Utility of modified multiple-locus variable-number tandemrepeat fingerprinting and repetitive-sequence-based PCR in comparison with pulsed-field gel electrophoresis for typing clinical isolates of Staphylococcus aureus
황색포도알균 임상 분리 균주 타이핑을 위한 변형 다중 유전자좌 일렬반복수변이 지문분석법 및 반복염기서열 기반 PCR의 유용성, 표준방법인 간헐영역겔전기이동과의 비교
- 의과대학 의학과
- Issue Date
- 서울대학교 대학원
- 학위논문 (석사)-- 서울대학교 대학원 : 의학과 검사의학 전공, 2013. 2. 김의종.
- Introduction: Multiple-locus variable-number tandem-repeat fingerprinting (MLVF) is based on multiplex PCR, utilizing variable number tandem repeat. Similarly, repetitive sequence-based PCR (rep-PCR) uses primers that target non-coding repetitive sequences interspersed in bacterial genomes. Our goal was to compare the performance of MLVF and rep-PCR in distinguishing clinical Staphylococcus aureus isolates with that of pulsed-field gel electrophoresis (PFGE), which has traditionally been the gold standard.
Methods: Sixty-three clinically significant S. aureus isolates were tested using MLVF, rep-PCR, and PFGE. Multiplex PCR for MLVF was performed using PCR primers for clfA, clfB, sdrCDE, sspA, and spa. Rep-PCR was performed using DiversiLab S. aureus kit for DNA fingerprinting. PFGE was performed with genomic DNA fragments generated by SmaI endonuclease digestion. Banding patterns of MLVF or PFGE were analyzed using InfoQuestFP software. The data obtained by rep-PCR was analyzed by internet-based DiversiLab software
Results: The hands-on time of our modified MLVF method and rep-PCR was about 3 h, on average, for each of 18 or 12 isolates. PFGE (80% cutoff) or MLVF (75% cutoff) or rep-PCR (95% cutoff) separated all of the 63 isolates into 13, 12, 12 types, respectively. Three types generated by PFGE were identical to those generated by MLVF. However, no identical types were found between PFGE and rep-PCR. Only two of the types clustered similar isolates between PFGE and rep-PCR. PFGE and MLVF yielded similar Simpsons diversity indices, indicating similar discriminatory power which is slightly higher than that of rep-PCR. The overall concordance between PFGE and MLVF was low, as represented by adjusted Rand indices (0.266‒0.278). PFGE predicted MLVF type better than MLVF predicted PFGE type, as reflected by Wallace coefficients (PFGE cutoff 80% vs. MLVF cutoff 75%, 0.389 vs. 0.233). Between PFGE and rep-PCR, similar result of the adjusted Rand indices (0.234–0.250) and Wallace coefficients (PFGE cutoff 80% vs. rep-PCR cutoff 95%, 0.628 vs. 0.156) were observed. Analysis of the relationship between a pair of isolates showed 91.0% concordance between the PFGE (80% cutoff) and MLVF (75% cutoff).
Conclusions: Our simple, low-cost, modified MLVF protocol can effectively discriminate between S. aureus clinical isolates. MLVF can replace PFGE for the hospital infection control of S. aureus.