Design of a New Universal Real-Time PCR System Targeting the tuf Gene for the Enumeration of Bacterial Counts in Food

2010 ◽  
Vol 73 (4) ◽  
pp. 670-679 ◽  
Author(s):  
YUICHIRO TANAKA ◽  
HAJIME TAKAHASHI ◽  
USIO SIMIDU ◽  
BON KIMURA
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Real Time Pcr ◽  
Bacterial Species ◽  
Linear Regression Analysis ◽  
Bacterial Counts ◽  
Real Time Quantitative Pcr ◽  
Tuf Gene ◽  
Wide Range ◽  
The Difference

A novel universal real-time PCR, consisting of newly designed oligonucleotide subsets, was designed for a bacterial housekeeping gene encoding the peptide elongation factor Tu. Specificity and universality were confirmed in 66 bacterial strains, including 51 genera and 63 species. The amplification kinetics of tuf gene–targeted real-time quantitative PCR were consistent in a wide range of bacterial species tested. A calibration curve (r2 = 0.97) was produced for the estimation of bacterial counts, based on measurements of representative inoculations with 10-fold serial dilutions of the cells of representative bacterial species. Linear regression analysis of the real-time PCR–derived bacterial counts and aerobic plate counts, in a total 149 samples consisting of 25 minced meat, 34 fresh-cut vegetables, and 90 fish, exhibited a high correlation (r2 = 0.84, 0.87, and 0.95, respectively) over the range of 3.0 to 9.0 log CFU/g. In total, the difference between the two methods was less than 0.5 log in 75 of these samples, and in the remaining 74 samples, the difference was 0.5 to 1.0 log. Presently, our tuf gene–targeted real-time quantitative PCR assay achieves a rapid (within 2 h) estimation of bacterial counts of 3.0 to 9.0 log CFU/g, in a practical manner.

scholarly journals Wolbachia pipientis Growth Kinetics and Susceptibilities to 13 Antibiotics Determined by Immunofluorescence Staining and Real-Time PCR

2003 ◽  
Vol 47 (5) ◽  
pp. 1665-1671 ◽  
Author(s):  
Florence Fenollar ◽  
Max Maurin ◽  
Didier Raoult
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Growth Kinetics ◽  
Real Time Pcr ◽  
Pcr Assay ◽  
Wolbachia Pipientis ◽  
Real Time Quantitative Pcr ◽  
Wide Range ◽  
Filarial Nematodes ◽  
Quantitative Pcr Assay

ABSTRACT Wolbachia spp. are strict intracellular bacteria that infect a wide range of arthropods and filarial nematodes. Filarial nematodes are important causes of human diseases. There is increasing evidence that Wolbachia spp. influence important functions in the biology of the hosts, specifically, infertility. Preliminary experiments with humans and animals have suggested that antibiotics with activity against Wolbachia may help to treat filariasis. In this study, we determined using a real-time quantitative PCR assay the growth kinetics of a strain of Wolbachia pipientis from a mosquito grown in Aa23 cells. The doubling time was estimated to be 14 h. We then determined the susceptibilities of this strain to 13 antibiotics by two methods: an immunofluorescent-antibody test and a real-time quantitative PCR assay. Both techniques gave similar results. Doxycycline and rifampin were the most effective compounds, with MICs of 0.125 and 0.06 to 0.125 μg/ml, respectively. Fluoroquinolones were less effective, with MICs of 2 to 4 μg/ml for ciprofloxacin, 2 μg/ml for ofloxacin, and 1 μg/ml for levofloxacin. β-Lactams (penicillin G, amoxicillin, ceftriaxone) were not effective at concentrations up to 128 μg/ml. The MIC of erythromycin was >32 μg/ml, whereas that of telithromycin was 8 μg/ml. Other antibiotic compounds were bacteriostatic only at high concentrations, including gentamicin, co-trimoxazole, and thiamphenicol. The real-time PCR assay was a convenient and reliable technique for determination of the antibiotic susceptibilities of Wolbachia. It may help in the future to simplify antibiotic susceptibility testing of strict intracellular pathogens.

