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Development of a High-Throughput Microfluidic qPCR System for the Quantitative Determination of Quality-Relevant Bacteria in Cheese.

ABSTRACT: The composition of the cheese microbiome has an important impact on the sensorial quality and safety of cheese. Therefore, much effort has been made to investigate the microbial community composition of cheese. Quantitative real-time polymerase chain reaction (qPCR) is a well-established method for detecting and quantifying bacteria. High-throughput qPCR (HT-qPCR) using microfluidics brings further advantages by providing fast results and by decreasing the cost per sample. We have developed a HT-qPCR approach for the rapid and cost-efficient quantification of microbial species in cheese by designing qPCR assays targeting 24 species/subspecies commonly found in cheese. Primer pairs were evaluated on the Biomark (Fluidigm) microfluidic HT-qPCR system using DNA from single strains and from artificial mock communities. The qPCR assays worked efficiently under identical PCR conditions, and the validation showed satisfying inclusivity, exclusivity, and amplification efficiencies. Preliminary results obtained from the HT-qPCR analysis of DNA samples of model cheeses made with the addition of adjunct cultures confirmed the potential of the microfluidic HT-qPCR system to screen for selected bacterial species in the cheese microbiome. HT-qPCR data of DNA samples of two downgraded commercial cheeses showed that this approach provides valuable information that can help to identify the microbial origin of quality defects. This newly developed HT-qPCR system is a promising approach that will allow simultaneous monitoring of quality-relevant species in fermented foods with high bacterial diversity, thereby opening up new perspectives for the control and assurance of high product quality.

SUBMITTER: Dreier M

PROVIDER: S-EPMC7817891 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Development of a High-Throughput Microfluidic qPCR System for the Quantitative Determination of Quality-Relevant Bacteria in Cheese.

Dreier Matthias M Berthoud Hélène H Shani Noam N Wechsler Daniel D Junier Pilar P

Frontiers in microbiology 20210107

The composition of the cheese microbiome has an important impact on the sensorial quality and safety of cheese. Therefore, much effort has been made to investigate the microbial community composition of cheese. Quantitative real-time polymerase chain reaction (qPCR) is a well-established method for detecting and quantifying bacteria. High-throughput qPCR (HT-qPCR) using microfluidics brings further advantages by providing fast results and by decreasing the cost per sample. We have developed a HT ...[more]

PMID: 33488561

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Project description:A rare viroid disease, Orange Spotting (OS), has been associated with Coconut cadang-cadang viroid variant (named OS-CCCVd) in oil palm. The low concentration of OS-CCCVd in oil palm leaf tissues makes it tedious to obtain high-quality RNA. Various conventional extraction protocols are available for extracting RNA; however, the bottleneck to the methods is the acquisition of good yields of high-quality RNA suitable for use in viroid detection. Studies looking into the automation of magnetic bead extraction systems for viroid detection in oil palm are limited. In this study, we have compared four extraction methods, namely the MagMAX™ mirVana Total RNA isolation kit (Mag-A), MagMAX™ plant RNA isolation kit (Mag-B), modification of MagMAX™ mirVana Total RNA isolation kit (Mag-Mod) and the convention method (NETME buffer). The KingFisher Flex System uses a 96-well plate format for the three automated approaches. The major modification in the Mag-Mod protocol is the inclusion of lithium chloride solution and NETME buffer to the lysis buffer (which enhances RNA recovery) and the reduction in the volume of reagents used per reaction and run conditions, including time. High-quality small RNA was produced as a result of altering the buffers and reagent volume in the Mag-Mod method, which also increased sample productivity (48 samples per day) and detection sensitivity. These effects indirectly decreased the number of technical replicates in a run. Importantly, the alteration permitted the use of ground plant tissue samples with a small sample amount of 0.05- 0.1 g as opposed to the 2-4 grequired with the conventional method. The development of the modified Mag-Mod method using the 96-deep well plate with a magnetic bead system vastly increased our sample throughput, complementing the detection methods (e.g. RT-PCR and HRM-qPCR). Aside from developing an improved extraction method (Mag-Mod method), a novel HRM-qPCR assay successfully differentiated OS-CCCVd variants and identified the presence of OS-CCCVd in samples, obviating the need for Sanger Sequencing.

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Dataset Information

Development of a High-Throughput Microfluidic qPCR System for the Quantitative Determination of Quality-Relevant Bacteria in Cheese.

Publications

Development of a High-Throughput Microfluidic qPCR System for the Quantitative Determination of Quality-Relevant Bacteria in Cheese.

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