• Journal of Microbiology and Biotechnology
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• 제20권3호
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• pp.569-573
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• 2010

A quantitative detection method for Salmonella in seafood was developed using a SYBR Green-based real-time PCR assay. The assay was developed using pure Salmonella DNA at different dilution levels [i.e., 1,000 to 2 genome equivalents (GE)]. The sensitivity of the real-time assay for Salmonella in seeded seafood samples was determined, and the minimum detection level was 20 CFU/g, whereas a detection level of 2 CFU/ml was obtained for pure culture in water with an efficiency of ${\geq}85%$. The real-time assay was evaluated in repeated experiments with seeded seafood samples and the regression coefficient ($R^2$) values were calculated. The performance of the real-time assay was further assessed with naturally contaminated seafood samples, where 4 out of 9 seafood samples tested positive for Salmonella and harbored cells <100 GE/g, which were not detected by direct plating on Salmonella Chromagar media. Thus, the method developed here will be useful for the rapid quantification of Salmonella in seafood, as the assay can be completed within 2-3 h. In addition, with the ability to detect a low number of Salmonella cells in seafood, this proposed method can be used to generate quantitative data on Salmonella in seafood, facilitating the implementation of control measures for Salmonella contamination in seafood at harvest and post-harvest levels.

• Genomics & Informatics
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• 제19권3호
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• pp.30.1-30.8
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• 2021

Salmonella species are among the major pathogens that cause foodborne illness outbreaks. In this study, we aimed to develop a loop-mediated isothermal amplification (LAMP) assay for the rapid and sensitive detection of Salmonella species. We designed LAMP primers targeting the hilA gene as a universal marker of Salmonella species. A total of seven Salmonella species strains and 11 non-Salmonella pathogen strains from eight different genera were used in this study. All Salmonella strains showed positive amplification signals with the Salmonella LAMP assay; however, there was no non-specific amplification signal for the non-Salmonella strains. The detection limit was 100 femtograms (20 copies per reaction), which was ~1,000 times more sensitive than the detection limits of the conventional polymerase chain reaction (PCR) assay (100 pg). The reaction time for a positive amplification signal was less than 20 minutes, which was less than one-third the time taken while using conventional PCR. In conclusion, our Salmonella LAMP assay accurately detected Salmonella species with a higher degree of sensitivity and greater rapidity than the conventional PCR assay, and it may be suitable for point-of-care testing in the field.

• 대한의생명과학회지
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• 제14권2호
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• pp.115-118
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• 2008

Salmonella typhi is frequent causes of foodborne illness and its detection is important for monitoring disease progression. In this study, by using general PCR and novel LAMP (Loop Mediated Isothermal Amplification) assay, we evaluated the usefulness of LAMP assay for detection of Salmonella typhi. In this LAMP assay, forward inner primer (FIP) and back inner primer (BIP) was specially designed for recognizing target invA gene. Target DNA was amplified and visualized as ladder-like pattern of bands on agarose gel within 60 min under isothermal conditions at $65^{\circ}C$. When the sensitivity and reproducibility of LAMP were compared to general PCR, there was no difference of reproducibility but sensitivity of LAMP assay was more efficient than PCR (the detection limit of LAMP assay was 30 fg, while the PCR assay was 3 pg). These results indicate that the LAMP assay is a potential and valuable means for detection of Salmonella typhi, especially for its rapidity, simplicity and low cost.

• 한국식품위생안전성학회지
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• 제34권3호
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• pp.290-295
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• 2019

최근 한국에서 발생한 Salmonella로 인한 식중독 사고는 2018년 9월 학교급식에서 제공된 초콜릿 무스 케이크가 원인이 되었다. 이 연구의 목적은 Salmonella Typhimurium이 인위적으로 접종된 무스케이크와 티라미수에서 3M Molecular Detection Assay 2 - Salmonella와 식품공전에 등재된 방법인 분리배지와 real-time PCR을 비교하는 것이었다. 무스케이크 2종과 티라미수 2종 25 g에 225 mL BPW를 넣고 $37^{\circ}C$에서 24시간 동안 증균 배양하였다. 배양 후, 3M Molecular Detection Assay 2 - Salmonella, 분리배지 그리고 real-time PCR로 분석하였다. 초콜릿 무스 케이크를 제외하고 3가지 방법은 유사한 결과를 보였다. 초콜릿 무스 케이크에서 분리배지와 3M Molecular Detection Assay 2 - Salmonella는 모든 접종수준에서 동일한 결과를 나타낸 반면 real-time PCR은 $10^4CFU/25g$ 수준에서 1번의 양성결과를 제외하고 모두 검출되지 않았다. 초콜릿 무스에 S. Typhimurium을 $10^2CFU/25g$ 수준으로 접종하였을때, real-time PCR를 이용한 검출은 15%에서는 부분적인 음성을 나타냈고, 20-100% 함량의 초콜릿 무스에서는 모두 음성이었다. Real-time PCR로는 chocolate이 15% 이상 함유된 식품에서의 Salmonella균 검출이 불가능하였지만, LMAP 기반의 3M Molecular Detection Assay 2으로는 chocolate 농도에 관계없이 검출이 가능하였다.

