• Title/Summary/Keyword: Salmonella assay

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Rapid Quantification of Salmonella in Seafood Using Real-Time PCR Assay

  • Kumar, Rakesh;Surendran, P.K.;Thampuran, Nirmala
    • Journal of Microbiology and Biotechnology
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    • v.20 no.3
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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.

Rapid and sensitive detection of Salmonella species targeting the hilA gene using a loop-mediated isothermal amplification assay

  • Chu, Jiyon;Shin, Juyoun;Kang, Shinseok;Shin, Sun;Chung, Yeun-Jun
    • Genomics & Informatics
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    • v.19 no.3
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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.

Detection of Salmonella typhi by Loop-mediated Isothermal Amplification Assay

  • Jo, Yoon-Kyung;Lee, Chang-Yeoul
    • Biomedical Science Letters
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    • v.14 no.2
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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.

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Comparison of Isolation Agar Method, Real-Time PCR and Loop-Mediated Isothermal Amplification-Bioluminescence for the Detection of Salmonella Typhimurium in Mousse Cake and Tiramisu (Mousse cake와 Tiramisu에 인위접종된 Salmonella Typhimurium의 식품공전 분리배지, Real-time PCR과 Loop-mediated isothermal amplification-bioluminescence의 검출 특성 비교)

  • Lee, So-Young;Gwak, Seung-Hae;Kim, Jin-Hee;Oh, Se-Wook
    • Journal of Food Hygiene and Safety
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    • v.34 no.3
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    • pp.290-295
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    • 2019
  • Salmonella spp. are frequently associated with food and are among the most important foodborne pathogens. The recent Salmonella out breaks in Korea was associated with chocolate mousse cakes served with school meals during September 2018. The objective of this research was to compare the 3M Molecular Detection Assay 2 - Salmonella and the Korean Standard Method of Salmonella in artificially inoculated mousse (chocolate and cheese) and tiramisu cakes. Mousse (chocolate and cheese) and tiramisu cakes were artificially inoculated with S. Typhimurium. Twenty five gram of sample was enriched with 225 mL buffered peptone water for incubation at $37^{\circ}C$ for 24 h. After enrichment, the cultures were analyzed by using the 3M Molecular Detection Assay 2 - Salmonella and the Korean Standard Method. Most of the inoculated samples showed similar results except the chocolate mousse cakes, in which real-time PCR was unable to detect S. Typhimurium even after $10^4CFU/25g$ of inoculation. However, S. Typhimurium inoculated at a concentration of $10^0CFU/25g$ was detected by using 3M Molecular Detection Assay 2 - Salmonella. In chocolate mousse, detection of S. Typhimurium using real-time PCR was partially successful when dark chocolate was added at less than 15%. Negative results in real-time PCR and 3M Molecular Detection Assay 2 - Salmonella were confirmed by gel electrophoresis. The data indicated that dark chocolate could inhibit amplification of the target gene in the PCR reactions. In conclusion, the 3M Molecular Detection Assay 2 - Salmonella was better than the Korean Standard Method (real-time PCR) for the detection of S. Typhimurium in chocolate mousse cakes and chocolate mousse.

Detection of Salmonella in Milk by Sandwich ELISA using Anti-Outer Membrane Protein Immunoglobulins (Anti-Outer Membrane Protein 면역단백질을 이용한 Sandwich ELISA 방법에 의한 우유 내 Salmonella의 검출)

  • 최석호
    • Food Science of Animal Resources
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    • v.24 no.2
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    • pp.176-181
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    • 2004
  • The specificity of sandwich enzyme-linked immunosorbent assay (ELISA) to detect Salmonella in milk was determined in this study. The antibodies used in sandwich ELISA were egg yolk immunoglobulin G (IgY) obtained after immunization of hen with outer membrane protein (OMP) fraction from Salmonella typhimurium and rabbit IgG obtained after immunization of rabbit with the purified OMP with the molecular weight of 40,000. The immunoblot assay showed that the IgY reacted strongly with OMP with the molecular weight of 6,000 and the rabbit IgG reacted strongly with OMP with the molecular weights of 40,000, 35,000, and 6,000 from the bacteria including Salmonella which belongs to Enterobacteriaceae. The IgY and rabbit IgG also reacted with other proteins from Salmonella typhimurium in immunoblot assay. Competitive ELISA showed that IgY showed specifity to react with two strains of Salmonella typhimurium and Salmonella cholerasuis but not with Escherichia coli and Yersinia enterocolitica. Two strains of Salmonella typhimurium added to UHT milk showed the highest absorbance of all the bacteria used in the sandwich ELISA. Some strains of Salmonella cholerasuis showed higher absorbances than non-Salmonella bacteria.

Rapid and Sensitive Detection of Salmonella spp. by Using a Loop-Mediated Isothermal Amplification Assay in Duck Carcass Sample (오리 도체에서 등온유전자증폭기법을 이용한 Salmonella spp. 신속 고감도 검출 기법 연구)

  • Cho, Ae-Ri;Dong, Hee-Jin;Cho, Seongbeom
    • Food Science of Animal Resources
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    • v.33 no.5
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    • pp.655-663
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    • 2013
  • In this study, a rapid and sensitive detection tool for screening Salmonella spp. by using a loop-mediated isothermal amplification (LAMP) assay targeting the genomic sequence of the invA gene was developed. The inclusivity and exclusivity were both at 100% in the analysis, and the limit of detection (LOD) in a pure S. Enteritidis culture suspended in saline was $3.2{\times}10^3$ CFU/mL at 18.17 min ($R^2$ = 0.9446). The LODs of the LAMP assay in buffered peptone water with Salmonella (BPW) and duck carcass swab sample enriched in BPW with Salmonella (BPWS) after 0 and 12 h incubation were $3.2{\times}10^3$ CFU/mL and $3.2{\times}10^0$ CFU/mL, respectively. Comparing the LODs in BPW with those in BPWS, the LAMP assay was less influenced by compounds of duck carcass swab sample than the PCR assay. Sensitivity and specificity of the LAMP assay in 50 duck carcass swab samples enriched in BPW for 6 h were 96% and 84%, respectively, indicating that the LAMP assay is a rapid, simple and sensitive assay, which can be utilized as a potential screening tool of Salmonella spp. in duck carcass sample.

Loop-mediated isothermal amplification assay for the detection of Salmonella spp. in pig feces

  • Kim, Yong Kwan;Kim, Ha-Young;Jeon, Albert Byungyun;Lee, Myoung-Heon;Bae, You-Chan;Byun, Jae-Won
    • Korean Journal of Veterinary Research
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    • v.54 no.2
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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.

Development of the rapid detection kit for Salmonella spp. using immunochromatographic assay (면역크로마토그라피 기법을 이용한 Salmonella 속균 신속 검출킷트 개발)

  • Jung, Byeong-yeal;Jung, Suk-chan
    • Korean Journal of Veterinary Research
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    • v.45 no.2
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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.

Evaluation of New Metallized Direct Dyes for Mutagenicity Using the Salmonella Mammalian Mutagenicity Assay

  • Rae Jin-Seok;Freeman Harold S.
    • Fibers and Polymers
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    • v.6 no.3
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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.

Evaluation of New Direct Dyes for Mutagenicity Using the Salmonella Mammalian Mutagenicity Assay

  • Bae Jin-Seok;Freeman Harold S.
    • Fibers and Polymers
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    • v.6 no.4
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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.