[논문]비브리오 콜레라 신속 검출을 위한 펩티드 핵산 기반 비대칭 real-time PCR 방법의 적용

강민경; 이택견

doi:10.5762/kais.2019.20.12.117

비브리오 콜레라 신속 검출을 위한 펩티드 핵산 기반 비대칭 real-time PCR 방법의 적용
Application of a Peptide Nucleic Acid-Based Asymmetric Real-Time PCR Method for Rapid Detection of Vibrio cholerae 원문보기

한국산학기술학회논문지 = Journal of the Korea Academia-Industrial cooperation Society, v.20 no.12, 2019년, pp.117 - 124

강민경 (한국해양과학기술원 생태위해성 연구부) , 이택견 (한국해양과학기술원 생태위해성 연구부)

초록
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비브리오 콜레라는 수산물과 선박평형수 내에서 모니터링되고 있는 중요 병원성 박테리아이다. 이를 검출하기 위한 여러 방법들이 개발되어 왔으나, 시간 소모가 크고 민감도에서 한계가 있었다. 본 연구는 비브리오 콜레라를 보다 정확하게 검출하기 위한 방법을 개발하는 목적으로 수행하였다. PNA 기반 비대칭 real-time PCR 기술에 적용하기 위하여 펩티드 핵산(Peptide nucleic acid, PNA) 프로브를 개발하였다. 독성 유전자인 Cholera enterotoxin subunit B (ctxB)를 비브리오 콜레라 검출을 위한 타겟 유전자로 선정하고, conventional PCR과 real-time PCR을 위한 positive template를 합성하였다. Real-time PCR 프라이머와 PNA 프로브를 디자인하여, 정량 분석을 위한 표준곡선 실험을 수행하였다. 선택된 PNA 프로브는 비브리오 콜레라에 특이적으로 반응하였으며, 검출한계는 0.1 cfu/100 mL이었다. 종합해 보면, 본 연구에서 개발된 PNA 프로브와 비대칭 real-time PCR 방법은 수산물과 선박평형수 뿐만 아니라 해양환경에 있는 비브리오 콜레라를 신속하고 정확하게 모니터링할 수 있는 기술로 판단된다.

Abstract ▼ AI-Helper

Vibrio cholerae is a very important pathogenic bacterium that has to be monitored in seafood and ships' ballast water. Various methods have been developed to identify this bacterium, yet these methods are time-consuming and have limitations for their sensitivity to detect contamination. The purpose of the present study was to develop a robust and reliable method for identifying V. cholerae. Peptide nucleic acid (PNA) probes were developed to use for PNA-based asymmetrical real-time PCR techniques. The toxigenic Cholera enterotoxin subunit B (ctxB) gene was selected as a target for detecting V. cholerae and the gene was synthesized as a positive template for conventional and real-time PCR. Real-time PCR primers and PNA probes were designed and standard curves were produced for the quantitative analysis. The selected PNA probes reacted specifically to V. cholerae without any ambiguity, even among closely related species, and the detection limit was 0.1 cfu/100 mL. Taken together, the PNA probes and asymmetrical qPCR methods developed in this present study could contribute to the rapid, accurate monitoring of V. cholerae in marine environments, and as well as in seafood and ships' ballast waters.

주제어

표/그림 (6)

표 Table 1. Synthesized V. cholerae ctxB gene fragment.
그림 Fig. 1. Real-time PCR and standard curve for V. cholera ctxB gene.
표 Table 2. Cross reactivity with various Vibrio species.
표 Table 3. The nucleotide sequences of the PNA probes for the detection of ctxB genes of V. cholerae.
그림 Fig. 2. Melting peak for Tm values of PNA1, PNA2, and PNA3 at each temperature.
그림 Fig. 3. Standard curve for quantitative analysis of V. cholerae using PNA probes.

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문제 정의

cholerae. The objective of the present work was to develop highly specific and sensitive molecular methods based on PNA for the rapid identification and monitoring of V.cholerae in marine products and in ballast water. Using the synthesized V.
1cfu/100 mL, which is more sensitive thannon-PNA probes. This study provided useful diagnostic method for the detection of V.Cholerae in seafoods and ship ballast water.

제안 방법

Although Vibrio cholerae is a critical pathogenic bacterium, diagnostic techniques are time-consuming and have limitations insensitivity. In this study, a detection method was developed for the identification of V. cholerae with PNA-based asymmetrical real-time PCR. We selected the ctxB gene as the target for the detection and designed the real-time PCR primers and PNA probes containing a fluorescent reporter dye.
The Tm values of PNA1,PNA2, and PNA3 were 53°C, 70.2°C, and 63.2°C,respectively, and subsequent experiments, such as detection limit analysis, were performed using PNA2 with the highest Tm value.
The detection limit of real-time PCR of the V.cholerae ctxB gene-specific PNA probes was analyzed. The limit of detection was determined using the ctxB PNA2 probe, which shows the highest Tm value among the selected probes.
We selected the ctxB gene as the target for the detection and designed the real-time PCR primers and PNA probes containing a fluorescent reporter dye. Three different PNAs with different Tm values were designed for asymmetrical PCR reactions, and performed using PNA2 with the highest Tm value. The selected PNA probes detected specifically to V.
The detection limit of the ctxB gene obtained from the real-time PCR reaction and standard curves was 1×10² copies/mL. To confirm that the selected primers reacted specifically with toxic V.cholerae, cross-reactivity analysis with other species (V. xuii, V. sinaloensis, V. litoralis, V.zureus, and V natriegens) was performed. PCR results show that the primers for the selected ctxB gene did not react with other Vibrio species, only with V.
To the best of our knowledge, this study is the first application of the PNA-based asymmetrical real-time PCR method for the detection of V.cholerae. The PNA-based qPCR method proposed in the present study significantly reduced the detection time as compared with culture-based methods and improved sensitivity as compared with previous gene-based detection methods.

이론/모형

Melting curve analysis using the selected primer sets and PNA probes was performed by MyGo mini real-time PCR (IT-IS Life Science Ltd,Ireland). Asymmetrical PCR was performed using primer sets and PNA probes to generate single-stranded target nucleic acids.

성능/효과

We designed various primers for conventional andreal-time PCR, selected optimal primer sets by evaluating product size and reaction intensity,and finally designed PNA probes containing a fluorescent reporter dye (Table 3). The optimal reaction conditions for asymmetrical real-time PCR with PNA probes were established and the detection limits were calculated as 0.1 cfu/100mL when applied to the standard curve, which is consistent with seafoods and the detection criteria for ballast water (Fig. 3). The detection limits for multiplex PCR using the ctxA, chxA,and vopF genes [10] and for the LAMP assay using the ctxA gene [8] were 10 cfu/PCR tube and 10 cfu/mL, respectively.
The nucleotide sequence of the ctxB gene was selected as 536bp, and synthesis by Bioneer, South Korea was requested (Table 1). To confirm whether the synthesized gene was inserted properly into the pBHA vector, the nucleotide sequence obtained from EcoRI restriction enzyme digestion and sequencing was compared with the synthesized nucleotide sequence. The synthesized ctxB gene was used as a template for the design of primersand PNA probes for the detection of V.

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