A new methodology for rapid detection of Lactobacillus delbrueckii subsp. bulgaricus based on multiplex PCR

Nikolaou, Anastasios; Saxami, Georgia; Kourkoutas, Yiannis; Galanis, Αlex

doi:10.1016/j.mimet.2010.12.010

Cited by 8 publications

(10 citation statements)

References 11 publications

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“…To this end, several polymerase chain reaction (PCR)-based molecular methods have been presented with the power to detect, identify, and distinguish the microorganisms of interest among closely related species and strains [ 18 ]. For example, multiplex PCR assays have been developed for accurate and efficient detection of specific LAB strains in probiotic products [ 19 , 20 , 21 ]. The design of strain specific primers was performed by comparative sequence analysis.…”

Section: Introductionmentioning

confidence: 99%

Molecular Detection of Two Potential Probiotic Lactobacilli Strains and Evaluation of Their Performance as Starter Adjuncts in Yogurt Production

Saxami

Papadopoulou

Chorianopoulos

et al. 2016

IJMS

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A molecular method for efficient and accurate detection and identification of two potential probiotic lactobacilli strains isolated from fermented olives, namely Lactobacillus pentosus B281 and Lb. plantarum B282, was developed in the present study. Random Amplified Polymorphic DNA (RAPD) analysis was performed, and strain specific primers were designed and applied in a multiplex polymerase chain reaction (PCR) assay. The specificity of the assay was tested and successfully confirmed in 27 and 22 lactobacilli strains for Lb. pentosus B281 and Lb. plantarum B282, respectively. Moreover, the two strains were used as starter cultures in yogurt production. Cell enumeration followed by multiplex PCR analysis demonstrated that the two strains were present in yogurt samples at levels ≥6 log CFU/g even after 35 days of storage at 4 °C. Microbiological analysis showed that lactobacilli and streptococci were present within usual levels, whereas enterobacteriaceae and yeast/mold counts were not detected as expected. Although the pH values of the novel products were slightly lower than the control ones, the yogurt containing the probiotic cultures scored similar values compared to the control in a series of sensory tests. Overall, these results demonstrated the possible use of the two strains as starter adjuncts in the production of yogurt with potential probiotic properties.

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Section: Introductionmentioning

confidence: 99%

Molecular Detection of Two Potential Probiotic Lactobacilli Strains and Evaluation of Their Performance as Starter Adjuncts in Yogurt Production

Saxami

Papadopoulou

Chorianopoulos

et al. 2016

IJMS

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“…delbrueckii ssp. bulgaricus in food products have been demonstrated (Karapetsas et al, 2010;Nikolaou et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

Monitoring survival of Lactobacillus casei ATCC 393 in probiotic yogurts using an efficient molecular tool

Sidira

Saxami

Dimitrellou

et al. 2013

Journal of Dairy Science

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The aim of the present study was to monitor the survival of the probiotic strain Lactobacillus casei ATCC 393 during refrigerated storage of natural regular yogurts compared with Lactobacillus delbrueckii ssp. bulgaricus. Both free and immobilized cells on supports of high industrial interest, such as fruits and oat pieces, were tested. Microbiological and strain-specific multiplex PCR analysis showed that both free and immobilized Lb. casei ATCC 393 were detected in the novel products at levels required to confer a probiotic effect (at least 6 log cfu/g) for longer periods than required by the dairy industry (≥ 30 d) during storage at 4°C. In contrast, the viable bacterial density of Lb. delbrueckii ssp. bulgaricus decreased to levels <6 log cfu/g after 14 d of cold storage. Of note, the final pH of all products was 4.2 to 4.3. Acid resistance or cold tolerance of Lb. casei ATCC 393 apparently allows for increased survival compared with Lb. delbrueckii ssp. bulgaricus in these yogurt formulations.

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“…In previous studies, genes such as 16S rRNA, 16S-23S rRNA intergenic spacer region, and the elongation factor Tu (tuf) gene have been used to distinguish microorganisms at the species or subspecies level [4,9,[26][27][28][29]. However, some studies have reported that these genes share high sequence similarities without showing sufficient variabilities to allow for the differentiation between L. delbrueckii subspecies [4,13]. In contrast, we selected genetic markers specific to the genomes of each subspecies using pangenome analysis.…”

Section: Genetic Marker Specificity Testmentioning

confidence: 99%

“…Molecular-based methods for the identification and typing of lactic acid bacteria have been reported, including amplified fragment length polymorphism (AFLP), DNA-DNA hybridization (DDH), multi-locus sequence analysis (MLST), and restriction fragment length polymorphism (RFLP) [6,[10][11][12]. However, these techniques are labor-intensive, expensive, and time-consuming with low reproducibility whereas PCR-based methods are rapid, sensitive, and reliable for identifying lactic acid bacteria [4]. Of these methods, genetic markers such as the 16S rRNA gene and 16S-23S rRNA intergenic spacer region have been used to distinguish L. delbrueckii used in PCR-based methods [13].…”

Section: Introductionmentioning

confidence: 99%

Identification and Monitoring of Lactobacillus delbrueckii Subspecies Using Pangenomic-Based Novel Genetic Markers

Kim

Cho

Yang

et al. 2021

J. Microbiol. Biotechnol.

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Genetic markers currently used for the discrimination of Lactobacillus delbrueckii subspecies have low efficiency for identification at subspecies level. Therefore, our objective in this study was to select novel genetic markers for accurate identification and discrimination of six L. delbrueckii subspecies based on pangenome analysis. We evaluated L. delbrueckii genomes to avoid making incorrect conclusions in the process of selecting genetic markers due to mislabeled genomes. Genome analysis showed that two genomes of L. delbrueckii subspecies deposited at NCBI were misidentified. Based on these results, subspecies-specific genetic markers were selected by comparing the core and pangenomes. Genetic markers were confirmed to be specific for 59,196,562 genome sequences via in silico analysis. They were found in all strains of the same subspecies, but not in other subspecies or bacterial strains. These genetic markers also could be used to accurately identify genomes at the subspecies level for genomes known at the species level. A real-time PCR method for detecting three main subspecies (L. delbrueckii subsp. delbrueckii, lactis, and bulgaricus) was developed to cost-effectively identify them using genetic markers. Results showed 100% specificity for each subspecies. These genetic markers could differentiate each subspecies from 44 other lactic acid bacteria. This real-time PCR method was then applied to monitor 26 probiotics and dairy products. It was also used to identify 64 unknown strains isolated from raw milk samples and dairy products. Results confirmed that unknown isolates and subspecies contained in the product could be accurately identified using this real-time PCR method.

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A new methodology for rapid detection of Lactobacillus delbrueckii subsp. bulgaricus based on multiplex PCR

Cited by 8 publications

References 11 publications

Molecular Detection of Two Potential Probiotic Lactobacilli Strains and Evaluation of Their Performance as Starter Adjuncts in Yogurt Production

Molecular Detection of Two Potential Probiotic Lactobacilli Strains and Evaluation of Their Performance as Starter Adjuncts in Yogurt Production

Monitoring survival of Lactobacillus casei ATCC 393 in probiotic yogurts using an efficient molecular tool

Identification and Monitoring of Lactobacillus delbrueckii Subspecies Using Pangenomic-Based Novel Genetic Markers

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