Khristine B. Balaga , Rance Derrick N. Pavon , Alyzza Marie B. Calayag, Christine Aubrey C. Justo, Davin Edric V. Adao, Windell L. Rivera
{"title":"开发一种基于钙蓝蛋白的闭管环介导等温扩增测定法,用于检测生肉样本中的沙门氏菌属。","authors":"Khristine B. Balaga , Rance Derrick N. Pavon , Alyzza Marie B. Calayag, Christine Aubrey C. Justo, Davin Edric V. Adao, Windell L. Rivera","doi":"10.1016/j.mimet.2024.106922","DOIUrl":null,"url":null,"abstract":"<div><p>Foodborne pathogens compromise food safety and public health, and <em>Salmonella</em> spp. are among the major pathogenic bacteria that cause outbreaks worldwide. Proper surveillance through timely and cost-effective detection methods across the food animal production chain is crucial to prevent <em>Salmonella</em> outbreaks and agricultural losses. Traditional culture methods are labor- and resource-intensive, with lengthy turnaround times. Meanwhile, conventional molecular tools, such as PCR and qPCR, are expensive and require technical skills and equipment. Loop-mediated isothermal amplification (LAMP) is a simple, rapid, inexpensive, highly sensitive, and specific molecular assay that does not require expensive equipment. Hence, this study developed and optimized a closed-tube, calcein-based LAMP assay to detect <em>Salmonella</em> using the <em>invA</em> gene and performed evaluation and validation against conventional PCR. The LAMP assay showed high specificity and sensitivity. It showed 10-fold higher sensitivity than conventional PCR, at <1 ng/μL DNA concentrations. Meanwhile, for CFU/mL, LAMP assay showed 1000-fold higher sensitivity than conventional PCR at 4.8 × 10<sup>3</sup> cells/mL than 4.8 × 10<sup>7</sup> cells/mL, respectively. For parallel testing of 341 raw meat samples, after conventional culture enrichment (until Rappaport-Vassiliadis broth), the optimized LAMP assay showed 100% detection on all samples while conventional PCR showed 100%, 99.04%, and 96.64% for raw chicken, beef, and pork samples, respectively. Meanwhile, a shortened enrichment protocol involving 3-h incubation in buffered peptone water only, showed lower accuracy in tandem with the optimized LAMP assay ranging from 55 to 75% positivity rates among samples. These suggest that the optimized LAMP assay possesses higher sensitivity over conventional PCR for <em>invA</em> gene detection when coupled with conventional enrichment culture methods. Hence, this assay has potential as a powerful complementary or alternative <em>Salmonella</em> detection method to increase surveillance capacity and protect consumer food safety and public health worldwide.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":1.7000,"publicationDate":"2024-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Development of a closed-tube, calcein-based loop-mediated isothermal amplification assay to detect Salmonella spp. in raw meat samples\",\"authors\":\"Khristine B. Balaga , Rance Derrick N. Pavon , Alyzza Marie B. Calayag, Christine Aubrey C. Justo, Davin Edric V. Adao, Windell L. Rivera\",\"doi\":\"10.1016/j.mimet.2024.106922\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>Foodborne pathogens compromise food safety and public health, and <em>Salmonella</em> spp. are among the major pathogenic bacteria that cause outbreaks worldwide. Proper surveillance through timely and cost-effective detection methods across the food animal production chain is crucial to prevent <em>Salmonella</em> outbreaks and agricultural losses. Traditional culture methods are labor- and resource-intensive, with lengthy turnaround times. Meanwhile, conventional molecular tools, such as PCR and qPCR, are expensive and require technical skills and equipment. Loop-mediated isothermal amplification (LAMP) is a simple, rapid, inexpensive, highly sensitive, and specific molecular assay that does not require expensive equipment. Hence, this study developed and optimized a closed-tube, calcein-based LAMP assay to detect <em>Salmonella</em> using the <em>invA</em> gene and performed evaluation and validation against conventional PCR. The LAMP assay showed high specificity and sensitivity. It showed 10-fold higher sensitivity than conventional PCR, at <1 ng/μL DNA concentrations. Meanwhile, for CFU/mL, LAMP assay showed 1000-fold higher sensitivity than conventional PCR at 4.8 × 10<sup>3</sup> cells/mL than 4.8 × 10<sup>7</sup> cells/mL, respectively. For parallel testing of 341 raw meat samples, after conventional culture enrichment (until Rappaport-Vassiliadis broth), the optimized LAMP assay showed 100% detection on all samples while conventional PCR showed 100%, 99.04%, and 96.64% for raw chicken, beef, and pork samples, respectively. Meanwhile, a shortened enrichment protocol involving 3-h incubation in buffered peptone water only, showed lower accuracy in tandem with the optimized LAMP assay ranging from 55 to 75% positivity rates among samples. These suggest that the optimized LAMP assay possesses higher sensitivity over conventional PCR for <em>invA</em> gene detection when coupled with conventional enrichment culture methods. Hence, this assay has potential as a powerful complementary or alternative <em>Salmonella</em> detection method to increase surveillance capacity and protect consumer food safety and public health worldwide.