Identification of porcine intestinal spirochetes by PCR-restriction fragment length polymorphism analysis of ribosomal DNA encoding 23S rRNA.
The Brachyspira (formerly Serpulina) species rrl gene encoding 23S ribosomal RNA (rRNA) was used as a target for amplification of a 517bp DNA fragment by polymerase chain reaction (PCR). The primers for PCR amplification had sequences that were conserved among Brachyspira 23S rRNA gene and were designed from nucleotide sequences of Brachyspira hyodysenteriae, Serpulina intermedia, Brachyspira innocens and Brachyspira pilosicoli available from the GenBank database. Digestion of PCR-generated products from reference and field isolates of swine intestinal spirochetes with restriction enzymes Taq I and Alu I revealed five restriction fragment length polymorphism (RFLP) patterns. Each RFLP pattern corresponded to previously established genetic groups including B. hyodysenteriae (I), S. intermedia/B. innocens (II), Brachyspira murdochii (III), B. pilosicoli (IV) and B. alvinipulli (V). The 23S rRNA PCR/RFLP provided a relatively simple genotypic method for identification of porcine pathogenic B. hyodysenteriae and B. pilosicoli.
Barcellos
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DE
Instituto de Microbiologia 'Paulo de Góes', Universidade Federal do Rio de Janeiro, R.J., Rio de Janeiro, Brazil.
de Uzeda
M
M
Ikuta
N
N
Lunge
V R
VR
Fonseca
A S
AS
Kader
I I
II
Duhamel
G E
GE
D000818:Animals; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry; D004587:Electrophoresis, Agar Gel / Q000662:veterinary; D004591:Electrophoresis, Polyacrylamide Gel; D007410:Intestinal Diseases / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012150:Polymorphism, Restriction Fragment Length; D012335:RNA, Ribosomal / Q000737:chemistry*; D012338:RNA, Ribosomal, 23S / Q000737:chemistry / Q000235:genetics*; D016415:Sequence Alignment; D013143:Spirochaetaceae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D013145:Spirochaetales Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000382:microbiology
D017931:DNA Primers; D004269:DNA, Bacterial; D012335:RNA, Ribosomal; D012338:RNA, Ribosomal, 23S; C024669:endodeoxyribonuclease AluI; D015252:Deoxyribonucleases, Type II Site-Specific; C027046:TCGA-specific type II deoxyribonucleases