scholarly journals Differentiation of Lacticaseibacillus zeae Using Pan-Genome Analysis and Real-Time PCR Method Targeting a Unique Gene

Foods ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2112
Author(s):  
Eiseul Kim ◽  
Seung-Min Yang ◽  
Hae-Yeong Kim
Keyword(s):  
Real Time ◽  
Genome Analysis ◽  
Real Time Pcr ◽  
Raw Milk ◽  
Rrna Gene ◽  
Probiotic Properties ◽  
The Real ◽  
Unique Gene ◽  
Pan Genome ◽  
Pcr Method

Lacticaseibacillus zeae strains, isolated from raw milk and fermented dairy products, are closely related to the Lacticaseibacillus species that has beneficial probiotic properties. However, it is difficult to distinguish those using conventional methods. In this study, a unique gene was revealed to differentiate L. zeae from other strains of the Lacticaseibacillus species and other species by pan-genome analysis, and a real-time PCR method was developed to rapidly and accurately detect the unique gene. The genome analysis of 141 genomes yielded an 17,978 pan-genome. Among them, 18 accessory genes were specifically present in five genomes of L. zeae. The glycosyltransferase family 8 was identified as a unique gene present only in L. zeae and not in 136 other genomes. A primer designed from the unique gene accurately distinguished L. zeae in pure and mixed DNA and successfully constructed the criterion for the quantified standard curve in real-time PCR. The real-time PCR method was applied to 61 strains containing other Lacticaseibacillus species and distinguished L. zeae with 100% accuracy. Also, the real-time PCR method was proven to be superior to the 16S rRNA gene method in the identification of L. zeae.

scholarly journals Real-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine

2003 ◽  
Vol 69 (12) ◽  
pp. 7430-7434 ◽  
Author(s):  
Trevor G. Phister ◽  
David A. Mills
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Primers ◽  
Rrna Gene ◽  
Dekkera Bruxellensis ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Spoilage Yeast ◽  
Pcr Method ◽  
26S Rrna

ABSTRACT Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis.

Relationship among the potato rot nematode, Ditylenchus destructor, densities in soil, root and garlic (Allium sativum) bulbs, and rot damage in stored garlic bulbs

Nematology ◽  
2019 ◽  
Vol 21 (5) ◽  
pp. 547-555 ◽  
Author(s):  
Zejun Cheng ◽  
Koki Toyota ◽  
Rie Aoyama
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Significant Positive Correlation ◽  
Allium Sativum ◽  
Outer Skin ◽  
Positive Correlation ◽  
The Real ◽  
Pcr Method ◽  
Ditylenchus Destructor

Summary The potato rot nematode, Ditylenchus destructor, threatens garlic production in Japan. The objectives of this study were to determine the relationships of D. destructor densities in soil, garlic roots and outer skins of garlic bulbs, and damage to bulbs that rot during storage. Ditylenchus destructor densities were evaluated with the real-time PCR method. There was a significant positive correlation between D. destructor densities in soil at planting and those in the outer skin of garlic bulbs at harvest in 2016, but not in 2017. Ditylenchus destructor densities in outer skins at harvest were consistently low when those in roots at harvest were lower than 80 ind. (0.05 g)−1. No damage to garlic bulbs after storage was observed when D. destructor densities in outer skins were lower than 300 ind. (0.05 g)−1. These results indicate that D. destructor densities in roots and outer skins may be a good indicator to estimate nematode damage to garlic bulbs after storage.

scholarly journals Evaluation of the activity of thermostable DNA polymerases in the presence of heme, as a key inhibitor in the real time PCR method in diagnostics of sepsis.

2013 ◽  
Vol 60 (4) ◽  
Author(s):  
Tomasz Gosiewski ◽  
Monika Brzychczy-Włoch ◽  
Agata Pietrzyk ◽  
Agnieszka Sroka ◽  
Małgorzata Bulanda
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dna Polymerases ◽  
The Real ◽  
Pcr Method ◽  
Dna Template ◽  
Selection Of

The study aim was evaluation of the usefulness of several thermostable DNA polymerases in real time PCR conducted in the presence of the heme. Our study had the advantage of testing several different polymerases, one of which proved to be the least sensitive to heme activity. We also found that there is no need of supplementing the reaction mixture with protective substances like BSA. Selection of the appropriate polymerase can increase the efficiency of the PCR reaction which is very important for diagnosis of sepsis and for other analyses performed on DNA template isolated from the blood.

scholarly journals Contamination and Sensitivity Issues with a Real-Time Universal 16S rRNA PCR

