A Żaczek1, A Brzostek2, A Kuroń2, A Wojtasik2, A Sajduda3, J Dziadek4. 1. Department of Biochemistry and Cell Biology, University of Rzeszów, Rzeszów, Poland. 2. Institute of Medical Biology, Polish Academy of Science, Łódź, Poland. 3. Department of Microbial Genetics, Faculty of Biology and Environmental Protection, University of Łódź, Łódź, Poland. 4. Department of Biochemistry and Cell Biology, University of Rzeszów, Rzeszów, Poland; and Institute of Medical Biology, Polish Academy of Science, Łódź, Poland.
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. DESIGN: 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. CONCLUSION: FLAP proved highly discriminating, sensitive and specific and could be a valuable molecular tool, especially for analysing a limited number of M. tuberculosis strains.
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. DESIGN: 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. CONCLUSION: 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: B Molina-Moya; A Lacoma; N García-Sierra; S Blanco; L Haba; S Samper; J Ruiz-Manzano; C Prat; C Arnold; J Domínguez Journal: Sci Rep Date: 2017-07-28 Impact factor: 4.379