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A rapid novel visualized loop-mediated isothermal amplification method for Salmonella detection targeting at fimW gene.

ABSTRACT: Salmonella infection causes huge losses in the poultry industry worldwide. With the aim to prevent infectious diseases caused by Salmonella and to achieve rapid visualized Salmonella detection in poultry production, we used cresol red as an indicator to develop a novel visualized loop-mediated isothermal amplification method that targets the Salmonella fimW gene firstly in related field. The detection limit was 7.3 × 101 CFU/mL, and the method was highly specific and showed a high clinical detection rate. The entire reaction can be completed in about 40 min and only requires a water bath at 62°C, which makes the method extremely suitable for application to poultry production.

SUBMITTER: Wen J

PROVIDER: S-EPMC7597837 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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A rapid novel visualized loop-mediated isothermal amplification method for Salmonella detection targeting at fimW gene.

Wen Junping J Gou Hongchao H Zhan Zeqiang Z Gao Yuan Y Chen Zhengquan Z Bai Jie J Wang Shaojun S Chen Kaifeng K Lin Qijie Q Liao Ming M Zhang Jianmin J

Poultry science 20200425 7

Salmonella infection causes huge losses in the poultry industry worldwide. With the aim to prevent infectious diseases caused by Salmonella and to achieve rapid visualized Salmonella detection in poultry production, we used cresol red as an indicator to develop a novel visualized loop-mediated isothermal amplification method that targets the Salmonella fimW gene firstly in related field. The detection limit was 7.3 × 10<sup>1</sup> CFU/mL, and the method was highly specific and showed a high cli ...[more]

PMID: 32616260

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Project description:Vibrio cholerae is a leading waterborne pathogenic bacterium worldwide. It can cause human cholera that is still pandemic in developing nations. Detection of V. cholerae contamination in drinking water and aquatic products is imperative for assuring food safety. In this study, a simple, sensitive, specific, and visualized method was developed based on loop-mediated isothermal amplification (LAMP) (designated sssvLAMP) to detect virulence-associated (ctxA, tcpA, hapA, mshA, pilA, and tlh) and species-specific (lolB) genes of V. cholerae. Three pairs of oligonucleotide primers (inner, outer, and loop primers) were designed and or synthesized to target each of these genes. The optimal conditions of the sssvLAMP method was determined, and one-step sssvLAMP reaction was performed at 65°C for 40 min. Positive results were simply read by the naked eye via color change (from orange to light green) under the visible light, or by the production of green fluorescence under the UV light (260 nm). The sssvLAMP method was more efficient in detecting 6.50 × 101-6.45 × 104-fold low number of V. cholerae cells, and more sensitive in V. cholerae genomic DNA (1.36 × 10-2-4.42 × 10-6 ng/reaction) than polymerase chain reaction (PCR) method. Among 52 strains of V. cholerae and 50 strains of non-target species (e.g., other Vibrios and common pathogens) examined, the sensitivity and specificity of the sssvLAMP method were 100% for all the target genes. Similar high efficiency of the method was observed when tested with spiked samples of water and aquatic products, as well as human stool specimens. Water from various sources and commonly consumed fish samples were promptly screened by this simple and efficient visualized method and diversified variation in the occurrence of the target genes was observed. V. cholerae strains could be mostly detected by the presence of hapA and tlh alone or in combination with other genes, indicating a variable risk of potentially pathogenic non-O1/O139 strains in edible food products. This novel LAMP method can be a promising tool to address the increasing need of food safety control of aquatic products.

Project description:Background:Enterocytozoon hepatopenaei (EHP) is a newly emerged microsporidian parasite that causes retarded shrimp growth in many countries. But there are no effective approaches to control this disease to date. The EHP could be an immune risk factor for increased dissemination of other diseases. Further, EHP infection involves the absence of obvious clinical signs and it is difficult to identify the pathogen through visual examination, increasing the risk of disease dissemination. It is urgent and necessary to develop a specific, rapid and sensitive EHP-infected shrimp diagnostic method to detect this parasite. In the present study, we developed and evaluated a rapid real-time loop-mediated isothermal amplification (real-time LAMP) for detection of EHP. Methods:A rapid and efficient real-time LAMP method for the detection of EHP has been developed. Newly emerged EHP pathogens in China were collected and used as the sample, and three sets of specificity and sensitivity primers were designed. Three other aquatic pathogens were used as templates to test the specificity of the real-time LAMP assay. Also, we compared the real-time LAMP with the conventional LAMP by the serial dilutions of EHP DNA and their amplification curves. Application of real-time LAMP was carried out with clinical samples. Results:Positive products were amplified only from EHP, but not from other tested species, EHP was detected from the clinical samples, suggesting a high specificity of this method. The final results of this assay were available within less than 45 min, and the initial amplification curve was observed at about 6 min. We found that the amplification with an exponential of sixfold dilutions of EHP DNA demonstrated a specific positive signal by the real-time LAMP, but not for the LAMP amplicons from the visual inspection. The real-time LAMP amplification curves demonstrated a higher slope than the conventional LAMP. Discussion:In this study, pathogen virulence impacts have been increased in aquaculture and continuous observation was predominantly focused on EHP. The present study confirmed that the real-time LAMP assay is a promising and convenient method for the rapid identification of EHP in less time and cost. Its application greatly aids in the detection, surveillance, and prevention of EHP.

Dataset Information

A rapid novel visualized loop-mediated isothermal amplification method for Salmonella detection targeting at fimW gene.

Publications

A rapid novel visualized loop-mediated isothermal amplification method for Salmonella detection targeting at fimW gene.

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