2000 ◽  
Vol 38 (5) ◽  
pp. 1747-1752 ◽  
Author(s):  
C. E. Corless ◽  
M. Guiver ◽  
R. Borrow ◽  
V. Edwards-Jones ◽  
E. B. Kaczmarski ◽  
...  
Keyword(s):  
16S Rrna ◽  
Real Time ◽  
Dna Polymerase ◽  
Real Time Pcr ◽  
Rrna Gene ◽  
Restriction Digestion ◽  
Bacterial Dna ◽  
The Real ◽  
Log Reduction ◽  
Highly Sensitive

A set of universal oligonucleotide primers specific for the conserved regions of the eubacterial 16S rRNA gene was designed for use with the real-time PCR Applied Biosystems 7700 (TaqMan) system. During the development of this PCR, problems were noted with the use of this gene as an amplification target. Contamination of reagents with bacterial DNA was a major problem exacerbated by the highly sensitive nature of the real-time PCR chemistry. This was compounded by the use of a small amplicon of approximately 100 bases, as is necessary with TaqMan chemistry. In an attempt to overcome this problem, several methodologies were applied. Certain treatments were more effective than others in eliminating the contaminating DNA; however, to achieve this there was a decrease in sensitivity. With UV irradiation there was a 4-log reduction in PCR sensitivity, with 8-methoxypsoralen activity facilitated by UV there was between a 5- and a 7-log reduction, and with DNase alone and in combination with restriction digestion there was a 1.66-log reduction. Restriction endonuclease treatment singly and together did not reduce the level of contaminating DNA. Without the development of ultrapure Taq DNA polymerase, ultrapure reagents, and plasticware guaranteed to be free of DNA, the implementation of a PCR for detection of eubacterial 16S rRNA with the TaqMan system will continue to be problematical.

Analysis of community structures in anaerobic processes using a quantitative real-time PCR method

2005 ◽  
Vol 52 (1-2) ◽  
pp. 85-91 ◽  
Author(s):  
Y. Yu ◽  
C. Lee ◽  
S. Hwang
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rrna Gene ◽  
Quantitative Real Time Pcr ◽  
Community Structures ◽  
Specific Primer ◽  
Anaerobic Processes ◽  
Promising Tool ◽  
Pcr Method ◽  
Group Specific Primer

The methanogenic community structures of four different anaerobic processes were characterized using a quantitative real-time PCR with group-specific primer and probe sets targeting the 16S rRNA gene (rDNA). The group specific primer and probe sets were developed and used to detect the orders Methanosarcinales, and the families Methanosarcinaceae and Methanosaetaceae. Two separate sets targeting the domains Archaea and Bacteria were also used. Each microbial population in different anaerobic processes was determined and the relative abundance in the system was compared with each other. Dominant methanogenic populations and the community structures in the processes were varied by hydraulic retention time and acetate concentration. This indicates that the real-time PCR method with the primer and probe sets is a promising tool to analyze community structures in anaerobic processes.

scholarly journals New Approach Using the Real-Time PCR Method for Estimation of the Toxic Marine Dinoflagellate Ostreopsis cf. ovata in Marine Environment

PLoS ONE ◽  
2011 ◽  
Vol 6 (3) ◽  
pp. e17699 ◽  
Author(s):  
Federico Perini ◽  
Anna Casabianca ◽  
Cecilia Battocchi ◽  
Stefano Accoroni ◽  
Cecilia Totti ◽  
...  
Keyword(s):  
Real Time ◽  
Marine Environment ◽  
Real Time Pcr ◽  
New Approach ◽  
The Real ◽  
Marine Dinoflagellate ◽  
Pcr Method

scholarly journals Development and Validation of a Real-Time PCR Method To Quantify Rumen Protozoa and Examination of Variability between Entodinium Populations in Sheep Offered a Hay-Based Diet

2006 ◽  
Vol 72 (1) ◽  
pp. 200-206 ◽  
Author(s):  
Lucy C. Skillman ◽  
Andrew F. Toovey ◽  
Andrew J. Williams ◽  
André-Denis G. Wright
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Primers ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Advantages And Disadvantages ◽  
Pcr Efficiency ◽  
Rumen Protozoa ◽  
Pcr Method ◽  
Serial Dilutions

ABSTRACT PCR and real-time PCR primers for the 18S rRNA gene of rumen protozoa (Entodinium and Dasytricha spp.) were designed, and their specificities were tested against a range of rumen microbes and protozoal groups. External standards were prepared from DNA extracts of a rumen matrix containing known numbers and species of protozoa. The efficiency of PCR (ε) was calculated following amplification of serial dilutions of each standard and was used to calculate the numbers of protozoa in each sample collected; serial dilutions of DNA were used similarly to calculate PCR efficiency. Species of Entodinium, the most prevalent of the rumen protozoa, were enumerated in rumen samples collected from 100 1-year-old merino wethers by microscopy and real-time PCR. Both the counts developed by the real-time PCR method and microscopic counts were accurate and repeatable, with a strong correlation between them (R 2 = 0.8), particularly when the PCR efficiency was close to optimal (i.e., two copies per cycle). The advantages and disadvantages of each procedure are discussed. Entodinium represented on average 98% of the total protozoa, and populations within the same sheep were relatively stable, but greater variation occurred between different sheep (100 and 106 entodinia per gram of rumen contents). With this inherent variability, it was estimated that, to detect a statistically significant (P = 0.05) 20% change in Entodinium populations, 52 sheep per treatment group would be required.

