• 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.

• 한국미생물·생명공학회지
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• 제47권2호
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• pp.310-316
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• 2019

Distribution of Salmonella enterica serovars and their associated virulence determinants is wide-spread among food animals, which are continuously implicated in periodic salmonellosis outbreaks globally. The aim of this study was to determine and evaluate the diversity of five Salmonella serovar virulence genes (invA, pefA, cdtB, spvC and iroN) isolated from food animals and humans. Using standard microbiological techniques, Salmonella spp. were isolated from the feces of humans and three major food animals. Virulence determinants of the isolates were assayed using PCR. Clonal relatedness of the dominant serovar was determined via pulsed-field gel electrophoresis (PFGE) using the restriction enzyme, Xbal. Seventy one Salmonella spp. were isolated and serotyped into 44 serovars. Non-typhoidal Salmonella (NTS; 68) accounted for majority (95.8%) of the Salmonella serovars. Isolates from chicken (34) accounted for 47.9% of all isolates, out of which S. Budapest (14) was predominant (34.8%). However, the dominant S. Budapest serovars showed no genetic relatedness. The invA gene located on SPI-1 was detected in all isolates. Furthermore, 94% of the isolates from sheep harbored the spvC genes. The iroN gene was present in 50%, 100%, 88%, and 91% of isolates from human, chicken, sheep, and cattle, respectively. The pefA gene was detected in 18 isolates from chicken and a single isolate from sheep. Notably, having diverse Salmonella serovars containing plasmid encoded virulence genes circulating the food chain is of public health significance; hence, surveillance is required.

• Journal of Microbiology and Biotechnology
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• 제29권3호
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• pp.454-464
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• 2019

Salmonellosis is a highly contagious bacterial disease that threatens both human and poultry health. Tests that can detect Salmonella in the field are urgently required to facilitate disease control and for epidemiological investigations. Here, we combined loop-mediated isothermal amplification (LAMP) with a chromatographic lateral flow dipstick (LFD) to rapidly and accurately detect Salmonella. LAMP primers were designed to target the Salmonella invA gene. LAMP conditions were optimized by adjusting the ratio of inner to outer primers, $MgSO_4$ concentration, dNTP mix concentration, amplification temperature, and amplification time. We evaluated the specificity of our novel LAMP-LFD method using six Salmonella species and six related non-Salmonella strains. All six of the Salmonella strains, but none of the non-Salmonella strains, were amplified. LAMP-LFD was sensitive enough to detect concentrations of Salmonella enterica subsp. enterica serovar Pullorum genomic DNA as low as $89fg/{\mu}l$, which is 1,000 times more sensitive than conventional PCR. When artificially contaminated feed samples were analyzed, LAMP-LFD was also more sensitive than PCR. Finally, LAMP-LFD gave no false positives across 350 chicken anal swabs. Therefore, our novel LAMP-LFD assay was highly sensitive, specific, convenient, and fast, making it a valuable tool for the early diagnosis and monitoring of Salmonella infection in chickens.

• Journal of Microbiology and Biotechnology
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• 제15권1호
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• pp.175-182
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• 2005

Abstract Salmonella pathogenicity island 1 (SPI1) gene expression is regulated by many environmental signals such as oxygen, osmolarity, and pH. Here, we examined changes in the expression level of various regulatory proteins encoded within SPI1 in response to three different concentrations of NaCl, using primer extension analysis. Transcription of all the regulatory genes tested was activated most when Salmonella were grown in Luria Broth (LB) containing 0.17 M NaCl. The expression of hilA, invF, and hilD was decreased in the presence of 0.47 M NaCl or in the absence of NaCl, while hilC expression was almost constant regardless of the NaCl concentration when Salmonella were grown to exponential phase under low-oxygen condition. The reduced expression of hilA, invF, and hilD resulted in lower invasion of hilC mutant to the cultured animal cells when the mutant was grown in the presence of 0.47 M NaCl or in the absence of NaCl prior to infection. Among the proteins secreted via the SPI1-type III secretion system (TTSS), the level of sopE2 expression was not influenced by medium osmolarity. Various effects of osmolarity on virulence gene regulation observed in this study is one example of multiple regulatory pathways used by Salmonella to cause infection.

• 한국식품영양과학회지
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• 제39권4호
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• pp.595-601
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• 2010

본 연구는 국내 주요 식중독 원인균인 Staphylococcus aureus, Salmonella enterica subsp., Vibrio parahaemolyticus를 동시에 검출 및 동정할 수 있는 simultaneous multiplex PCR방법을 개발하고자 하였다. S. aureus의 23s rRNA 유전자(482 bp), V. Parahaemolyticus의 toxR 유전자(368 bp), S. enterica subsp.의 invA 유전자(284 bp)를 특이적으로 검출 및 동정할 수 있는 3개 primer set 즉, STA-5F/STA-5R, ToxR-F/ToxR-R, 139/141을 구축하였으며, 그 결과 정제되어진 각 식중독 원인균의 genomic DNA를 template로 하여 세 균주 모두 10 pg까지 다중동시검출이 가능하였다. 생균수(CFU)와 상응되는 검출한계 결과로써 $10^1\sim10^2$ CFU/reaction의 검출한계를 보였으며 이는 즉, S. aureus $6.0\times10^4$ CFU/mL, S. enterica subsp. $9.5\times10^4$ CFU/mL, V. parahaemolyticus $6.1\times10^5$ CFU/mL의 검출한계를 나타내었다. 균체회수부터 agarose gel 상에서 검출 및 동정까지 3~4 hr의 시간 소요로 single tube 반응으로 세 식중독 원인균의 다중동시검출이 가능하였다. 또한 추가적인 연구를 통하여 세 식중독 원인균주의 검출을 위한 향상된 민감도를 가지는 multiplex PCR법 및 real time PCR을 이용한 다중동시검출법 개발을 위한 기초자료로서 활용 가능할 것이라 사료된다.

