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

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          SUMMARY

          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.

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          Differentiation of Lactobacillus plantarum, L. pentosus, and L. paraplantarum by recA gene sequence analysis and multiplex PCR assay with recA gene-derived primers.

          Giovanna Felis, F Dellaglio, S Torriani (2001)
          In this study, we succeeded in differentiating Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum by means of recA gene sequence comparison. Short homologous regions of about 360 bp were amplified by PCR with degenerate consensus primers, sequenced, and analyzed, and 322 bp were considered for the inference of phylogenetic trees. Phylograms, obtained by parsimony, maximum likelihood, and analysis of data matrices with the neighbor-joining model, were coherent and clearly separated the three species. The validity of the recA gene and RecA protein as phylogenetic markers is discussed. Based on the same sequences, species-specific primers were designed, and a multiplex PCR protocol for the simultaneous distinction of these bacteria was optimized. The sizes of the amplicons were 318 bp for L. plantarum, 218 bp for L. pentosus, and 107 bp for L. paraplantarum. This strategy permitted the unambiguous identification of strains belonging to L. plantarum, L. pentosus, and L. paraplantarum in a single reaction, indicating its applicability to the speciation of isolates of the L. plantarum group.
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            High-resolution melting analysis (HRMA): more than just sequence variant screening.

            Anja Roos, Emmelien Aten, Rolf H. A. M. Vossen … (2009)
            Transition of the double-stranded DNA molecule to its two single strands, DNA denaturation or melting, has been used for many years to study DNA structure and composition. Recent technological advances have improved the potential of this technology, especially to detect variants in the DNA sequence. Sensitivity and specificity were increased significantly by the development of so-called saturating DNA dyes and by improvements in the instrumentation to measure the melting behavior (improved temperature precision combined with increased measurements per time unit and drop in temperature). Melt analysis using these new instruments has been designated high-resolution melting curve analysis (HRM or HRMA). Based on its ease of use, simplicity, flexibility, low cost, nondestructive nature, superb sensitivity, and specificity, HRMA is quickly becoming the tool of choice to screen patients for pathogenic variants. Here we will briefly discuss the latest developments in HRMA and review in particular other applications that have thus far received less attention, including presequence screening, single nucleotide polymorphism (SNP) typing, methylation analysis, quantification (copy number variants and mosaicism), an alternative to gel-electrophoresis and clone characterization. Together, these diverse applications make HRMA a multipurpose technology and a standard tool that should be present in any laboratory studying nucleic acids.
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              Culture independent methods to assess the diversity and dynamics of microbiota during food fermentation.

              Luca Cocolin, Valentina Alessandria, Paola Dolci … (2013)
              Culture independent methods first appeared in the food microbiology field at the end of the 90s and since then they have been applied extensively. These methods do not rely on cultivation and target nucleic acids (DNA and RNA) to identify and follow the changes that occur in the main populations present in a specific ecosystem. The method that has most often been used as a culture independent method in food microbiology is denaturing gradient gel electrophoresis (DGGE). The number of papers dealing with DGGE grew exponentially in the late nineties and, by analysing the studies available in the literature, it is possible to describe a trend in the subjects that have been investigated. DGGE was first used as a tool to monitor the ecology of fermented food, such as fermented sausage, cheese and sourdough, and later it also showed its potential in microbial spoilage process. In the last few years, the main application of DGGE has been to study fermented food from Asia, Africa and South America. The information collected using DGGE has made it possible to confirm the existing knowledge on food fermentation and spoilage. However, in some cases, new evidence that helps scientists to fully comprehend a specific microbial ecosystem has emerged. In this review, the roadmap of culture independent methods in food microbiology will be summarized, focusing on the DGGE technique. Examples of how this approach is useful to obtain a better understanding of microbial diversity are reported for several kinds of fermented food, such as fermented sausage, cheese and wine. The future of culture independent methods in food microbiology, with the increasing availability of next generation sequencing techniques, is also discussed.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Food Technol Biotechnol
                Journal ID (iso-abbrev): Food Technol. Biotechnol
                Journal ID (publisher-id): FTB
                Title: Food Technology and Biotechnology
                Publisher: University of Zagreb Faculty of Food Technology and Biotechnology
                ISSN (Print): 1330-9862
                ISSN (Electronic): 1334-2606
                Publication date (Print and electronic): March 2019
                Publication date (Print): March 2019
                Volume: 57
                Issue: 1
                Pages: 97-104
                Affiliations
                [1 ]V.M. Gorbatov Federal Research Center for Food Systems of Russian Academy of Sciences, Talalikhina 26, 109316, Moscow, Russian Federation
                [2 ]Vavilov Institute of General Genetics, Gubkina 3, 119333, Moscow, Russian Federation
                [3 ]Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases, Sechenov University, Malaya Pirogovskaya 20-1, 119435 Moscow, Russian Federation
                Author notes
                [* ]Corresponding author:
Phone: +79629529223
E-mail: homo_ludens@ 123456vniimp.ru
                Author information
                http://orcid.org/0000-0003-1348-860X
                http://orcid.org/0000-0001-8748-9117
                http://orcid.org/0000-0002-0038-9744
                http://orcid.org/0000-0002-7474-485X
                Article
                Publisher ID: FTB-57-097
                DOI: 10.17113/ftb.57.01.19.5983
                PMC ID: 6600297
                SO-VID: 4867ef1d-eba1-48f4-92da-654dc2466556
                Copyright statement: Copyright @ 2019
                License:

                This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial (CC BY-NC) 4.0 License.

                History
                Date received : 09 August 2018
                Date accepted : 17 January 2019
                Categories
                Subject: Preliminary Communications

                Keywords: real-time pcr,hrm,starter cultures,fermented sausages,lactobacillus sakei,lactobacillus curvatus
                Data availability:
                Keywords: real-time pcr, hrm, starter cultures, fermented sausages, lactobacillus sakei, lactobacillus curvatus

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