Real-time PCR identification of Listeria monocytogenes serotype 4c using primers for novel target genes obtained by comparative genomic analysis

ABSTRACT We developed and assessed real-time PCR (RTi-PCR) assays for the detection and quantification of the food-borne pathogen Listeria monocytogenes and the closely related nonpathogenic species L. innocua. The target genes were hly and iap for L. monocytogenes and lin02483 for L. innocua. The assays were 100% specific, as determined with 100 Listeria strains and 45 non-Listeria strains, and highly sensitive, with detection limits of one target molecule in 11 to 56% of the reactions with purified DNA and 3 CFU in 56 to 89% of the reactions with bacterial suspensions. Quantification was possible over a 5-log dynamic range, with a limit of 15 target molecules and R 2 values of >0.996. There was an excellent correspondence between the predicted and the actual numbers of CFU in the samples (deviations of <23%). The hly-based assay accurately quantified L. monocytogenes in all of the samples tested. The iap-based assay, in contrast, was unsuitable for quantification purposes, underestimating the bacterial counts by 3 to 4 log units in a significant proportion of the samples due to serovar-related target sequence variability. The combination of the two assays enabled us to classify L. monocytogenes isolates into one of the two major phylogenetic divisions of the species, I and II. We also assessed the new AmpliFluor technology for the quantitative detection of L. monocytogenes by RTi-PCR. The performance of this system was similar to that of the TaqMan system, although the former system was slightly less sensitive (detection limit of 15 molecules in 45% of the reactions) and had a higher quantification limit (60 molecules).

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Draft Genome Sequence of Listeria monocytogenes CIIMS-NV-3, a Strain Isolated from Vaginal Discharge of a Woman from Central India

Microbiology Resource Announcements ◽

10.1128/mra.01553-18 ◽

2019 ◽

Vol 8 (6) ◽

Author(s):

Pooja M. Kishnani ◽

Nitin V. Kurkure ◽

Sukhadeo B. Barbuddhe ◽

Swapnil P. Doijad ◽

Trinad Chakraborty ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Genome Sequence ◽

Draft Genome ◽

Genomic Analysis ◽

Central India ◽

Vaginal Swab ◽

Comparative Genomic Analysis ◽

Draft Genome Sequence ◽

Comparative Genomic ◽

Content Type

We present here the draft genome sequence of Listeria monocytogenes CIIMS-NV-3, a serovar 4b strain isolated from the vaginal swab of a female patient from central India. The availability of this genome may provide useful information on virulence characteristics for comparative genomic analysis.

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Simultaneous detection of 37 Lactobacillus species using a real-time PCR assay based on whole-genome sequence analysis

10.21203/rs.2.20588/v1 ◽

2020 ◽

Author(s):