</p></div>\",\"PeriodicalId\":16409,\"journal\":{\"name\":\"Journal of microbiological methods\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":1.7000,\"publicationDate\":\"2024-03-19\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of microbiological methods\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0167701224000344\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q4\",\"JCRName\":\"BIOCHEMICAL RESEARCH METHODS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of microbiological methods","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0167701224000344","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
Development of a closed-tube, calcein-based loop-mediated isothermal amplification assay to detect Salmonella spp. in raw meat samples
Foodborne pathogens compromise food safety and public health, and Salmonella spp. are among the major pathogenic bacteria that cause outbreaks worldwide. Proper surveillance through timely and cost-effective detection methods across the food animal production chain is crucial to prevent Salmonella outbreaks and agricultural losses. Traditional culture methods are labor- and resource-intensive, with lengthy turnaround times. Meanwhile, conventional molecular tools, such as PCR and qPCR, are expensive and require technical skills and equipment. Loop-mediated isothermal amplification (LAMP) is a simple, rapid, inexpensive, highly sensitive, and specific molecular assay that does not require expensive equipment. Hence, this study developed and optimized a closed-tube, calcein-based LAMP assay to detect Salmonella using the invA gene and performed evaluation and validation against conventional PCR. The LAMP assay showed high specificity and sensitivity. It showed 10-fold higher sensitivity than conventional PCR, at <1 ng/μL DNA concentrations. Meanwhile, for CFU/mL, LAMP assay showed 1000-fold higher sensitivity than conventional PCR at 4.8 × 103 cells/mL than 4.8 × 107 cells/mL, respectively. For parallel testing of 341 raw meat samples, after conventional culture enrichment (until Rappaport-Vassiliadis broth), the optimized LAMP assay showed 100% detection on all samples while conventional PCR showed 100%, 99.04%, and 96.64% for raw chicken, beef, and pork samples, respectively. Meanwhile, a shortened enrichment protocol involving 3-h incubation in buffered peptone water only, showed lower accuracy in tandem with the optimized LAMP assay ranging from 55 to 75% positivity rates among samples. These suggest that the optimized LAMP assay possesses higher sensitivity over conventional PCR for invA gene detection when coupled with conventional enrichment culture methods. Hence, this assay has potential as a powerful complementary or alternative Salmonella detection method to increase surveillance capacity and protect consumer food safety and public health worldwide.
期刊介绍:
The Journal of Microbiological Methods publishes scholarly and original articles, notes and review articles. These articles must include novel and/or state-of-the-art methods, or significant improvements to existing methods. Novel and innovative applications of current methods that are validated and useful will also be published. JMM strives for scholarship, innovation and excellence. This demands scientific rigour, the best available methods and technologies, correctly replicated experiments/tests, the inclusion of proper controls, calibrations, and the correct statistical analysis. The presentation of the data must support the interpretation of the method/approach.
All aspects of microbiology are covered, except virology. These include agricultural microbiology, applied and environmental microbiology, bioassays, bioinformatics, biotechnology, biochemical microbiology, clinical microbiology, diagnostics, food monitoring and quality control microbiology, microbial genetics and genomics, geomicrobiology, microbiome methods regardless of habitat, high through-put sequencing methods and analysis, microbial pathogenesis and host responses, metabolomics, metagenomics, metaproteomics, microbial ecology and diversity, microbial physiology, microbial ultra-structure, microscopic and imaging methods, molecular microbiology, mycology, novel mathematical microbiology and modelling, parasitology, plant-microbe interactions, protein markers/profiles, proteomics, pyrosequencing, public health microbiology, radioisotopes applied to microbiology, robotics applied to microbiological methods,rumen microbiology, microbiological methods for space missions and extreme environments, sampling methods and samplers, soil and sediment microbiology, transcriptomics, veterinary microbiology, sero-diagnostics and typing/identification.