• 한국축산식품학회지
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• 제24권2호
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• pp.176-181
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• 2004

우유내 Salmonella를 검출하기 위한 Sandwich ELISA의 특이성을 조사하였다. Sandwich ELISA에 사용한 항체들은 OMP 분획을 닭에 면역주사하여 얻은 IgY와 OMP 분획을 gel filtration하여 얻은 분자량 40,000의 OMP를 토끼에 면역 주사하여 얻은 토끼 IgG를 사용하였다. Immnoblot assay에서 IgY는 분자량 6,000의 OMP에 강하게 반응하였으며 토끼 IgG는 분자량 40,000, 35,000과 6,000의 OMP들에 강하게 반응하였다. IgY와 토끼 IgG는 Salmonella typhimurium의 다른 단백질에도 반응하였다. Competitive ELISA에서 IgY가 Salmonella의 두 개 균주에 대해 특이성을 나타냈으며 Eshcherchia coli와 Yersinia enterocolitica에 의하여서는 반응을 나타내지 않았다. 우유에 세균을 첨가하여 실시한 sandwich ELISA에서 Salmonella typhimurium 2균주가 가장 높은 흡광도를 보였다. Salmonella cholerasuis 균주들은 상대적으로 흡광도가 낮았으며 이루 일부 Salmonella cholerasuis 균주들은 비 Salmonella 균주들과 차이가 없었다.

• 한국축산식품학회지
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• 제33권5호
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• pp.655-663
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• 2013

본 연구에서는 Salmonella invA 유전자를 마커로 Salmonella 특이 LAMP primer를 제작하였고, inclusivity 및 exclusivity 실험 결과는 각각 100%로 관찰되었다. 순수 배양된 Salmonella 희석액에서의 검출한계는 18.17분에서 $3.2{\times}10^3$ CFU/mL ($R^2$ = 0.9446)으로 관찰되어 신속 간단하고 민감도가 높아 Salmonella 검출을 위한 방법으로 효과적일 뿐만 아니라 간접적인 정량기법으로서도 활용 가능함을 알 수 있었다. 1차 증균 배지인 BPW에 S. Enteritidis 접종 후 0시간 및 12시간 배양 후 LAMP의 검출한계는 각각 $3.2{\times}10^3$ CFU/mL 및 $3.2{\times}10^0$ CFU/mL으로 관찰되었고, 오리도체 시료가 포함된 BPW에 S. Enteritidis 접종 후 0시간 및 12시간 증균액에서의 LAMP 검출한계는 각각 $3.2{\times}10^3$ CFU/mL 및 $3.2{\times}10^0$ CFU/mL으로 관찰되어 PCR과 비교하였을 때 오리도체시료 성분에 의한 영향이 비교적 낮았음을 알 수 있었다. 또한, 실제 도압장에서 채취한 오리도체시료를 BPW에 6시간 배양한 1차 증균액에 개발한 LAMP기법을 적용한 결과, 96%의 민감도 및 84%의 특이도를 보여 오리도체에서의 Salmonella 신속 스크리닝 기법으로서 효과적으로 활용될 수 있음을 알 수 있었다.

• 대한수의학회지
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• 제54권2호
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• pp.113-115
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• 2014

Salmonella are causative agents of gastroenteritis and systemic disease in animals. The invA gene was selected as a target sequence of loop-mediated isothermal amplification (LAMP) assay for diagnosis of Salmonella infection. The detection limits for broth dilution, spiked feces and enrichment were $10^4$, $10^5$ and $10^2$ CFUs/mL, respectively. The LAMP assay developed in the present study may be a reliable method for detection of Salmonella spp. in pig feces.

• 대한수의학회지
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• 제45권2호
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• pp.191-197
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• 2005

An immunochromatographic (IC) strip for the rapid detection of Salmonella spp. in the enriched sample was developed. Affinity purified Salmonella polyclonal antibody was conjugated with 40 nm colloidal gold particles which were prepared by citrate method in our laboratory. The antigen-antibody-gold complex was captured by Salmonella antibody attached to test line of nitrocellulose membrane during the capillary migration of sample. Specificity of the IC strip was calculated to be 100% (12/12) and sensitivity was 97.6% (41/42) in the test with pure cultured bacteria. Salmonella was artificially inoculated into raw pork macerated with enrichment broth. And then it was 10-fold diluted from $5.2{\times}10^{8}CFU/ml$ to 5.2 CFU/ml. The IC strip could detect $5.2{\times}10^{6}CFU/ml$ before enrichment. However, the lowest limit of detection was 5.2 CFU/ml after overnight incubation. The results indicated that the IC assay was a rapid, economical and simple method with high specificity and sensitivity for the detection of Salmonella spp. without using any equipment.

• Fibers and Polymers
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• 제6권3호
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• pp.235-243
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• 2005

A series of new metallized direct dyes based on benzidine congeners, 2,2'-dimethyl-5,5'-dipropoxybenzidine and 5,5'-dipropoxybenzidine, were evaluated for mutagenicity in Salmonella typhimurium strains TA98 and TA 100. All of the dyes examined were judged to be non-mutagenic with and without metabolic activation while toxicity was seen in some dyes at high doses. The study also suggested that the standard Salmonella mutagenicity plate-incorporated assay was an excellent method for evaluation of dyes for mutagenicity.

• Fibers and Polymers
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• 제6권4호
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• pp.297-305
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• 2005

A series of new direct dyes based on benzidine congeners, 2,2'-dimethyl-5,5'-dipropoxybenzidine and 5,5'-dipropoxybenzidine, were evaluated for mutagenicity in Salmonella typhimurium strains TA98 and TA100. All of the dyes examined were judged to be non-mutagenic with and without metabolic activation while toxicity was seen in some dyes at high doses. The study also suggested that the standard Salmonella mutagenicity plate-incorporated assay was an excellent method for evaluation of direct dyes for mutagenicity.