title: Polymerase chain reaction-restriction fragment length polymorphism of the rpoB gene for identification of Mycobacterium avium subsp. paratuberculosis and differentiation of Mycobacterium avium subspecies.
abstract: Mycobacterial speciation by polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis (PRA) of the rpoB gene was evaluated for identification of Mycobacterium avium subsp. paratuberculosis (MAP) and other Mycobacterium avium complex (MAC) members to the species or subspecies level by comparison with conventional methods including hsp65 sequencing, high-performance liquid chromatography, and PCR for accepted species- or subspecies-specific genomic targets. A total of 185 type and clinical mycobacterial strains from humans, animals, and environments were tested. A 360-bp PCR product was subsequently digested with MspI, HaeIII, and SmaI restriction enzymes. The PRA using SmaI restriction showed a unique digestion pattern for MAP distinguishing it from other MAC members and other Mycobacterium spp. Moreover, HaeIII and MspI restriction of the rpoB gene enabled MAC-species and -subspecies discrimination. The rpoB-PRA using SmaI or MspI and HaeIII restriction of the rpoB gene is a simple, convenient, and reliable confirmatory assay for simultaneous identification of MAP and other MAC members.
authors.affiliation: Department of Microbiology and Research Institute for Medical Sciences, College of Medicine, Chungnam National University, Daejeon 301-747, South Korea.
authors.forename: Jake
authors.initials: J
authors.lastname: Whang
authors.forename: Byung Soo
authors.initials: BS
authors.lastname: Lee
authors.forename: Go-Eun
authors.initials: GE
authors.lastname: Choi
authors.forename: Sang-Nae
authors.initials: SN
authors.lastname: Cho
authors.forename: Park Young
authors.initials: PY
authors.lastname: Kil
authors.forename: Michael T
authors.initials: MT
authors.lastname: Collins
authors.forename: Sung Jae
authors.initials: SJ
authors.lastname: Shin
mesh_terms: D000818:Animals; D001431:Bacteriological Techniques / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D019003:Deoxyribonuclease HpaII / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004783:Environmental Microbiology; D006801:Humans; D015269:Mycobacterium avium Complex / Q000145:classification* / Q000235:genetics*; D015270:Mycobacterium avium-intracellulare Infection / Q000382:microbiology / Q000662:veterinary; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length
chemical_list: D004269:DNA, Bacterial; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D019003:Deoxyribonuclease HpaII; C025651:CCCGGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; C024403:GGCC-specific type II deoxyribonucleases