갯벌 퇴적물내 병원성 Vibrio vulnificus의 신속하고 특이적인 검출
KCI
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Title
- 갯벌 퇴적물내 병원성 Vibrio vulnificus의 신속하고 특이적인 검출
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Alternative Title
- Rapid and Specific Detection of Virulent V. vulnificus in Tidal Flat Sediments
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Author(s)
- 변기득; 이정현; 이계준; 김상진
- KIOST Author(s)
- Lee, Jung Hyun(이정현)
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Alternative Author(s)
- 이정현; 김상진
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Publication Year
- 2005-09
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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.
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ISSN
- 0440-2413
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URI
- https://sciwatch.kiost.ac.kr/handle/2020.kiost/5025
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Bibliographic Citation
- 미생물학회지, v.41, no.3, pp.168 - 176, 2005
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Publisher
- 한국미생물학회
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Keywords
- real-time TaqMan PCR; tidal flat sediment; Vibrio vulnificus
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Type
- Article
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