Rapid species identification within the Mycobacterium chelonae-abscessus group by high-resolution melting analysis of hsp65 PCR products.
Polymerase chain reaction (PCR) amplification of the heat shock protein 65 (hsp65) gene followed by high-resolution melting analysis with LCGreen I (Idaho Technology, Salt Lake City, UT) was used to differentiate the mycobacteria species Mycobacterium chelonae, Mycobacterium abscessus, and Mycobacterium immunogenum in less than 20 minutes. A 105-base-pair amplicon that clustered the different species by predicted melting temperature was found from available GenBank hsp65 sequences. We identified 24 clinical isolates within the M chelonae-abscessus group by proximal 16S ribosomal RNA and hsp65 gene sequencing. Rapid-cycle PCR followed by high-resolution melting analysis clustered these samples into the following groups: M abscessus, 12; M abscessus sequence variant, 2; M chelonae, 7; unexpected M chelonae sequence variant, 1; and M immunogenum, 2. The M chelonae variant had a single base change not found in reported GenBank sequences. Advantages of the method include speed, low risk of amplicon contamination (closed-tube), and no need for separation steps (sequencing, electrophoresis, high-performance liquid chromatography) or real-time monitoring.
Odell
Ian D
ID
Department of Pathology, University of Utah Medical School, Salt Lake City, 84132, USA.
Cloud
Joann L
JL
Seipp
Michael
M
Wittwer
Carl T
CT
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D018834:Chaperonin 60; D018833:Chaperonins / Q000235:genetics*; D008969:Molecular Sequence Data; D016926:Mycobacterium chelonae / Q000145:classification* / Q000235:genetics; D009170:Nontuberculous Mycobacteria / Q000145:classification* / Q000235:genetics; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*
D001426:Bacterial Proteins; D018834:Chaperonin 60; C074119:heat-shock protein 65, Mycobacterium; D018833:Chaperonins