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.

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.

Development of a Quantitative Real-Time PCR Method To Enumerate Total Bacterial Counts in Ready-to-Eat Fruits and Vegetables

2006 ◽  
Vol 69 (10) ◽  
pp. 2504-2508 ◽  
Author(s):  
HAJIME TAKAHASHI ◽  
HIROTAKA KONUMA ◽  
YUKIKO HARA-KUDO
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Fruits And Vegetables ◽  
Bacterial Species ◽  
Culture Method ◽  
Plate Count ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Cycle Number ◽  
Wide Range

A newly developed real-time PCR assay rapidly quantifies the total bacterial numbers in contaminated ready-to-eat vegetables and fruits compared with the standard plate count method. Primers targeting the rpoB gene, which encodes for the β subunit of the bacterial RNA polymerase and which is common to most bacterial species, was used instead of the 16S rRNA gene, which has multiple copies and varies among bacterial species. A primer pair specific for rpoB was confirmed to amplify rpoB in a wide range of bacterial species after we assessed 49 strains isolated from five kinds of fruits and vegetables. We purchased fruits and vegetables from retail shops and enumerated the bacteria associated with them by use of real-time PCR and compared this to the number found by the culture method. We found a high correlation between the threshold PCR cycle number when compared with the plate count culture number. The real-time PCR assay developed in this study can enumerate the dominant bacterial species in ready-to-eat fruits and vegetables.

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.

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 Identification of Lactobacillus delbrueckii and Streptococcus thermophilus Strains Present in Artisanal Raw Cow Milk Cheese Using Real-time PCR and Classic Plate Count Methods

2017 ◽  
Vol 66 (4) ◽  
pp. 491-499 ◽  
Author(s):  
Milena A. Stachelska
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Real Time Pcr ◽  
Streptococcus Thermophilus ◽  
Lactobacillus Delbrueckii ◽  
Cow Milk ◽  
Plate Count ◽  
Real Time Quantitative Pcr ◽  
Count Method ◽  
Plate Count Method

The aim of this paper was to detect Lactobacillus delbrueckii and Streptococcus thermophilus using real-time quantitative PCR assay in 7-day ripening cheese produced from unpasteurised milk. Real-time quantitative PCR assays were designed to identify and enumerate the chosen species of lactic acid bacteria (LAB) in ripened cheese. The results of molecular quantification and classic bacterial enumeration showed a high level of similarity proving that DNA extraction was carried out in a proper way and that genomic DNA solutions were free of PCR inhibitors. These methods revealed the presence of L. delbrueckii and S. thermophilus. The real-time PCR enabled quantification with a detection of 101–103 CFU/g of product. qPCR-standard curves were linear over seven log units down to 101 copies per reaction; efficiencies ranged from 77.9% to 93.6%. Cheese samples were analysed with plate count method and qPCR in parallel. Compared with the classic plate count method, the newly developed qPCR method provided faster and species specific identification of two dairy LAB and yielded comparable quantitative results.

scholarly journals Detection of Human Cytomegalovirus DNA by Real-Time Quantitative PCR

2000 ◽  
Vol 38 (7) ◽  
pp. 2734-2737 ◽  
Author(s):  
Andreas Nitsche ◽  
Nina Steuer ◽  
Christian Andreas Schmidt ◽  
Olfert Landt ◽  
Heinz Ellerbrok ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Real Time ◽  
Quantitative Pcr ◽  
Human Cytomegalovirus ◽  
Real Time Pcr ◽  
Blood Donors ◽  
Marrow Transplant ◽  
Pcr Assay ◽  
Real Time Quantitative Pcr ◽  
Transplant Patients

