One-tube cell lysis and DNA extraction procedure for PCR-based detection of Mycobacterium ulcerans in aquatic insects, molluscs and fish.
The purpose of this study was to develop a simple procedure for cell lysis and DNA extraction for direct detection of Mycobacterium ulcerans in aquatic insects, gills and intestinal contents of fish, molluscs and human tissue samples using a nested PCR method specific for the insertion sequence IS2404. The simultaneous action of sodium N-lauroyl sarcosine, guanidinium isothiocyanate, chloroform and Tris-saturated phenol on mycobacteria, followed by a DNA purification method using mini-columns fitted with silica-cellulose membranes was successfully employed to extract DNA from cultured bacteria, environmental and human tissue samples. All specimens were collected from Buruli ulcer endemic regions. M. ulcerans DNA was detected in 11 of 57 aquatic insects, one of six molluscs and three of 15 fish, supporting the hypothesis that the fauna of major Buruli ulcer endemic foci in swampy terrain of tropical and subtropical regions can be a source of M. ulcerans infection.
Prince Leopold Institute of Tropical Medicine, Mycobacteriology Unit, Nationalestraat 155, B-2000 Antwerpen, Belgium.
Roman
R
Kotlowski
Prince Leopold Institute of Tropical Medicine, Mycobacteriology Unit, Nationalestraat 155, B-2000 Antwerpen, Belgium.
Anandi
A
Martin
Prince Leopold Institute of Tropical Medicine, Mycobacteriology Unit, Nationalestraat 155, B-2000 Antwerpen, Belgium.
Anthony
A
Ablordey
Prince Leopold Institute of Tropical Medicine, Mycobacteriology Unit, Nationalestraat 155, B-2000 Antwerpen, Belgium.
Karim
K
Chemlal
Prince Leopold Institute of Tropical Medicine, Mycobacteriology Unit, Nationalestraat 155, B-2000 Antwerpen, Belgium.
Pierre-Alain
PA
Fonteyne
Prince Leopold Institute of Tropical Medicine, Mycobacteriology Unit, Nationalestraat 155, B-2000 Antwerpen, Belgium.
Françoise
F
Portaels
D000818:Animals; D001433:Bacteriolysis; D004251:DNA Transposable Elements / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification*; D005393:Fish Diseases / Q000382:microbiology; D005399:Fishes / Q000382:microbiology; D005880:Gills / Q000382:microbiology; D006801:Humans; D007313:Insecta / Q000382:microbiology; D007422:Intestines / Q000382:microbiology; D008974:Mollusca / Q000382:microbiology; D009165:Mycobacterium Infections, Nontuberculous / Q000382:microbiology*; D019911:Mycobacterium ulcerans / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D016133:Polymerase Chain Reaction / Q000379:methods*
D004251:DNA Transposable Elements; D004269:DNA, Bacterial