scholarly journals New Real-Time Quantitative PCR Procedure for Quantification of Bifidobacteria in Human Fecal Samples

2004 ◽  
Vol 70 (7) ◽  
pp. 4165-4169 ◽  
Author(s):  
Miguel Gueimonde ◽  
Satu Tölkkö ◽  
Teemu Korpimäki ◽  
Seppo Salminen
Keyword(s):  
In Situ Hybridization ◽  
Detection Limit ◽  
Real Time ◽  
Quantitative Pcr ◽  
Real Time Pcr ◽  
Fluorescent In Situ Hybridization ◽  
The Real ◽  
Real Time Quantitative Pcr ◽  
Lanthanide Chelate

ABSTRACT The application of a real-time quantitative PCR method (5′ nuclease assay), based on the use of a probe labeled at its 5′ end with a stable, fluorescent lanthanide chelate, for the quantification of human fecal bifidobacteria was evaluated. The specificities of the primers and the primer-probe combination were evaluated by conventional PCR and real-time PCR, respectively. The results obtained by real-time PCR were compared with those obtained by fluorescent in situ hybridization, the current gold standard for intestinal microbiota quantification. In general, a good correlation between the two methods was observed. In order to determine the detection limit and the accuracy of the real-time PCR procedure, germfree rat feces were spiked with known amounts of bifidobacteria and analyzed by both methods. The detection limit of the method used in this study was found to be about 5 × 104 cells per g of feces. Both methods, real-time PCR and fluorescent in situ hybridization, led to an accurate quantification of the spiked samples with high levels of bifidobacteria, but real-time PCR was more accurate for samples with low levels. We conclude that the real-time PCR procedure described here is a specific, accurate, rapid, and easy method for the quantification of bifidobacteria in feces.

Detection and Enumeration of Salmonella Enteritidis in Homemade Ice Cream Associated with an Outbreak: Comparison of Conventional and Real-Time PCR Methods

2006 ◽  
Vol 69 (3) ◽  
pp. 639-643 ◽  
Author(s):  
K. H. SEO ◽  
I. E. VALENTIN-BON ◽  
R. E. BRACKETT
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Real Time Pcr ◽  
Salmonella Enteritidis ◽  
Direct Detection ◽  
Ice Cream ◽  
Plate Count ◽  
Pcr Assay ◽  
The Real ◽  
Real Time Quantitative Pcr

Salmonellosis caused by Salmonella Enteritidis (SE) is a significant cause of foodborne illnesses in the United States. Consumption of undercooked eggs and egg-containing products has been the primary risk factor for the disease. The importance of the bacterial enumeration technique has been enormously stressed because of the quantitative risk analysis of SE in shell eggs. Traditional enumeration methods mainly depend on slow and tedious most-probable-number (MPN) methods. Therefore, specific, sensitive, and rapid methods for SE quantitation are needed to collect sufficient data for risk assessment and food safety policy development. We previously developed a real-time quantitative PCR assay for the direct detection and enumeration of SE and, in this study, applied it to naturally contaminated ice cream samples with and without enrichment. The detection limit of the real-time PCR assay was determined with artificially inoculated ice cream. When applied to the direct detection and quantification of SE in ice cream, the real-time PCR assay was as sensitive as the conventional plate count method in frequency of detection. However, populations of SE derived from real-time quantitative PCR were approximately 1 log higher than provided by MPN and CFU values obtained by conventional culture methods. The detection and enumeration of SE in naturally contaminated ice cream can be completed in 3 h by this real-time PCR method, whereas the cultural enrichment method requires 5 to 7 days. A commercial immunoassay for the specific detection of SE was also included in the study. The real-time PCR assay proved to be a valuable tool that may be useful to the food industry in monitoring its processes to improve product quality and safety.

