title: Development of a new ligation-mediated PCR method for the differentiation of Mycobacterium tuberculosis strains.
abstract: BACKGROUND
  There is a need for rapid, inexpensive methods for analysing a limited number of Mycobacterium tuberculosis strains. The ligation-mediated polymerase chain reaction (LM-PCR) method appears to be sufficiently discriminative and reproducible to be considered as a molecular tool for the initial evaluation of hospital outbreaks, laboratory cross-contamination, and family or small community transmission.
  
  
  OBJECTIVE
  To develop a new LM-PCR method based on PCR amplification of the 5'-flanking region of insertion sequence (IS) 6110 consisting of SalI/PvuII digestion of chromosomal DNA, ligation of a SalI linker and differentiation of IS6110-carrying restriction fragments by suppression subtractive hybridisation.
  
  
  METHODS
  The fast ligation amplification polymorphism (FLAP) method was applied in the analysis of 62 M. tuberculosis clinical isolates and compared with IS6110-restriction fragment length polymorphism (RFLP) and mycobacterial interspersed repetitive units-variable number of tandem repeat (MIRU-VNTR) analyses of the same strains.
  
  
  RESULTS
  The sensitivity of FLAP was estimated at 0.25 ng/l. FLAP yielded 32 patterns among the 62 M. tuberculosis strains compared to respectively 28 and 36 patterns obtained using MIRU-VNTR and IS6110-RFLP. Its Hunter-Gaston discriminatory index value (0.973) is similar to that of MIRU-VNTR (0.966) and IS6110-RFLP (0.971). The specificity of the FLAP patterns was also confirmed.
  
  
  CONCLUSIONS
  FLAP proved highly discriminating, sensitive and specific and could be a valuable molecular tool, especially for analysing a limited number of M. tuberculosis strains.
authors.affiliation: Department of Biochemistry and Cell Biology, University of Rzeszów, Rzeszów, Poland.
authors.forename: A
authors.initials: A
authors.lastname: Żaczek
authors.affiliation: Institute of Medical Biology, Polish Academy of Science, Łódź, Poland.
authors.forename: A
authors.initials: A
authors.lastname: Brzostek
authors.affiliation: Institute of Medical Biology, Polish Academy of Science, Łódź, Poland.
authors.forename: A
authors.initials: A
authors.lastname: Kuroń
authors.affiliation: Institute of Medical Biology, Polish Academy of Science, Łódź, Poland.
authors.forename: A
authors.initials: A
authors.lastname: Wojtasik
authors.affiliation: Department of Microbial Genetics, Faculty of Biology and Environmental Protection, University of Łódź, Łódź, Poland.
authors.forename: A
authors.initials: A
authors.lastname: Sajduda
authors.affiliation: Department of Biochemistry and Cell Biology, University of Rzeszów, Rzeszów, Poland; and Institute of Medical Biology, Polish Academy of Science, Łódź, Poland.
authors.forename: J
authors.initials: J
authors.lastname: Dziadek
mesh_terms: D015373:Bacterial Typing Techniques; D004269:DNA, Bacterial / Q000235:genetics*; D005838:Genotype; D006801:Humans; D018598:Minisatellite Repeats; D009169:Mycobacterium tuberculosis / Q000145:classification / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D011237:Predictive Value of Tests; D015203:Reproducibility of Results; D013183:Sputum / Q000382:microbiology; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis / Q000382:microbiology*
chemical_list: D004269:DNA, Bacterial