[Analysis of PCR products from various mycobacteria using a primer pair for the nucleotide sequence of 65 kilodalton antigen].
Sequences of the DNA fragments amplified in PCR using a primer pair YNP-1 and YNP-2 and template DNAs from various mycobacteria were analysed with the kit Sequenase Ver. 2.0. The size of each PCR product was as follows; 164 bp for M. tuberculosis and M. bovis, 137 bp for M. kansasii, 109 bp for M. intracellulare, 136 bp for M. gordonae. Homology of the sequences to M. tuberculosis was 100% to M. bovis, 76% to M. kansasii, 64% to M. intracellulare, and 74% to M. gordonae. Only 2 of 12 strains belonging to M. avium were positive in PCR in this experiment. The sequence of these PCR products was 100% homologous to that of M. intracellulare. RFLP using Mbo I and BstE II was examined in each PCR product. Theoretically, it is expected that the product of M. tuberculosis complex is cut into 119 bp and 45 bp fragments by BstE II and 140 bp and 24 bp fragments by Mbo I; M. kansasii is cut by only Mbo I into 102 bp and 35 bp fragments; M. gordonae 92 bp and 44 bp fragments by BstE II and 112 bp and 24 bp fragments by Mbo I; Neither enzyme can cut the product of M. intracellulare. The results of the enzyme digestion were consistent with the expectation. Thus, it is suggested that the combination of PCR and RFLP would become a powerful tool for the detection and identification of mycobacteria from the clinical isolates within a short time as 48 hours.
Yamazaki
T
T
Department of Bacteriology, National Institute of Health, Tokyo, Japan.
Nakamura
R M
RM
D001483:Base Sequence; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D012689:Sequence Homology, Nucleic Acid