Eiseul Kim ◽

Seung-Min Yang ◽

Bora Lim ◽

Si Hong Park ◽

Bryna Rackerby ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Real Time ◽

Real Time Pcr ◽

Genomic Analysis ◽

23S Rrna ◽

Comparative Genomic ◽

Rrna Gene ◽

Gene Sequences ◽

Species Specific

Abstract BackgroundLactobacillus species are used as probiotics and play an important role in fermented food production. However, use of 16S rRNA gene sequences as standard markers for the differentiation of Lactobacillus species offers a very limited scope, as several species of Lactobacillus share similar 16S rRNA gene sequences. In this study, we developed a rapid and accurate method based on comparative genomic analysis for the simultaneous identification of 37 Lactobacillus species that are commonly used in probiotics and fermented foods. ResultsTo select species-specific sequences or genes, a total of 143 Lactobacillus complete genome sequences were compared using Python scripts. In 14 out of 37 species, species-specific sequences could not be found due to the similarity of the 16S–23S rRNA gene. Selected unique genes were obtained using comparative genomic analysis and all genes were confirmed to be specific for 52,478,804 genomes via in silico analysis; they were found not to be strain-specific, but to exist in all strains of the same species. Species-specific primer pairs were designed from the selected 16S–23S rRNA gene sequences or unique genes of species. The specificity of the species-specific primer pairs was confirmed using reference strains, and the accuracy and efficiency of the real-time polymerase chain reaction (PCR) with the standard curve were confirmed. The real-time PCR method developed in this study is able to accurately differentiate species that were not distinguishable using the 16S rRNA gene alone. This Real-time PCR method was designed to detect 37 Lactobacillus species in a single reaction. The developed method was then applied in the monitoring of 19 probiotics and 12 dairy products. The applied tests confirmed that the species detected in 17 products matched those indicated on their labels, whereas the remaining products contained species other than those appearing on the label. ConclusionsThe method developed in this study is able to rapidly and accurately distinguish different species of Lactobacillus, and can be used to monitor specific Lactobacillus species in foods such as probiotics and dairy products.

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Protocol - Comparative genomic analysis of Listeria monocytogenes isolated from environments of salmon processing plants and human cases in Norway 2005-2016 - DNA isolation, WGS, and cgMLST v1 (protocols.io.bcuciwsw)

protocols.io ◽

10.17504/protocols.io.bcuciwsw ◽

2020 ◽

Author(s):

Trond Lovdal

Keyword(s):

Listeria Monocytogenes ◽

Dna Isolation ◽

Genomic Analysis ◽

Comparative Genomic Analysis ◽

Comparative Genomic ◽

Processing Plants

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Validation of the TadpoleTMCampylobacter jejuni Real-Time PCR Identification Kit

Journal of AOAC International ◽

10.5740/jaoacint.18-0323 ◽

2019 ◽

Vol 102 (3) ◽

pp. 842-854

Author(s):

Yuan Jiang ◽

Jinling Shen ◽

Feng Xue ◽

Lina Zhao ◽

Jielin Yang ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Target Genes ◽

Accurate Method ◽

Common Species ◽

Stability Study ◽

Time Saving ◽

Columbia Blood Agar ◽

Negative Results ◽

Pcr Identification

Abstract Background: The gene-based real-time PCR method for identification of Campylobacter jejuni is more simple, rapid and accurate than the traditional biochemical method. Objective: A performance validation of the TadpoleTMCampylobacter jejuni Real-Time PCR Identification Kit was performed. Method: The assay uses TaqMan Real-time PCR technology to amplify target genes from isolated colonies. Bacterial deoxyribonucleic acid (DNA) from inclusivity and exclusivity organisms cultured on Columbia Blood Agar, Campy-Cefex agar and modified Charcoal Cefoperazone Deoxycholate was extracted and analyzed on three instruments: Applied Biosystems (ABI) 7500 Fast, ABI StepOne Plus and Bio-Rad CFX96. Results: When 57 distinct strains of C. jejuni were tested for inclusivity, all 57 strains produced positive results on the three instruments. In exclusivity testing, all 35 strains of related organisms, including 7 non-target Campylobacter strains and other common species, produced negative results on the three instruments. The Independent Laboratory validation consisting of an inclusivity and exclusivity evaluation for 10 C. jejuni isolates and 10 nontarget Campylobacter isolates also showed 100% expected results on the three instruments. In addition, in robustness testing, small, deliberate changes to the assay parameters, including cell suspension turbidity, heat lysis time, and DNA template volume in the PCR reaction, did not affect the kit performance. Finally, the combined lot-to-lot and stability study on both the ABI 7500 Fast and the ABI StepOne Plus showed that the 11 C. jejuni strains and 5 nontarget Campylobacter strains can be correctly identified by the three independently manufactured, lots and it supported a shelf life of 9 months when stored at –20°C. Conclusions: The Tadpole method offers a rapid, accurate, and robust alternative for C. jejuni identification. Highlights: Rapid and accurate method to identify C. jejuni, which has a good robustness and high stability. It is flexible and offers the advantages of reduced labor and time saving.

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