• 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.

• Microbiology and Biotechnology Letters
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• v.47 no.2
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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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• v.29 no.3
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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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• v.15 no.1
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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.

• Journal of the Korean Society of Food Science and Nutrition
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• v.39 no.4
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• pp.595-601
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• 2010

This study was conducted to detect and identify Staphylococcus aureus, Vibrio parahaemolyticus, and Salmonella enterica subsp. using simultaneous multiplex polymerase chain reaction (multiplex PCR) assay. 23S rRNA partial gene (S. aureus), tox R gene (V. parahaemolyticus), and inv A gene (S. enterica subsp.) as diagnostic marker gene were suggested, and their amplicon sizes were 482 bp, 368 bp, and 284 bp, respectively. Non specific amplicons by STA-5F/STA-5R primer, ToxR-F/ToxR-R primer, and 139/141 primer were not observed in genomic DNA of pathogen bacteria as Bacillus cereus, Listeria monocytogenes, Escherichia coli, Proteus vulgaris, Streptococcus pyogenes, Candida albicans, and Shigella sonnei. The extracted crude DNA of targeted bacteria was detected as PCR template successfully. The detection limits were $10^5\sim10^4$ CFU/mL and 10 pg of purified genomic DNA of S. aureus, V. parahaemolyticus, and S. enterica subsp. by using simultaneous multiplex PCR.

• Korean Journal of Veterinary Research
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• v.53 no.1
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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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• v.29 no.10
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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.

• Korean Journal of Microbiology
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• v.37 no.1
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• pp.9-14
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• 2001

In order to investigate the pathogenicity and development of differential identification technique in the Yersinia species and other entericbacteria, we isolated 5 strains of Y.pseudotuberculosis from spring water sites in Seoul. The biochemical characteristics of isolated strains revealed that indole, VP($25^{\circ}C$, $37^{\circ}C$), $H_2S$, phenylalanine, lysine, arginine, ornithine, gas from glucose, lactose, sucrose, sorbitol, oxidase and motility($37^{\circ}C$) were all negative and urease, glucose, mannitol, salicin, catalase and motility($25^{\circ}C$) were all positive. To detect the causative agent of pseudotuberculosis(Y.pseudotuberculosis), we carried out a study using a PCR with inv primers complementary to the pathogenic region and found that all strains were positive, this revealed that strains from spring waters were pathogenic. Also 16S rDNA for total 5 strains of Y. pseudotuberculosis were amplified and a stretch of approximately 1,450 nucleotides were sequenced and analyzed. The 16S rDNA nucleotide sequence homologies among Yersinia species ranged 97.5% to 100% and between Y.pseudotuberculosis and other entericbacteria they ranged 93.0% to 95.1%. The Phylogenetic tree generated from the sequence analysis of the 16S rDNA gene showed 3 coherent clusters that could be separated into Y.pseudotuberculsis strains, some Yersinia species strains and other entericbacteria strains.

• 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.

• KSBB Journal
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• v.18 no.3
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• pp.190-196
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• 2003

An analytical system detecting DNA particularly utilizing a concept of membrane strip chromatography initially applied to home-version tests for, such as, pregnancy and ovulation has been developed. We have chosen S. typhimurium as model analyte among food-contaminating microorganisms that occurred in high frequencies, and invA gene, as a detection target, specific to Salmonella species. This gene was able to be amplified by PCR under optimal conditions employing newly designed primers in our laboratory. The PCR product was specifically measured via hybridization between the analyte and a DNA probe, which was a totally different feature from the conventional gel electrophoresis detecting the products based only on the molecular size. It is notable thar the DNA probe sequence was specially designed such that no separation of excess primers present after PCR was required. This was immobilized on a nitrocellulose (NC) membrane via streptavidin-biotin linkage minimizing a steric effect when the hybridization with the amplified DNA took place. The analyrical system detected the microorganism in a concentration of minimum $10^3$ cfu/mL (i.e., 10 cells per system), estimated from the standard curve, 20 to 40 minutes after adding the sample. This sneitivity was approximately 10 times higher than that of gel electrophoresis as an analytical tool conventionally used. Furthermore, the assay was able to be run at room temperature, which would ofter an extra advantage to users.