갯벌 퇴적물내 병원성 Vibrio vulnificus의 신속하고 특이적인 검출 KCI

Title
갯벌 퇴적물내 병원성 Vibrio vulnificus의 신속하고 특이적인 검출
Alternative Title
Rapid and Specific Detection of Virulent V. vulnificus in Tidal Flat Sediments
Author(s)
변기득; 이정현; 이계준; 김상진
KIOST Author(s)
Lee, Jung Hyun(이정현)
Publication Year
2005-09
Abstract
갯벌 퇴적물에 존재하는 병원성 해양미생물인 Vibrio vulnificus를 신속하고 정확하게 검출하기 위해 PCR, Southern hybridization 방법과 real-time PCR을 수행하여 검출 민감도를 비교하였다. 갯벌 퇴적물로부터 bead beater를 이용한 물리적 방법으로 DNA 조추출액을 얻고 상용화된 키트 (Geneclean turbo Kit)를 이용하여 부식물 질(humic substances)을 제거하였다. 병원성에 관련된 3 종의 유전자(hemolysin, vvhA; phosphomannomutase, pmm; metalloprotease, vvpE)를 대상으로 설계한 프라이머 셋을 동시에 사용하는 multiplex PCR 방법과 Southern hybridization과 병행한 방법(PCR/Southern hybridization)을 수행하였다. Real-time PCR은 hemolysin 유전자 (vvhA)에 특이한 프라이머와 TaqMan 탐침을 사용하였다. 전처리하지 않은 갯벌 퇴적물의 경우, PCR/Southern hybridization과 real-time PCR 방법의 검출 민감도는 퇴적물 1 g 당 약 102 개의 세포 수준이었다. 농후처리액 (APW; alkaline peptone water)으로 35oC에서 2~3시간, 8시간 증균 배양할 경우 갯벌 퇴적물 1 g당 2~10개 세포가 존재할때 PCR/Southern hybridization 방법과 real-time PCR 방법으로 각각 검출할 수 있었다. 전처리 과정을 포 함하여 real-time PCR은 6~7시간, PCR/Southern hybridization은 약 36시간이 소요되었다.

Vibrio vulnificus, one of the marine bacterial pathogens causing septicemia, was detected using molecular methods,
namely, PCR and/or Southern hybridization, and real-time PCR. Extracted and purified total DNAs by using commercial kits were used as templates for PCR. Multiplex-PCR was conducted by employing three sets of primers for the genes, hemolysin (vvhA), phosphomannomutase (pmm), and metalloprotease (vvpE), for V. vulnificus virulence. The presence of DMSO (5%) and BSA (0.1%) in PCR reaction mixture improved a detection
efficiency by higher PCR band intensities. TaqMan real-time PCR was carried out by using gene segment of vvhA as a target. Detection limit of PCR/Southern hybridization without enrichments was to be around 102cells g-1 of sample. However, those three methods using the enrichment at 35oC in APW showed high sensitivity (2~10 cells g-1 of sediments). Highly sensitive detection of V. vulnificus by real-time PCR was achieved within 5~6 hr, whereas the detection by PCR/Southern hybridization required about 36 hr. Thus, it was evident that real-time PCR is the most rapid and efficient method for detecting V. vulnificus in tidal flat sediments.
ISSN
0440-2413
URI
https://sciwatch.kiost.ac.kr/handle/2020.kiost/5025
Bibliographic Citation
미생물학회지, v.41, no.3, pp.168 - 176, 2005
Publisher
한국미생물학회
Keywords
real-time TaqMan PCR; tidal flat sediment; Vibrio vulnificus
Type
Article
Publisher
한국미생물학회
Related Researcher
Research Interests

marine biotechnology,molecular microbiology,해양생명공학,분자미생물학

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