Dataset Information

Multiplex Real-Time PCR with HRM for Detection of Lactobacillus sakei and Lactobacillus curvatus in Food Samples.

ABSTRACT: Optimization of fermentation processes requires monitoring the species composition of starter cultures and their growth during fermentation. Most starter cultures contain closely related species. Nowadays, high-resolution melting (HRM) analysis is extensively used for multiplex identification of closely related species. In the present paper, we applied real-time polymerase chain reaction (PCR) with HRM analysis for the detection and differentiation of Lactobacillus sakei and L. curvatus. A primer pair was selected for the site of the rpoA gene of Lactobacillus spp. Eleven starter cultures and fifteen fermented sausages with a known bacterial composition were successfully tested using real-time PCR with HRM analysis with the developed primer pair.

SUBMITTER: Kurbakov KA

PROVIDER: S-EPMC6600297 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Multiplex Real-Time PCR with HRM for Detection of Lactobacillus sakei and Lactobacillus curvatus in Food Samples.

Kurbakov Konstantin A KA Konorov Evgenii A EA Minaev Mikhail Y MY Kuznetsova Oksana A OA

Food technology and biotechnology 20190301 1

Optimization of fermentation processes requires monitoring the species composition of starter cultures and their growth during fermentation. Most starter cultures contain closely related species. Nowadays, high-resolution melting (HRM) analysis is extensively used for multiplex identification of closely related species. In the present paper, we applied real-time polymerase chain reaction (PCR) with HRM analysis for the detection and differentiation of Lactobacillus sakei and L. curvatu ...[more]

PMID: 31316281

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Project description:BackgroundReverse transcription quantitative real-time PCR (RT-qPCR) tests support personalized cancer treatment through more clinically meaningful diagnosis. However, samples obtained through standard clinical pathology procedures are formalin-fixed, paraffin-embedded (FFPE) and yield small samples with low integrity RNA containing PCR interfering substances. RT-qPCR tests able to assess FFPE samples with quality control and inter-laboratory reproducibility are needed.MethodsWe developed an RT-qPCR method by which 1) each gene was measured relative to a known number of its respective competitive internal standard molecules to control for interfering substances, 2) two-color fluorometric hydrolysis probes enabled analysis on a real-time platform, 3) external standards controlled for variation in probe fluorescence intensity, and 4) pre-amplification maximized signal from FFPE RNA samples. Reagents were developed for four genes comprised by a previously reported lung cancer diagnostic test (LCDT) then subjected to analytical validation using synthetic native templates as test articles to assess linearity, signal-to-analyte response, lower detection threshold, imprecision and accuracy. Fitness of this method and these reagents for clinical testing was assessed in FFPE normal (N = 10) and malignant (N = 10) lung samples.ResultsReagents for each of four genes, MYC, E2F1, CDKN1A and ACTB comprised by the LCDT had acceptable linearity (R(2)>0.99), signal-to-analyte response (slope 1.0 ± 0.05), lower detection threshold (<10 molecules) and imprecision (CV <20%). Poisson analysis confirmed accuracy of internal standard concentrations. Internal standards controlled for experimentally introduced interference, prevented false-negatives and enabled pre-amplification to increase signal without altering measured values. In the fitness for purpose testing of this two-color fluorometric LCDT using surgical FFPE samples, the diagnostic accuracy was 93% which was similar to that previously reported for analysis of fresh samples.ConclusionsThis quality-controlled two-color fluorometric RT-qPCR approach will facilitate the development of reliable, robust RT-qPCR-based molecular diagnostic tests in FFPE clinical samples.

Dataset Information

Multiplex Real-Time PCR with HRM for Detection of Lactobacillus sakei and Lactobacillus curvatus in Food Samples.

Publications

Multiplex Real-Time PCR with HRM for Detection of <i>Lactobacillus sakei</i> and <i>Lactobacillus curvatus</i> in Food Samples.

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