A real-time PCR assay was developed to quantify human cytomegalovirus (CMV) DNA. This assay was used to demonstrate a higher CMV DNA load in plasma of bone marrow transplant patients than in that of blood donors. The CMV load was higher in CMV antigen-positive patients than in antigen-negative patients.

scholarly journals Molecular identification of Anoplophora glabripennis (Coleoptera: Cerambycidae) and detection from frass samples based on real-time quantitative PCR

2021 ◽  
Author(s):  
Andrea Taddei ◽  
Matthias Becker ◽  
Beatrice Berger ◽  
Daniele Da Lio ◽  
Stephanie Feltgen ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Molecular Identification ◽  
Life Stage ◽  
Diagnostic Tools ◽  
Morphological Identification ◽  
Anoplophora Glabripennis ◽  
Real Time Quantitative Pcr ◽  
Non Invasive ◽  
Wide Range

AbstractAnoplophora glabripennis (Motschulsky 1853) (Coleoptera: Cerambycidae), the Asian Longhorned Beetle, is native to temperate and subtropical areas of China and the Korean peninsula. Due to its wide range of host plants, it is considered among the most economically important invasive plant pests. The morphological identification of A. glabripennis larvae can be confirmed by DNA barcoding, but obtaining the specimens from infested trees can be a demanding and challenging task. Therefore, non-invasive diagnostic tools based on DNA extracted from frass samples can be of key importance in phytosanitary surveys. In this study, an in silico generated real-time quantitative PCR test was developed for the detection of A. glabripennis DNA from frass material, which is naturally extruded from larval tunnels through cracks in the bark. Specificity was confirmed against a wide range of other wood-boring insect species frequently encountered during phytosanitary surveys and inclusivity was demonstrated for different populations of A. glabripennis from all main European outbreak areas. The test proved sensitive and reliable in detecting A. glabripennis DNA extracted from woody frass material of Acer saccharinum and Aesculus hippocastanum at least up to the 100-fold dilution. Furthermore, the test allowed the molecular identification of any life stage of the insect, including eggs and young larvae, whose morphological identification is impossible or very challenging. This study provides a reliable and sensitive molecular tool to detect A. glabripennis DNA in woody frass material, thus allowing a non-invasive sampling approach.

Rapid and direct quantitative detection of viable bifidobacteria in probiotic yogurt by combination of ethidium monoazide and real-time PCR using a molecular beacon approach

2010 ◽  
Vol 77 (4) ◽  
pp. 498-504 ◽  
Author(s):  
XC Meng ◽  
R Pang ◽  
C Wang ◽  
LQ Wang
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Molecular Beacon ◽  
Linear Regression Analysis ◽  
Quantitative Detection ◽  
Ethidium Monoazide ◽  
Real Time Quantitative Pcr ◽  
Probiotic Yogurt ◽  
Culture Independent

The potential of ethidium monoazide (EMA) real-time PCR method based on molecular beacon probe for rapid detection of viable bifidobacteria present in probiotic yogurt was evaluated in this work. A real-time PCR with molecular beacon assay was developed to determine genusBifidobacteriumquantitatively in order to increase the sensitivity and specificity of assay. EMA was used to treat probiotic yogurt prior to DNA extraction and real-time PCR detection to allow detection of only viable bacteria. The primer set of Bif-F/Bif-R which is genus-specific forBifid. was designed. The specificity of the probes ensures that no signal is generated by non-target amplicons. Linear regression analysis demonstrated a good correlation (R2=0·9948) between the EMA real-time PCR results and the plate counting, and real-time quantitative PCR results correlated adequately with enumeration of bifidobacteria by culture for commercial probiotic yogurt. This culture-independent approach is promising for the direct and rapid detection of viable bifidobacteria in commercial probiotic yogurt, and the detection can be carried out within 4 h. The detection limit for this method is about 104cell/ml. In conclusion, the direct quantitative EMA real-time PCR assay based on molecular beacon described in this research is a rapid and quantitative method.

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