PCR restriction fragment length polymorphism analysis (PRA)-algorithm targeting 644 bp Heat Shock Protein 65 (hsp65) gene for differentiation of Mycobacterium spp.
A method based on PCR-restriction fragment length polymorphism analysis (PRA) using a novel region of the hsp65 gene was developed for the rapid and exact identification of mycobacteria to the species level. A 644 bp region of hsp65 in 62 mycobacteria reference strains, and 4 related bacterial strains were amplified, and the amplified DNAs were subsequently digested with restriction enzymes, namely, AvaII, HphI, and HpaII. Most of the mycobacteria species were easily differentiated at the species level by the developed method. In particular, the method enabled the separation of M. avium, M. intracellulare and M. tuberculosis to the species level by AvaII digestion alone. An algorithm was constructed based on the results and a blind test was successfully performed on 251 clinical isolates, which had been characterized by conventional biochemical testing. Our results suggest that this novel PRA offers a simple, rapid, and accurate method for the identification of mycobacteria culture isolates at the species level.
Kim
Hong
H
Department of Microbiology and Liver Research Institute, College of Medicine, Seoul National University, 28 Yongon-dong, Chongno-gu, Seoul 110-799, South Korea.
Kim
Sun-Hyun
SH
Shim
Tae-Sun
TS
Kim
Mi-na
MN
Bai
Gill-Han
GH
Park
Young-Gil
YG
Lee
Sueng-Hyun
SH
Cha
Chang-Yong
CY
Kook
Yoon-Hoh
YH
Kim
Bum-Joon
BJ
D000465:Algorithms; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D018834:Chaperonin 60; D018833:Chaperonins / Q000737:chemistry / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D006801:Humans; D009161:Mycobacterium / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D009164:Mycobacterium Infections / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D018834:Chaperonin 60; D004269:DNA, Bacterial; C074119:heat-shock protein 65, Mycobacterium; D018833:Chaperonins