Development of a RAPD-PCR method for identification of Bacillus species isolated from Cheonggukjang.
A RAPD-PCR (Randomly Amplified Polymorphic DNA-PCR) method was developed for rapid identification of Bacillus species, especially B. subtilis, B. licheniformis, and B. amyloliquefaciens, the most frequently isolated organisms from fermented soy foods such as Cheonggukjang, a Korean traditional food. A RAPD-PCR using a 10-mer (S-30) produced species specific bands reproducibly. All B. subtilis strains tested produced common bands of 0.5 and 0.88 kb in size. All B. amyloliquefaciens strains generated 1.1 and 1.5 kb bands together with 0.5 kb fragment whereas B. licheniformis strains produced 1.25, 1.70, and 1.9 kb bands with an occasional 0.5 kb band. Using the RAPD-PCR protocol, six bacilli strains isolated from Cheonggukjang were identified to the species level, which was difficult by 16S rRNA gene and recA gene sequencing for some isolates. The 0.5 kb fragment, the major band for B. subtilis strains, was an internal part of a ytcP gene encoding a hypothetical ABC-type transporter. A B. subtilis species specific primer pair was designed based on ytcP sequences and PCR using the primer pair produced a 0.46 kb fragment only from B. subtilis strains.
Kwon
Gun-Hee
GH
Division of Applied Life Science (BK21 Program), Graduate School, Gyeongsang National University, Jinju 660-701, Republic of Korea.
Lee
Hwang-A
HA
Park
Jae-Young
JY
Kim
Jong Sang
JS
Lim
Jinkyu
J
Park
Cheon-Seok
CS
Kwon
Dae Young
DY
Kim
Yong-Suk
YS
Kim
Jeong Hwan
JH
D001407:Bacillus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D001426:Bacterial Proteins; D001483:Base Sequence; D005285:Fermentation; D005798:Genes, Bacterial; D007723:Korea; D019105:Random Amplified Polymorphic DNA Technique / Q000379:methods*; D011938:Rec A Recombinases / Q000235:genetics; D013025:Glycine max / Q000382:microbiology*
D001426:Bacterial Proteins; D011938:Rec A Recombinases