[논문]Detection of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp. and Staphylococcus aureus using duplex real-time PCR assay with melting curve analysis on fresh lettuce

Lee, Na-Ri; Kwon, Kyung-Yoon; Choi, Sung-Wook; Koo, Min-Seon; Chun, Hyang-Sook

Detection of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp. and Staphylococcus aureus using duplex real-time PCR assay with melting curve analysis on fresh lettuce 원문보기

한국식품위생안전성학회지 = Journal of food hygiene and safety, v.26 no.2, 2011년, pp.114 - 119

Lee, Na-Ri (Food Safety Research Division, Korea Food Research Institute) , Kwon, Kyung-Yoon (Food Safety Research Division, Korea Food Research Institute) , Choi, Sung-Wook (Food Safety Research Division, Korea Food Research Institute) , Koo, Min-Seon (Food Safety Research Division, Korea Food Research Institute) , Chun, Hyang-Sook (Food Safety Research Division, Korea Food Research Institute)

초록
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본 연구에서는 신선편의 채소와 과일에서 주로 검출되는 대장균(E. coli Ol577:H7), 리스테리아(L. monocytogenes), 살모넬라(Salmonella, spp.), 형색포도상구균(S. aureus)을 검출하고 동정할 수 있는 real time PCR 방법을 melting curve 분석을 활용하여 single tube 반응으로 두 종의 식중독균을 동시 검출하는 방법을 개발하고자 하였다. 대장균의 ${\beta}$-glucuronidase (uidA), 황색포도상구균의 thermonuclease (nuc), 리스테리아의 hemolycin (hly), 살모넬라의 tetrahionate reductase (ttr) 를 특이적으로 검출할 수 있는 4종의 프라이머 세트에 대한 real-time PCR의 melting curve 분석을 통하여 황색포도상구균과 대장균 동시분석 시 $T_m$ 값이 $80.6{\pm}0.9^{\circ}C$와 $86.9{\pm}0.5^{\circ}C$, 리스테리아와 살모넬라 동시분석 시 Tm 값이 $80.4{\pm}0.6^{\circ}C$와 $88.1{\pm}0.11^{\circ}C$로 확인하였고, 그 결과 정제되어진 각 식중독균의 genomic DNA를 주형으로 한 duplex real-time PCR 방법이 uniplex real-time PCR 방법과 마찬가지로 10 genome equivalents 까지 검출할 수 있는 민감도를 나타내었다. 또한, 양배추에 네 종의 식중독균을 접종하고, 증균배양 없이 DNA를 추출하여 duplex real-time PCR 을 수행한 결과 모든 식종목균에서 $10^3$ CFU/g의 검출 한계를 나타내었다. 결과적으로 개발된 melting curve 분석을 이용한 duplex real-time PCR 방법은 식품안전 증진을 위한 시간, 노동력, 비용 절감에 있어서 유효한 방법이 될 것으로 판단된다.

Abstract ▼ AI-Helper

In this study, two duplex real-time PCR approach with melting curve analysis is presented for the detection of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp. and Staphylococcus aureus, which are important food-borne bacterial pathogens usually present in fresh and/or minimally processed vegetables. Reaction conditions were adjusted for the simultaneous amplification and detection of specific fragments in the ${\beta}$-glucuronidase (uidA, E. coli), thermonuclease (nuc, S. aureus), hemolycin (hly, L. monocytogenes) and tetrathionate reductase (ttr, Salmonella spp.) genes. Melting curve analysis using a SYBR Green I real-time PCR approach showed characteristic $T_m$ values demonstrating the specific and efficient amplification of the four pathogens; $80.6{\pm}0.9^{\circ}C$, $86.9{\pm}0.5^{\circ}C$, $80.4{\pm}0.6^{\circ}C$ and $88.1{\pm}0.11^{\circ}C$ for S. aureus, E. coli O157:H7, L. monocytogenes and Salmonella spp., respectively. For all the pathogens, the two duplex, real-time PCR was equally sensitive to uniplex real-time PCR, using same amounts of purified DNA, and allowed detection of 10 genome equivalents. When our established duplex real-time PCR assay was applied to artificially inoculated fresh lettuce, the detection limit was $10^3$ CFU/g for each of these pathogens without enrichment. The results from this study showed that the developed duplex real-time PCR with melting curve analysis is promising as a rapid and cost-effective test method for improving food safety.

