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Development of a real-time nucleic acid sequence-based amplification assay for the rapid detection of Salmonella spp. from food.

ABSTRACT: Salmonella spp. is one of the most common foodborne infectious pathogen. This study aimed to develop a real-time nucleic acid sequence-based amplification (NASBA) assay for detecting Salmonella in foods. Primers and a molecular beacon targeting the Salmonella-specific xcd gene were designed for mRNA transcription, and 48 Salmonella and 18 non-Salmonella strains were examined. The assay showed a high specificity and low detection limit for Salmonella (7?×?10-1 CFU/mL) after 12 h of pre-enrichment. Importantly, it could detect viable cells. Additionally, the efficacy of the NASBA assay was examined in the presence of pork background microbiota; it could detect Salmonella cells at 9.5?×?103 CFU/mL. Lastly, it was successfully used to detect Salmonella in pork, beef, and milk, and its detection limit was as low as 10 CFU/25 g (mL). The real-time NASBA assay developed in this study may be useful for rapid, specific, and sensitive detection of Salmonella in food of animal origin.

SUBMITTER: Zhai L

PROVIDER: S-EPMC6863195 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Development of a real-time nucleic acid sequence-based amplification assay for the rapid detection of Salmonella spp. from food.

Zhai Ligong L Liu Hongxia H Chen Qiming Q Lu Zhaoxin Z Zhang Chong C Lv Fengxia F Bie Xiaomei X

Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology] 20181203 1

Salmonella spp. is one of the most common foodborne infectious pathogen. This study aimed to develop a real-time nucleic acid sequence-based amplification (NASBA) assay for detecting Salmonella in foods. Primers and a molecular beacon targeting the Salmonella-specific xcd gene were designed for mRNA transcription, and 48 Salmonella and 18 non-Salmonella strains were examined. The assay showed a high specificity and low detection limit for Salmonella (7 × 10<sup>-1</sup> CFU/mL) after 12 h of pre ...[more]

PMID: 30637640

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Project description:Equine piroplasmosis (EP) is a severe disease of horses caused by the tick-borne protozoa Theileria equi (T. equi) and Babesia caballi (B. caballi). Infectious carriers are not always symptomatic, meaning there is a risk to non-enzootic areas. Regulatory tests for EP include sero-epidemiological methods for equine babesiosis, but these lack specificity due to cross-reactivity with other Babesia species. In this study, we present a real-time quantitative recombinase polymerase amplification (qRPA) method for fast simultaneous detection of both T. equi and B. caballi. In this method, primers and probes targeting the 18S rRNA gene of both T. equi and B. caballi, the ema-1 gene of T. equi and the bc48 gene of B. caballi were designed and evaluated. The sensitivity of qRPA was evaluated using the pUC57 plasmid DNA containing the target gene. For the pUC57-bc48 gene DNA, the R2 value was 0.983 for the concentration range 0.2 ng (4.1 × 107 DNA copies) to 2.0 fg (4.1 × 101 DNA copies). For the pUC57-ema gene DNA, the R2 value was 0.993 for the concentration range 0.2 ng (5.26 × 107 DNA copies) to 2.0 fg (5.26 × 102 DNA copies). For the pUC57-Bc18S gene DNA the R2 value was 0.976 for the concentration range 2.0 ng (4.21 × 108 DNA copies) to 2.0 fg (4.21 × 102 DNA copies). For the pUC57-Te18S gene DNA, the R2 value was 0.952 (Fig. S3b) for the concentration range 2.0 ng (4.16 × 108 DNA copies) to 2.0 fg (4.16 × 102 DNA copies). Furthermore, a duplex qRPA analysis was developed and optimized and the results showed that primers and probes targeting for the bc48 gene of B. caballi and the 18S rRNA gene of T. equi is the best combination for a duplex qRPA analysis in one reaction. The developed duplex qRPA assay has good specificity, and had negative amplification for several similar parasite. For DNA extracted from real horse blood specimens, this qRPA method has comparable sensitivity to traditional qPCR, but a simpler and more rapid operating process to obtain positive amplification. The qRPA, including the duplex strategy described here, could allow fast identification of the EP-causing T. equi and B. caballi, showing great potential for on-site EP screening of horses.

Dataset Information

Development of a real-time nucleic acid sequence-based amplification assay for the rapid detection of Salmonella spp. from food.

Publications

Development of a real-time nucleic acid sequence-based amplification assay for the rapid detection of Salmonella spp. from food.

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