Rapid and Simultaneous Detection of Vibrio parahaemolyticus, Salmonella spp. and Escherichia coli in Fish by Multiplex PCR

Q3 Environmental Science
R. Triwibowo, Novalia Rachmawati, D. Dwiyitno
{"title":"Rapid and Simultaneous Detection of Vibrio parahaemolyticus, Salmonella spp. and Escherichia coli in Fish by Multiplex PCR","authors":"R. Triwibowo, Novalia Rachmawati, D. Dwiyitno","doi":"10.15578/squalen.v15i2.444","DOIUrl":null,"url":null,"abstract":"Pathogenic bacteria are commonly found as natural contaminants in seafood and fish products. Globally, several countries have been imposing strict regulations on the maximum levels of pathogens and consequently require microbial testing of pathogens before the products can be marketed. A culture-based method with biochemical assay has been widely used to detect pathogenic bacteria in food, despite its long and extensive process. Meanwhile, the alternative molecular-based method to overcome this problem, cannot differentiate between viable and nonviable cells, which may lead to underestimation. This study aimed to develop a multiplex PCR (mPCR) method as a confirmatory assay for the culture-based method to detect pathogens in fish products simultaneously. This method applied a pre-enrichment step to ensure the growth of low-level pathogens and the injured cells in the sample. The target genes were ToxR, InvA, and UidA for Vibrio parahaemolyticus, Salmonella spp. and Escherichia coli, respectively. This assay also amplified the 16S rDNA gene of bacteria as an internal control for the PCR reaction. By implementing liquid-based DNA extraction during analysis, the developed-mPCR was comparable to detect the targeted bacteria in artificially-contaminated samples. The method was more sensitive in naturally-contaminated samples, where the number of E. coli, Salmonella spp. and V. parahaemolyticus detected were 28, 7, and 22, respectively. While the conventional method only detected 26, 5, and 19 of the respective pathogens. With a relatively shorter time and lower operation cost, the mPCR method is potential as an alternative for the culture-based method.","PeriodicalId":21935,"journal":{"name":"Squalen Bulletin of Marine and Fisheries Postharvest and Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2020-08-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"4","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Squalen Bulletin of Marine and Fisheries Postharvest and Biotechnology","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.15578/squalen.v15i2.444","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"Environmental Science","Score":null,"Total":0}
引用次数: 4

Abstract

Pathogenic bacteria are commonly found as natural contaminants in seafood and fish products. Globally, several countries have been imposing strict regulations on the maximum levels of pathogens and consequently require microbial testing of pathogens before the products can be marketed. A culture-based method with biochemical assay has been widely used to detect pathogenic bacteria in food, despite its long and extensive process. Meanwhile, the alternative molecular-based method to overcome this problem, cannot differentiate between viable and nonviable cells, which may lead to underestimation. This study aimed to develop a multiplex PCR (mPCR) method as a confirmatory assay for the culture-based method to detect pathogens in fish products simultaneously. This method applied a pre-enrichment step to ensure the growth of low-level pathogens and the injured cells in the sample. The target genes were ToxR, InvA, and UidA for Vibrio parahaemolyticus, Salmonella spp. and Escherichia coli, respectively. This assay also amplified the 16S rDNA gene of bacteria as an internal control for the PCR reaction. By implementing liquid-based DNA extraction during analysis, the developed-mPCR was comparable to detect the targeted bacteria in artificially-contaminated samples. The method was more sensitive in naturally-contaminated samples, where the number of E. coli, Salmonella spp. and V. parahaemolyticus detected were 28, 7, and 22, respectively. While the conventional method only detected 26, 5, and 19 of the respective pathogens. With a relatively shorter time and lower operation cost, the mPCR method is potential as an alternative for the culture-based method.
多重PCR快速同时检测鱼类中副溶血性弧菌、沙门氏菌和大肠杆菌
致病菌通常是海产品和鱼类产品中的天然污染物。在全球范围内,一些国家对病原体的最大限量实施了严格的规定,因此要求在产品上市之前对病原体进行微生物检测。以培养为基础的生化检测方法已广泛应用于食品中致病菌的检测,但其过程漫长而繁琐。同时,克服这一问题的另一种基于分子的方法无法区分有活力和无活力的细胞,这可能导致低估。本研究旨在建立一种多重PCR (mPCR)方法,作为基于培养的方法同时检测鱼类产品中病原体的验证性方法。该方法采用预富集步骤,确保样品中低水平病原体和损伤细胞的生长。靶基因分别为副溶血性弧菌、沙门氏菌和大肠杆菌的ToxR、InvA和UidA。该实验还扩增了细菌的16S rDNA基因作为PCR反应的内控。通过在分析过程中进行基于液体的DNA提取,所开发的mpcr可用于检测人工污染样品中的目标细菌。该方法对自然污染样品较为敏感,检出大肠杆菌28株,沙门氏菌7株,副溶血性弧菌22株。而传统方法分别只检测到26、5和19种病原菌。由于时间相对较短,操作成本较低,mPCR方法有可能成为基于培养的方法的替代方案。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 求助全文
来源期刊
CiteScore
1.40
自引率
0.00%
发文量
10
审稿时长
16 weeks
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信