Rapid, Specific, and Sensitive Detection of Spoilage Molds in Orange Juice Using a Real-Time Taqman PCR Assay

2006 ◽  
Vol 69 (2) ◽  
pp. 385-390 ◽  
Author(s):  
KAI WAN ◽  
AHMED E. YOUSEF ◽  
STEVE J. SCHWARTZ ◽  
HUA H. WANG
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Orange Juice ◽  
Raw Materials ◽  
Pcr Primers ◽  
Cross Reactivity ◽  
Rrna Gene ◽  
Control Analysis ◽  
Financial Loss ◽  
Food Ingredients

The outgrowth of spoilage organisms, including molds and yeasts, results in significant financial loss to the food industry and wastes natural resources. The objective of this study was to develop a rapid, specific, and sensitive real-time PCR method for detecting spoilage molds during screening of raw materials and final product quality control analysis. The 18S rRNA gene was used to develop PCR primers and probe. With this set of primers and probe, less than 1,000 mold cells per milliliter of orange juice (10 cells per reaction) were detected with the real-time PCR system within 6 to 7 h. No cross-reactivity was found with other common foodborne bacteria, yeasts, or food ingredients. This technique is significantly faster than current detection and identification procedures, which take from days to weeks.

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.

scholarly journals Molecular diagnosis of Kingella kingae osteoarticular infections by specific real-time PCR assay

2009 ◽  
Vol 58 (1) ◽  
pp. 65-68 ◽  
Author(s):  
Abdessalam Cherkaoui ◽  
Dimitri Ceroni ◽  
Stéphane Emonet ◽  
Yan Lefevre ◽  
Jacques Schrenzel
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Cell Types ◽  
Cross Reactivity ◽  
Toxin Gene ◽  
Rrna Gene ◽  
Putative Virulence Factor ◽  
Pcr Assay ◽  
Kingella Kingae ◽  
Rtx Toxin

Kingella kingae is an emerging pathogen that is recognized as a causative agent of septic arthritis and osteomyelitis, primarily in infants and children. The bacterium is best detected by rapid inoculation in blood culture systems or by real-time PCR assays. Pathogenesis of the agent was linked recently to the production of a potent cytotoxin, known as RTX, which is toxic to a variety of human cell types. The locus encoding the RTX toxin is thought to be a putative virulence factor, and is, apparently, essential for inducing cytotoxic effects on respiratory epithelial, synovial and macrophage-like cells. Herein, we describe a novel real-time PCR assay that targets the RTX toxin gene and illustrate its use in two clinical cases. The assay exhibited a sensitivity of 30 c.f.u., which is 10-fold more sensitive than a previously published semi-nested broad-range 16S rRNA gene PCR, and showed no cross-reactivity with several related species and common osteoarticular pathogens.

Design and testing of real-time PCR primers for the quantification ofMethanoculleus,Methanosarcina,Methanothermobacter, and a group of uncultured methanogens

2009 ◽  
Vol 55 (5) ◽  
pp. 611-616 ◽  
Author(s):  
Ingrid H. Franke-Whittle ◽  
Marta Goberna ◽  
Heribert Insam
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Biogas Production ◽  
Pcr Primers ◽  
Sybr Green ◽  
Rrna Gene ◽  
Pcr Assays ◽  
Design And Testing ◽  
Uncultured Archaea ◽  
Detection And Quantification

In this study, 16S rRNA gene primers were designed to complement the suite of already available PCR primers for the detection of different methanogens involved in biogas production through anaerobic digestion by SYBR Green real-time PCR. Primers designed for use in TaqMan real-time PCR for the organisms Methanosaeta , Methanosarcina , and Methanoculleus have been described previously; however, we found that (i) the Methanoculleus primers were not specific to members of the genus and that (ii) the Methanosarcina primers did not work specifically with SYBR Green real-time PCR. Thus, we designed new primers for these and other methanogens, and we optimized SYBR Green real-time PCR assays. Primers were tested by end-point and real-time PCR, and they were found to work specifically and sensitively. Application of these primers will allow the detection and quantification of Methanoculleus, Methanosarcina, Methanothermobacter , and a group of yet uncultured archaea from anaerobic habitats.