scholarly journals SIMPLIFYING CELIAC DISEASE PREDISPOSING HLA-DQ ALLELES DETERMINATION BY THE REAL TIME PCR METHOD

2015 ◽  
Vol 52 (2) ◽  
pp. 143-146 ◽  
Author(s):  
Nicole SELLESKI ◽  
Lucas Malta ALMEIDA ◽  
Fernanda Coutinho de ALMEIDA ◽  
Lenora GANDOLFI ◽  
Riccardo PRATESI ◽  
...  
Keyword(s):  
Celiac Disease ◽  
Real Time ◽  
Internal Control ◽  
Real Time Pcr ◽  
Melting Curve ◽  
Melting Curve Analysis ◽  
Specific Primers ◽  
The Real ◽  
Hla Dq ◽  
Pcr Method

Background Celiac disease is an autoimmune enteropathy triggered by the ingestion of gluten in genetically susceptible individuals. Genetic susceptibility is associated with two sets of alleles, DQA1*05 - DQB1*02 and DQA1*03 - DQB1*03:02, which code for class II MHC DQ2 and DQ8 molecules, respectively. Approximately 90%-95% of celiac patients are HLA-DQ2 positive, and half of the remaining patients are HLA-DQ8 positive. In fact, during a celiac disease diagnostic workup, the absence of these specific DQA and DQB alleles has a near perfect negative predictive value. Objective Improve the detection of celiac disease predisposing alleles by combining the simplicity and sensitivity of real-time PCR (qPCR) and melting curve analysis with the specificity of sequence-specific primers (SSP). Methods Amplifications of sequence-specific primers for DQA1*05 (DQ2), DQB1*02 (DQ2), and DQA1*03 (DQ8) were performed by the real time PCR method to determine the presence of each allele in independent reactions. Primers for Human Growth Hormone were used as an internal control. A parallel PCR-SSP protocol was used as a reference method to validate our results. Results Both techniques yielded equal results. From a total of 329 samples the presence of HLA predisposing alleles was determined in 187 (56.8%). One hundred fourteen samples (61%) were positive for a single allele, 68 (36.3%) for two alleles, and only 5 (2.7%) for three alleles. Conclusion Results obtained by qPCR technique were highly reliable with no discordant results when compared with those obtained using PCR-SSP.

Detection of Yersinia Enterocolitica Species in Pig Tonsils and Raw Pork Meat by the Real-Time Pcr and Culture Methods

2017 ◽  
Vol 20 (3) ◽  
pp. 477-484 ◽  
Author(s):  
M.A. Stachelska
Keyword(s):  
Real Time ◽  
Yersinia Enterocolitica ◽  
Real Time Pcr ◽  
Culture Method ◽  
Molecular Techniques ◽  
Pork Meat ◽  
Culture Methods ◽  
The Real ◽  
Pcr Method ◽  
Classical Culture

AbstractThe aim of the present study was to establish a rapid and accurate real-time PCR method to detect pathogenic Yersinia enterocolitica in pork. Yersinia enterocolitica is considered to be a crucial zoonosis, which can provoke diseases both in humans and animals. The classical culture methods designated to detect Y. enterocolitica species in food matrices are often very time-consuming. The chromosomal locus _tag CH49_3099 gene, that appears in pathogenic Y. enterocolitica strains, was applied as DNA target for the 5’ nuclease PCR protocol. The probe was labelled at the 5’ end with the fluorescent reporter dye (FAM) and at the 3’ end with the quencher dye (TAMRA). The real-time PCR cycling parameters included 41 cycles. A Ct value which reached a value higher than 40 constituted a negative result. The developed for the needs of this study qualitative real-time PCR method appeared to give very specific and reliable results. The detection rate of locus _tag CH49_3099 - positive Y. enterocolitica in 150 pig tonsils was 85 % and 32 % with PCR and culture methods, respectively. Both the Real-time PCR results and culture method results were obtained from material that was enriched during overnight incubation. The subject of the study were also raw pork meat samples. Among 80 samples examined, 7 ones were positive when real-time PCR was applied, and 6 ones were positive when classical culture method was applied. The application of molecular techniques based on the analysis of DNA sequences such as the Real-time PCR enables to detect this pathogenic bacteria very rapidly and with higher specificity, sensitivity and reliability in comparison to classical culture methods.

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