PCR detection of Clostridium chauvoei in pure cultures and in formalin-fixed, paraffin-embedded tissues.
The polymerase chain reaction (PCR) was used to amplify specific segments of the 16S ribosomal RNA gene of Clostridium chauvoei, a major pathogen of ruminants. Three sets of primers were used to produce amplicons of 159, 836 and 959 base pairs (bp), respectively. The PCR was evaluated by testing clinically important strains of Clostridium, including 21 strains of C. chauvoei, five strains each of Clostridium septicum and Clostridium perfringens and two strains each of Clostridium novyi, Clostridium histolyticum and Clostridium sordellii. Both purified DNA and biomass from pure cultures of each of these microorganisms were evaluated as templates in the PCR. In addition, extracts of formalin-fixed, paraffin-embedded tissues of eight sheep experimentally inoculated with C. chauvoei or C. septicum (four animals each) were also tested by the PCR using the three sets of primers. Purified DNA template of all C. chauvoei strains produced PCR amplicons of the expected size for all three primer pairs. However, when biomass from pure cultures of C. chauvoei or tissue extracts were used as templates, only the primer pair designed to produce the 159bp amplicon gave consistently positive results. No positive results were obtained with any primer pair when purified DNA or biomass from pure cultures of non-target clostridial species were used as templates. Therefore, the PCR primer sets appear to be very specific for identifying C. chauvoei in both cultures and tissues.
Uzal
F A
FA
Instituto Nacional de Tecnologi;a Agropecuaria (INTA), CC 277 (8400), Bariloche, Argentina. fuzal@cahfs.ucdavis.edu
Hugenholtz
P
P
Blackall
L L
LL
Petray
S
S
Moss
S
S
Assis
R A
RA
Fernandez Miyakawa
M
M
Carloni
G
G
D000818:Animals; D001483:Base Sequence; D018533:Biomass; D003013:Clostridium / Q000235:genetics / Q000302:isolation & purification*; D003015:Clostridium Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D009132:Muscles / Q000382:microbiology; D016612:Paraffin Embedding / Q000662:veterinary; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis / Q000382:microbiology*; D016707:Tissue Fixation / Q000662:veterinary
D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S