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문제 정의

The aim of this study was to develop a multiplex real-time PCR assay with melting curve analysis for the simultaneous detection of Escherichia coli O157:H7, Listeria monocytogenes. Salmonella spp.

제안 방법

In order to establish detection limits of the duplex Real-Time PCR, amplifications were carried out for each pathogen using both uniplex and the duplex reaction. To this end, 10-fbld serial dilutions of the reference type strains S.
13°C, respectively. In this study, to develop efficient multiplex PCR, reaction conditions were optimized using the SYBR Greenl. The real-time PCR methods are the simplest and least expensive method and enables products to be identified by their different Tm, by analysing the melting curve of the amplicon post-PCR besides detection of target DNA during amplification24).
4 μM of each primer, and 1 μl (50 ng) of template made up to 20 μl with filter sterilized water. Real-time PCR amplification was performed in a ABI 7500 (Applied Biosystems, Foster City, CA, USA) using the following conditions: 5 min at 95°C, followed by 40 cycles of 15 s at 95°C, 15 s at 62°C and 30 s at 72°C. The reaction carried out without DNA sample was used as negative control.
Tenfold dilutions of DNA from the four pathogens, covering five log orders, were used for duplex and uniplex real-time PCR assays in order to evaluate the efficiency of the duplex real-time PCR compared to the uniplex real-time PCR. The CT values are shown in Table 2.
The 103 CFU/g detection scored by the duplex SYBR Green real-time PCR used in this study, is at the same level that other uniplex TaqMan real-time PCR assays applied to food, without prior enrichment. Fu et al.

이론/모형

A real-time PCR assay was performed using the SYBR® Green Realtime PCR Master Mix (Toyobo, Tokyo, Japan). Each reaction contained 10 μ1 of SYBR® Green Realtime PCR Master Mix, 0.

성능/효과

Amplification products corresponding to the four pathogens were obtained in the reactions used as external amplification control thus confirming the absence of PCR inhibitors. Detection limit, estimated as the highest dilution from which amplification is obtained and derived from inoculation experiments, was established at 102 CFU/g for all four pathogens by uniplex real-time PCR, although CT values over 39 were obtained in S. aureus (Table 3).
These results indicate a small decrease in sensitivity when using the two sets of primers, at the lowest concentration tested. Nevertheless, our results demonstrate that amplification of the two loci is efficient in the presence of the other competing primers and the detection limit was kept 10 genome equivalents per reaction for all of them.
The real-time PCR methods are the simplest and least expensive method and enables products to be identified by their different Tm, by analysing the melting curve of the amplicon post-PCR besides detection of target DNA during amplification24). The presence of a single peak indicates the specificity of the reaction and when more than one amplicon is obtained, they can be distinguished by diteences of at least 1°C in Z^25, 26\ Both of duplex real-time PCR developed in this study yielded only two peaks showing clearly different Tm values for each pathogen without nonspecific amplification products and primer dimers: 80.6 ± 0.9 and 86.9 士 0, 5°C for S. aureus and E. coli O157:H7, and 80.4 노 0, 6 and 88.1 ±0.H°C for L monocytogenes, and Salmonella spp.

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Detection of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp. and Staphylococcus aureus using duplex real-time PCR assay with melting curve analysis on fresh lettuce 원문보기

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Detection of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp. and Staphylococcus aureus using duplex real-time PCR assay with melting curve analysis on fresh lettuce 원문보기

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