimicrob Agents Chemother, 38, pp.1788~1793 (1994)
- Paule SM, "Comparison of PCR assay to culture for surveillance detection of vancomycin-resistant enterococci", J Clin Microbiol, 41, pp.4805~4807 (2003)
- Sloan LM, "Comparison of the Roche LightCycler vanA", vanB detection assay and culture for detection of vancomycin-resistant enterococci from perianal swabs, 2004, pp.42, 2636~2643
- Stamper PD, "Comparison of the BD GeneOhm VanR assay to culture for identification of vancomycin-resistant enterococci in rectal and stool specimens", J Clin Microbiol, 45, pp.3360~3365 (2007)
- Sahm DF, "Rapid charac terization schemes for surveillance isolates of vancomycin-resistant enterococci", J Clin Microbiol, 35, pp.2026~2030 (1997)
- Petrich A, "Effect of routine use of a multiplex PCR for detection of vanA- and vanB- mediated enterococcal resistance on accuracy, costs and earlier reporting", Diagn Microbiol Infect Dis, 41, pp.215~220 (2001)
- Drews SJ, "A 24-hour screening protocol for identification of vancomycin-resistant Enterococcus faecium", J Clin Microbiol, 44, pp.1578~1580 (2006)
- Angeletti S, "Routine molecular identification of enterococci by genespecific PCR and 16S ribosomal DNA sequencing", J Clin Microbiol, 39, pp.794~797 (2001)
- Song JH, "High frequency of vancomycin-resistant Enterococcus faecium isolates with VanB phenotype and vanA genotype in Korean hospitals", Diagn Microbiol Infect Dis, 56, pp.401~406 (2006)
- Park IJ, "VanB phenotype-vanA genotype Enterococcus faecium with heterogeneous expression of teicoplanin resistance", J Clin Microbiol, 46, pp.3091~3093 (2008)