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Development of a rapid detection method for Photobacterium spp. using Loop-mediated isothermal amplification (LAMP).
International Journal of Food Microbiology ( IF 5.4 ) Pub Date : 2020-08-06 , DOI: 10.1016/j.ijfoodmicro.2020.108805
Sandra Fuertes-Perez 1 , Maik Hilgarth 1 , Rudi F Vogel 1
Affiliation  

While the abundance of photobacteria has previously been exclusively associated with marine environments and spoilage of seafood, several recent studies have demonstrated their status as pervasive constituents of the microbiota on packaged meats. Since their ubiquitous nature has been revealed, detection of their presence on meat, their entry route into meat processing environments and prevention of their growth is a novel emerging challenge for the food industry.

In this study, we have developed a highly sensitive and specific loop-mediated isothermal amplification (LAMP) assay for the detection of relevant species of photobacteria on foods, and tested its efficacy on meats. The gene encoding trimethylamine-N-oxide reductase (torA) was chosen as the target for this assay. Designed primers based on the gene sequence proved their specificity by testing 67 isolates of 5 species of photobacteria (positive) as well as 63 strains of 16 species of other common meat spoilers (negative). The optimized assay takes 2 h including sample preparation and has a detection limit of only 10–11 copies (50 fg/reaction) of the average Photobacterium (P.) genome per reaction. Its applicability could be successfully demonstrated on naturally and artificially contaminated chicken, beef and pork samples and evaluated by comparison with a culture-dependent approach using selective media and MALDI-TOF MS for identification. The developed LAMP assay revealed presence of photobacteria on one naturally contaminated chicken sample stored at 4 °C long before (3 days) confirmation by the culture-dependent approach. This study demonstrates that the developed LAMP assay represents a reliable and sensitive method for rapid detection of photobacteria on meats. However, its specificity would allow the applicability of the methodology to be extended to other foods, e.g. fish and seafood where presence of photobacteria is directly linked to their shelf life. The method has no requirement for specialized equipment or specially trained personal allowing an easy implementation within the quality control of the food industry. Considering the lot-to-lot variations observed on meats regarding the presence of photobacteria and the impracticality of implementing quantitative methods within the routine control, the LAMP method can simplify and reduce the workload for detection of photobacteria on high sample numbers. Consequently, producers can identify batches/plants that need more stringent control, and are provided with a tool to determine the entry route of photobacteria into the processing and distribution chain of raw meats.



中文翻译:

细菌快速鉴定方法的开发。使用回路介导的等温扩增(LAMP)。

尽管以前大量的细菌细菌仅与海洋环境和海鲜变质有关,但最近的一些研究表明它们已成为包装肉类中微生物群落的普遍成分。由于已经揭示了它们的普遍性,因此检测它们在肉上的存在,进入肉类加工环境的途径以及防止其生长是食品工业面临的新挑战。

在这项研究中,我们已经开发了一种高灵敏度和特异性的环介导的等温扩增(LAMP)分析法,用于检测食品中相关的细菌种类,并测试了其在肉类上的功效。选择编码三甲胺-N-氧化物还原酶(tor A)的基因作为该测定的靶标。根据基因序列设计的引物通过测试5种细菌(阳性)的67种分离物以及16种其他常见肉类破坏物(阴性)的63个菌株来证明其特异性。经过优化的测定需要2小时(包括样品制备),并且检出限仅为普通细菌的10-11份(50 fg /反应)(P。)每个反应的基因组。它的适用性可以在天然和人工污染的鸡肉,牛肉和猪肉样品上成功地证明,并可以通过使用选择性培养基和MALDI-TOF MS进行鉴定的培养依赖性方法进行比较来评估。发达的LAMP测定法揭示了在一种通过培养依赖性方法确认之前(3天)很久之前(4天)存储在4°C的自然污染鸡样品中存在细菌。这项研究表明,开发的LAMP分析方法代表了一种快速检测肉类细菌细菌的可靠且灵敏的方法。但是,其特异性将使该方法的适用范围扩展到其他食品,例如鱼类和海鲜,其中细菌的存在直接与其货架寿命相关。该方法不需要专门的设备或经过专门培训的人员,从而可以在食品工业的质量控制范围内轻松实施。考虑到在肉上观察到的有关细菌中细菌存在的批次差异以及在常规控制中实施定量方法的不现实性,LAMP方法可以简化并减少大量样品中细菌检测的工作量。因此,生产者可以识别需要更严格控制的批次/工厂,并为他们提供确定细菌进入生肉加工和分销链的途径的工具。考虑到在肉上观察到的有关细菌中细菌存在的批次差异以及在常规控制中实施定量方法的不现实性,LAMP方法可以简化并减少大量样品中细菌检测的工作量。因此,生产者可以识别需要更严格控制的批次/工厂,并为他们提供确定细菌进入生肉加工和分销链的途径的工具。考虑到在肉上观察到的有关细菌中细菌存在的批次差异以及在常规控制中实施定量方法的不现实性,LAMP方法可以简化并减少大量样品中细菌检测的工作量。因此,生产者可以识别需要更严格控制的批次/工厂,并为他们提供确定细菌进入生肉加工和分销链的途径的工具。

更新日期:2020-08-12
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