Quantification and Differentiation of Campylobacter jejuni and Campylobacter coli in Raw Chicken Meats Using a Real-Time PCR Method

2007 ◽  
Vol 70 (9) ◽  
pp. 2015-2022 ◽  
Author(s):  
JOONBAE HONG ◽  
WOO KYUNG JUNG ◽  
JUN MAN KIM ◽  
SO HYUN KIM ◽  
HYE CHEONG KOO ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
High Specificity ◽  
Campylobacter Coli ◽  
Pcr Assay ◽  
The Real ◽  
Real Time Quantitative Pcr ◽  
Campylobacter Species

Campylobacter species are one of the most common causes of bacterial diarrhea in humans worldwide. The consumption of foods contaminated with two Campylobacter species, C. jejuni and C. coli, is usually associated with most of the infections in humans. In this study, a rapid, reliable, and sensitive multiplex real-time quantitative PCR was developed for the simultaneous detection, identification, and quantification of C. jejuni and C. coli. In addition, the developed method was applied to the 50 samples of raw chicken meat collected from retail stores in Korea. C. jejuni and C. coli were detected in 88 and 86% of the samples by real-time quantitative PCR and the conventional microbiological method, respectively. The specificity of the primer and probe sets was confirmed with 30 C. jejuni, 20 C. coli, and 35 strains of other microbial species. C. jejuni and C. coli could be detected with high specificity in less than 4 h, with a detection limit of 1 log CFU/ml by the developed real-time PCR. The average counts (log CFU per milliliter) of C. jejuni or C. coli obtained by the conventional methods and by the real-time PCR assay were statistically correlated with a correlation coefficient (R2) between 0.73 and 0.78. The real-time PCR assay developed in this study is useful for screening for the presence and simultaneous differential quantification of C. jejuni and C. coli.

scholarly journals Effects of DNA Extraction and Purification Methods on Real-Time Quantitative PCR Analysis of Roundup Ready Soybean

2009 ◽  
Vol 92 (4) ◽  
pp. 1136-1144 ◽  
Author(s):  
Tigst Demeke ◽  
Indira Ratnayaka ◽  
Anh Phan
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Quantitative Pcr ◽  
Real Time Pcr ◽  
Roundup Ready ◽  
Dna Recovery ◽  
Real Time Quantitative Pcr ◽  
Extraction And Purification ◽  
Purification Methods ◽  
Accuracy And Repeatability

Abstract The quality of DNA affects the accuracy and repeatability of quantitative PCR results. Different DNA extraction and purification methods were compared for quantification of Roundup Ready (RR) soybean (event 40-3-2) by real-time PCR. DNA was extracted using cetylmethylammonium bromide (CTAB), DNeasy Plant Mini Kit, and Wizard Magnetic DNA purification system for food. CTAB-extracted DNA was also purified using the Zymo (DNA Clean & Concentrator 25 kit), Qtip 100 (Qiagen Genomic-Tip 100/G), and QIAEX II Gel Extraction Kit. The CTAB extraction method provided the largest amount of DNA, and the Zymo purification kit resulted in the highest percentage of DNA recovery. The Abs260/280 and Abs260/230 ratios were less than the expected values for some of the DNA extraction and purification methods used, indicating the presence of substances that could inhibit PCR reactions. Real-time quantitative PCR results were affected by the DNA extraction and purification methods used. Further purification or dilution of the CTAB DNA was required for successful quantification of RR soybean. Less variability of quantitative PCR results was observed among experiments and replications for DNA extracted and/or purified by CTAB, CTAB+Zymo, CTAB+Qtip 100, and DNeasy methods. Correct and repeatable results for real-time PCR quantification of RR soybean were achieved using CTAB DNA purified with Zymo and Qtip 100 methods.

Quantification of an Eikelboom type 021N bulking event with fluorescence in situ hybridization and real-time PCR

2005 ◽  
Vol 68 (5) ◽  
pp. 695-704 ◽  
Author(s):  
Han Vervaeren ◽  
Kurt De Wilde ◽  
Jorg Matthys ◽  
Nico Boon ◽  
Lutgarde Raskin ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Real Time ◽  
Real Time Pcr
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