Development of duplex PCR-ELISA for simultaneous detection of Salmonella spp. and Escherichia coli O157: H7 in food

Hu, Jinqiang; Huang, Runna; Wang, Yi; Wei, Xiangke; Wang, Zhangcun; Geng, Yangyang; Jianzhou, Jing; Gao, Hui; Sun, Xiaoyan; Dong, Caiwen; Chun-peng, Jiang

doi:10.1016/j.mimet.2018.10.017

Cited by 24 publications

(4 citation statements)

References 35 publications

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“…However, these methods are relatively expensive and require instrumentation. qPCR is a specific molecular PCR-based assay commonly used to quantify different bacterial strains (DNA) in food samples (Wei et al, 2018).…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, in a clinical setting, it would enable a rapid treatment response that does safe life particularly when patients have to travel from remote areas to seek treatment and where on presentation toxic symptoms and systemic infection are often in an advanced state (Chen et al, 2021 ). However, the main limitation of this method is its inability to differentiate live from dead cell DNA (Hu et al, 2018 ). qPCR assays were used to detect and quantify E. coli serogroup O157 strains in different clinical, environmental, and food samples (Kim & Oh, 2021 ).…”

Section: Discussionmentioning

confidence: 99%

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A quantitative prevalence of Escherichia coliO157 in different food samples using real‐time qPCR method

Pakbin

Brück

et al. 2022

Food Science & Nutrition

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Escherichia coli serogroup O157 is the main causative agent of several intestinal and extra‐intestinal foodborne diseases in humans through consumption of low‐dose contaminated foods such as milk, beef, and vegetables. To date, studies regarding the quantitative prevalence of E. coli O157 in foods are so limited. Therefore, this study aimed to evaluate the quantitative prevalence rate of E. coli serogroup O157 in raw milk (n = 144), vegetable salad (n = 174), and minced beef samples (n = 108) using the real‐time qPCR SYBR green melting curve method targeting the rfbA gene. First, we evaluated the method and found a sensitive and specific qPCR assay with 1 log of CFU/ml detection limit to detect E. coli O157 (Tm = 80.3 ± 0.1°C). About 2.77%, 10.18%, and 9.19% of raw milk, minced beef, and vegetable salad samples, respectively, were contaminated with E. coli O157. Minced beef and vegetable salad samples were significantly more contaminated than raw milk samples. Population average of E. coli O157 in raw milk, minced beef, and vegetable salad samples were 2.22 ± 0.57, 3.30 ± 0.40, and 1.65 ± 0.44 log CFU/ml or gr, respectively. Significantly higher levels of population of E. coli O157 were observed in minced beef samples. Minced beef can be regarded as the main food in the transmission of this foodborne pathogen. Routine quantitative rapid monitoring is strongly suggested to be carried out to prevent foodborne diseases caused by E. coli O157.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A quantitative prevalence of Escherichia coliO157 in different food samples using real‐time qPCR method

Pakbin

Brück

et al. 2022

Food Science & Nutrition

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“…Enzyme-linked immunosorbent assay (ELISA) has been applied for identification of various targets such as antigens, antibodies, toxic or functional proteins, bacteria, and viruses due to its high selectivity and quantitative/qualitative capability. [1][2][3][4][5][6] ELISA is typically performed in a 96-well plate, which allows high-throughput detection of a variety of samples. [7][8][9] However, the nature and composition of a target substance might necessitate a multistep complicated ELISA procedure to ensure its accurate and highsensitive detection.…”

Section: Introductionmentioning

confidence: 99%

Development of Metal Nanoparticle-immobilized Microplate for High-throughput and Highly Sensitive Fluorescence Analysis

et al. 2020

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Enzyme-linked immunosorbent assay (ELISA) is a widespread analytical biochemistry assay. In this work, a direct ELISA method using a metallic nanoparticle (NP)immobilized 96-well plate was developed for high-throughput, highly sensitive fluorescence analysis. Immobilization of metallic NPs on a 96-well plate effectively amplified fluorescence signals of the assay. The silver (Ag) NP-immobilized plate showed the best fluorescence enhancement effect of all the metal-immobilized plates tested. We used the Ag NP-immobilized plate to detect biomolecules and bacteria and found that both the fluorescence intensity and the limit of detection (LOD) were strongly enhanced by more than 100 times compared with those of the unmodified 96-well plates. Quantitative and qualitative considerations for target bacteria regarding the impact of autofluorescence on detection were successfully obtained for several strains. Our results demonstrate the potential of applying Ag NPs for enhancing the efficiency of direct and indirect ELISA assays.

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“…Most Salmonella surveillance programs rely on polymerase chain reaction (PCR)-based assays for rapid and accurate detection (1,2). Among these molecular tools, the invA-PCR assay has been accepted as the conventional method for Salmonella detection (2)(3)(4). This PCR protocol amplifies a fragment of the invA gene, a Salmonella-specific locus (5,6) proposed as an international standard tool for the accurate detection of this pathogen (7).…”

Section: Introductionmentioning

confidence: 99%

PCR Assays Based on invA Gene Amplification are not Reliable for Salmonella Detection

Reséndiz-Nava

Esquivel-Hernandez

Alcaraz-Gonzalez

et al. 2019

Jundishapur J Microbiol

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Background: Salmonella surveillance relies on invA polymerase chain reaction (PCR) assays for the rapid detection of Salmonella; however, false-positive results have been reported using this method. Objectives: To evaluate the performance and specificity of the published and validated PCR protocols targeting invA gene for the detection of Salmonella. Methods: The performance and specificity of 11 different PCR primer sets were evaluated using Salmonella type strains and Citrobacter spp., Escherichia coli and Serratia spp. isolates recovered during a Salmonella surveillance program. Results: It was revealed that the published PCR protocols using validated primers targeting invA and 16S rRNA genes generated falsepositive signals. Importantly, a protocol targeting the ttrA/C genes was able to discriminate Salmonella and non-Salmonella isolates. Conclusions: Detection of Salmonella spp. by means of invA PCR amplification is not reliable. In fact, false-positive results are commonly obtained from Citrobacter, E. coli and Serratia isolates. It is recommended to use other loci, such as ttrA/C genes, for the accurate and reliable detection of Salmonella.

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Development of duplex PCR-ELISA for simultaneous detection of Salmonella spp. and Escherichia coli O157: H7 in food

Cited by 24 publications

References 35 publications

A quantitative prevalence of Escherichia coliO157 in different food samples using real‐time qPCR method

A quantitative prevalence of Escherichia coliO157 in different food samples using real‐time qPCR method

Development of Metal Nanoparticle-immobilized Microplate for High-throughput and Highly Sensitive Fluorescence Analysis

PCR Assays Based on invA Gene Amplification are not Reliable for Salmonella Detection

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