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 Detection of Biological Threat Agents by Real-Time PCR: Comparison of Assay Performance on the R.A.P.I.D., the LightCycler, and the Smart Cycler Platforms

2006 ◽  
Vol 52 (1) ◽  
pp. 141-145 ◽  
Author(s):  
Deanna R Christensen ◽  
Laurie J Hartman ◽  
Bonnie M Loveless ◽  
Melissa S Frye ◽  
Michelle A Shipley ◽  
...  
Keyword(s):  
Real Time ◽  
Sensitivity And Specificity ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Dna Amplification ◽  
Pcr Primers ◽  
Cross Reactivity ◽  
Dna Templates ◽  
Assay Performance ◽  
Fluorogenic Probes

Abstract Background: Rapid detection of biological threat agents is critical for timely therapeutic administration. Fluorogenic PCR provides a rapid, sensitive, and specific tool for molecular identification of these agents. We compared the performance of assays for 7 biological threat agents on the Idaho Technology, Inc. R.A.P.I.D.®, the Roche LightCycler®, and the Cepheid Smart Cycler®. Methods: Real-time PCR primers and dual-labeled fluorogenic probes were designed to detect Bacillus anthracis, Brucella species, Clostridium botulinum, Coxiella burnetii, Francisella tularensis, Staphylococcus aureus, and Yersinia pestis. DNA amplification assays were optimized by use of Idaho Technology buffers and deoxynucleotide triphosphates supplemented with Invitrogen Platinum® Taq DNA polymerase, and were subsequently tested for sensitivity and specificity on the R.A.P.I.D., the LightCycler, and the Smart Cycler. Results: Limit of detection experiments indicated that assay performance was comparable among the platforms tested. Exclusivity and inclusivity testing with a general bacterial nucleic acid cross-reactivity panel containing 60 DNAs and agent-specific panels containing nearest neighbors for the organisms of interest indicated that all assays were specific for their intended targets. Conclusion: With minor supplementation, such as the addition of Smart Cycler Additive Reagent to the Idaho Technology buffers, assays for DNA templates from biological threat agents demonstrated similar performance, sensitivity, and specificity on all 3 platforms.

scholarly journals Detection, Distribution, and Genetic Variability of ‘Candidatus Liberibacter’ Species Associated with Zebra Complex Disease of Potato in North America

Plant Disease ◽  
2009 ◽  
Vol 93 (11) ◽  
pp. 1102-1115 ◽  
Author(s):  
A. Wen ◽  
I. Mallik ◽  
V. Y. Alvarado ◽  
J. S. Pasche ◽  
X. Wang ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Complex Disease ◽  
Pcr Primers ◽  
23S Rrna ◽  
Rrna Gene ◽  
Gene Sequences ◽  
Candidatus Liberibacter ◽  
Total Dna ◽  
Pcr Assays

The specificity and sensitivity of polymerase chain reaction (PCR) primers developed for ‘Candidatus Liberibacter solanacearum’ and ‘Candidatus Liberibacter psyllaurous’ were evaluated in conventional and real-time PCR assays. All PCR primers were specific for ‘Ca. L. psyllaurous’ and ‘Ca. L. solanacearum’ insomuch as they did not detect other prokaryotic plant pathogens that affect potato except for the putative pathogens associated with psyllid-yellows and haywire. Conventional PCR assays were capable of detecting 0.19 to 1.56 ng of total DNA per reaction, and real-time PCR was found capable of detecting 1.56 to 6.25 ng of total DNA per reaction, depending on the specific PCR primer set used. ‘Ca. Liberibacter’ species associated with zebra complex disease (ZC) was confirmed in plants affected by this disease throughout Texas from 2005 to 2008, in seed tubers produced in Wyoming in 2007, and in Colorado, Kansas, Nebraska, and Mexico in 2008. A multiplex PCR assay using ‘Ca. L. solanacearum’–specific primers and primers specific for the β-tubulin DNA regions from potato was developed, providing possible utility of the multiplex assay for ‘Ca. Liberibacter’ detection in different solanaceous plant species. Preliminary studies suggest silverleaf nightshade (Solanum elaeagnifolium), wolfberry (Lycium barbarum), black nightshade (S. ptychanthum), and jalapeno pepper (Capsicum annuum) as additional solanaceous hosts for the ZC-associated bacterium. The ‘Ca. Liberibacter’ species detected in all samples divided into two clusters sharing similarity of 99.8% in their partial 16S rRNA gene sequences and 99.3% in their partial intergenic spacer region (ISR)-23S rRNA gene sequences. Genetic variation in the 16S rDNA region consistently matched that of the ISR-23S rDNA region. In this partial 16S-ISR-23S rDNA region, there was a total of eight single nucleotide polymorphisms among ‘Ca. L. psyllaurous’ and ‘Ca. L. solanacearum’ “strains” investigated in this study. ‘Ca. L. solanacearum’ and ‘Ca. L. psyllaurous’ were shown to be very closely related bacteria, if not the same, by successful amplification using a combination of forward primer of ‘Ca. L. solanacearum’ and reverse primer of ‘Ca. L. psyllaurous’ in ZC-affected potato samples. This finding clarifies the current taxonomic status of ‘Ca. L. solanacearum’ and ‘Ca. L. psyllaurous’. The detection of ‘Ca. L. solanacearum’ from haywire-symptomatic potato samples demonstrates that this bacterium might also be associated with this disease.

