Fast ligation-mediated PCR, a fast and reliable method for IS6110-based typing of Mycobacterium tuberculosis complex.
IS6110 restriction fragment length polymorphism (RFLP) analysis is the most widely applied method for strain differentiation of Mycobacterium tuberculosis complex. We have previously described mixed-linker PCR, an IS6110-based PCR method that favorably compared with other typing methods for M. tuberculosis complex according to reproducibility and ability to differentiate between strains. Here we report the further development of this method, called fast ligation-mediated PCR (FLiP), which allows analysis of strains within one working day and starting from less than 1 ng of mycobacterial DNA or a crude cell lysate. Blinded analysis of a standard set of 131 M. tuberculosis complex and nontuberculous isolates showed the ability to differentiate 81 types among 90 M. tuberculosis complex isolates with 84 different IS6110 RFLP fingerprint patterns and detected 97% of the 31 duplicate samples. We suggest that FLiP can serve to rapidly detect chains of transmission prior to starting high-throughput analysis or standard IS6110 RFLP. It may as well serve as a secondary typing technique for other, non-IS6110-based methods.
Reisig
Florian
F
Children's Hospital, University of Heidelberg, Heidelberg, Germany.
Kremer
Kristin
K
Amthor
Beate
B
van Soolingen
Dick
D
Haas
Walter H
WH
D015373:Bacterial Typing Techniques; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D005784:Gene Amplification; D009169:Mycobacterium tuberculosis / Q000145:classification* / Q000235:genetics; D009841:Oligonucleotides / Q000096:biosynthesis; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D009841:Oligonucleotides