• 대한수의학회지
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• 제53권1호
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• pp.25-28
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• 2013

The aim of this study was to applicate and evaluate a SYBR Green real-time PCR for the specific detection of Salmonella spp. Specificity of the PCR method was confirmed with 48 Salmonella spp. and 5 non-Salmonella strains using invA gene primer. The average threshold cycle ($C_T$) of Salmonella spp. was $11.83{\pm}0.78$ while non-Salmonella spp. was $30.86{\pm}1.19$. Correlation coefficients of standard curves constructed using $C_T$ versus copy number of Salmonella Enteritidis ATCC 13076 showed good linearity ($R^2=0.993$; slope = 3.563). Minimum level of detection with the method was > $10^2$ colony forming units (CFU)/mL. These results suggested that the SYBR Green real-time PCR might be applicable for the specific detection of Salmonella spp. isolates.

• Journal of Microbiology and Biotechnology
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• 제29권10호
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• pp.1543-1552
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• 2019

Salmonella is a common zoonotic and foodborne pathogen that causes high morbidity and mortality in developing countries. In this study, we established and validated a polymerase spiral reaction (PSR) assay which targeted the conserved invasion gene (invA) of Salmonella by SYBR Green I indicator methods. Subsequently, assays for determination of the optimal conditions for optimal specificity and sensitivity of PSR were performed. We performed comprehensive evaluations using loop-mediated isothermal amplification (LAMP) and real-time PCR. A total number of 532 samples of daily food were analyzed by PSR. Twenty-seven bacterial strains were tested in the specificity assay, from which positive results were obtained only for 14-Salmonella strains. However, none of the 13 non-Salmonella strains was amplified. Similarly with LAMP and real-time PCR, the detection limit of the PSR assay was 50 CFU/ml. The PSR method was also successfully applied to evaluate the contamination with Salmonella in 532 samples of daily food, corroborating traditional culture method data. The novel PSR method is simple, sensitive, and rapid and provides new insights into the prevention and detection of foodborne diseases.

• 미생물학회지
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• 제37권1호
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• pp.9-14
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• 2001

서울시내 25개 자치구에 산재한 약수터에서 5주의 Yersinia pseudotuberculosis를 분리하여 생화학적 특성, 병원성의 유무 및 16S rDNA 분석을 실시하였다. 분리된 Y. pseudotuberculosis는 모두 병원성 유전자인 inv를 소유하고 있었으며, 16S rDNA를 증폭한 후 염기서열을 분석하여 NCBI Genbank에 등록된 다른 Yersinia 속 및 장내세균 등과 비교해 본 결과 Yersinia 속 등과는 97.5%에서 100%의 높은 상동성(Similarity)을 나타내었고, 다른 장내세균 등과는 93.0%에서 95.1%의 낮은 상동성을 나타내었다. 165 rDNA 염기서열을 기초로 계통수를 작성한 결과 크게 3개의 cluster를 형성하였는데 특히 Y.enterocolitica (Z49830)은 Y.pseudotuberculasis (Z21939)와의 상동성(97.7%)보다 Y.intermedia (X75279)와의 상동성(97.9%)이 더 높게 나타났으며, E. coli (Z83205)는 Proteus vulgaris (AJ233425) 와의 상동성(93.2%)보다 Salmonella enteritidis (U90318)와의 상동성(97.7%)이 더 밀접한 연관성을 나타내었다.

• 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.

• KSBB Journal
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• 제18권3호
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• pp.190-196
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• 2003

임신이나 배란진단과 같이 가정에서 직접 사용할 수 있는 형태의 membrane strip 크로마토그래피 방법을 이용하여 식중독 균 DNA 분석시스템을 개발하였다. 분석물질로서는 빈번하게 발생하고 있는 식중독 미생물 중에서 S. typhimurium을 선택하였으며, Salmonella 종에 특이한 유전자 부위인 invA 유전자 분석을 목표로 하였다. 우선 이 유전자는 본 실험실 내에서 설계한 primer 쌍을 사용한 PCR 공정을 통하여 증폭되었다. 이렇게 증폭한 산물을 본 연구자들이 설계한 DNA probe와의 hybridization을 통하여 분석함으로써 전통적으로 사용하고 있는 전기영동 분석법의 단순히 분자크기에 의한 분리법과 비교하여 특이한 분석을 할 수 있었다. 이때 PCR 후 과량으로 잔존하는 primer를 별도로 제거하지 않고 hybridization을 수행할 수 있도록 특별하게 DNA probe를 설계하였다. 또한, probe가 증폭된 DNA와 hybridization 하였을 때 고체표면에 의한 간섭효과가 최소화되도록 설계 시 반영되었고 더욱이 streptavidin-비오틴의 결합을 이용하여 probe를 고정함으로써 고상에서의 상호작용이 더욱 용이하도록 배려하였다. 이러한 분석방법을 이용하여 분석한 결과 시료첨가 후 20-40분 정도에 최소 $10^3$cfu/mL (10 cells/system) 농도의 박테리아를 분석할 수 있었다. 이러한 결과는 일반적으로 사용하는 전기영동법보다 약 10배정도 더 민감한 결과일 뿐만 아니라 실험실 기기를 사용하지 않고 분석을 수행함으로써 분석시료가 제공되는 현장에서도 식중독 균의 탐지가 가능하게 되었다.