Xu, Wenli
(College of Life and Health Sciences, Northeastern University)
,
Gao, Jun
(College of Life and Health Sciences, Northeastern University)
,
Zheng, Haoyue
(College of Life and Health Sciences, Northeastern University)
,
Yuan, Chaowen
(College of Life and Health Sciences, Northeastern University)
,
Hou, Jinlong
(College of Life and Health Sciences, Northeastern University)
,
Zhang, Liguo
(Center for Animal Disease Emergency of Liaoning Province)
,
Wang, Guoqing
(College of Life and Health Sciences, Northeastern University)
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 meth...
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.
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.
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제안 방법
1A, the spiral primers (Ft and Bt) that targeted the invA gene sequence consisted of a forward primer (F) and a reverse primer (B), whose Nr and N sequences were abstracted from a botanic gene [19]. Additionally, two auxiliary accelerated primers IF and IB were designed by Primer Premier 5 (PREMIER Biosoft International Co., USA) to enhance the reaction utilized in this study. The specificity of the designed primers was checked by BLAST, available in the NCBI sequence database.
In this study, we established a PSR method targeting the invasion gene (invA), considered to trigger the invasion of Salmonella into cultured epithelial cells and detected in all Salmonella isolates. The primer design in PSR was simpler than that in LAMP, as the sequence of the target region was selected in the same way as in conventional PCR.
The specificity of the PSR assay, compared with that of LAMP, was evaluated by using 27 different DNA extracts (14 Salmonella strains and 13 non-Salmonella strains as listed in Table 1) and sterilized deionized water as the blank control. The reactions were performed as described above under corresponding conditions; all tests were repeated three times.
To evaluate the sensitivity of the PSR assay, the concentrations of Salmonella Enteritidis ATCC 13076 DNA, extracted from serially diluted bacterial culture, ranging from 106 CFU/ml-5 CFU/ml, were subjected to PSR, LAMP, and qPCR amplification in triplicate. The reactions were performed under the corresponding aforementioned amplification conditions.
대상 데이터
To standardize and evaluate the PSR system, we used a total of 27 bacterial strains in this study, including 23 standard strains and 4 clinical isolates (Table 1). Salmonella enteritidis ATCC 13076 was employed as a reference strain.
성능/효과
In conclusion, the invA-based PSR method developed and described in current study has many advantages, for detection of Salmonella in food, including rapidity, reliability, sensitivity, and specificity, not to mention lacking a requirement for expensive equipment and reagents.
The best results of the PSR assay were obtained at 63°C for 60 min, which were considered as standardized optimal PSR assay conditions (Figs. 3A and 3F).
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