scholarly journals Optimized genotyping method for identification of bacterial contaminants in pharmaceutical industry

2016 ◽  
Vol 66 (2) ◽  
pp. 289-295
Author(s):  
Borche Stamatoski ◽  
Miroslava Ilievska ◽  
Hristina Babunovska ◽  
Nikola Sekulovski ◽  
Sasho Panov
Keyword(s):  
Pharmaceutical Industry ◽  
Dna Sequencing ◽  
Bacterial Contamination ◽  
Raw Materials ◽  
Pcr Primers ◽  
Rrna Gene ◽  
Bacterial Identification ◽  
Bacterial Contaminants ◽  
Microbiological Control ◽  
Selection Of

AbstractMicrobiological control is of crucial importance in the pharmaceutical industry regarding the possible bacterial contamination of the environment, water, raw materials and finished products. Molecular identification of bacterial contaminants based on DNA sequencing of the hypervariable 16SrRNA gene has been introduced recently. The aim of this study is to investigate the suitability of gene sequencing using our selection of PCR primers and conditions for rapid and accurate bacterial identification in pharmaceutical industry quality control.DNA was extracted from overnight incubated colonies from 10 bacterial ATCC strains, which are common contaminants in the pharmaceutical industry. A region of bacterial 16SrRNA gene was analyzed by bidirectional DNA sequencing. Bacterial identification based on partial sequencing of the 16SrRNA gene is the appropriate method that could be used in the pharmaceutical industry after adequate validations. We have successfully identified all tested bacteria with more than 99 % similarity to the already published sequences.

O-091 Semen microbiota in patients with astenozoospermia and healthy controls: cluster analysis of real-time PCR data

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
E Panacheva ◽  
D Pochernikov ◽  
E Voroshilina
Keyword(s):  
Cluster Analysis ◽  
Real Time ◽  
Real Time Pcr ◽  
Detection Rate ◽  
Bacterial Load ◽  
Rrna Gene ◽  
Rt Pcr ◽  
Silhouette Index ◽  
Enterococcus Spp ◽  
Lactobacillus Spp

Abstract Study question What are the differences in the semen microbiota composition of patients with asthenozoospermia and normospermia according to cluster analysis of PCR data? Summary answer The detection rate of 4 stable semen microbiota clusters and the dominant bacteria groups varied in patients with asthenozoospermia and normospermia. What is known already Most of the research dedicated to analyzing normal and pathological semen microbiota is based on 16S rRNA gene specific Next generation sequencing (NGS). It has shown that microbiota is represented by polymicrobial communities (clusters) that consist of microorganisms from different genera and bacteria phyla. Despite it being highly informative, NGS has several weaknesses: complex sample preparation, difficult sample intake control, long analysis process, complicated results interpretation, high cost of equipment and reagents. These factors make it virtually impossible to use this approach in routine medical practice. Quantitative real-time PCR (RT-PCR) is far more suitable for this. Study design, size, duration Patients included in the study (n = 301) came to the “Garmonia” Medical Center (Yekaterinburg, Russia) either seeking preconception care or for infertility treatment. Depending on the spermiogram results, they were divided into two groups. Group 1 (n = 171) — asthenozoospermia, Group 2 (n = 130) — normospermia. Participants/materials, setting, methods Semen microbiota was analyzed using RT-PCR kit Androflor (DNA-Technology, Russia). Cluster analysis was performed for 201 samples with the total bacterial load (TBL) of at least 103 GE/ml (asthenozoospermia = 96, normospermia = 105). Cluster analysis was conducted using the k-means ++ algorithm, scikit-learn. The Silhouette index and the Davies–Bouldin index (DBI) were used to confirm the stability of clusters. Main results and the role of chance Both in the samples with normospermia and asthenozoospermia, four stable microbiota clusters were distinguished. Cluster I was characterized by the prevalence of obligate anaerobes, Lactobacillus spp. were prevalent in Cluster II, Gram-positive facultative anaerobes were prevalent in Cluster III, Enterobacteriaceae/Enterococcus spp. were prevalent in Cluster IV. Cluster I was detected the most often in both groups. However, in normospermia it was represented by various obligate anaerobes without pronounced quantitative predominance of any bacteria group. In samples with asthenozoospermia one of the bacteria groups were prevalent in Cluster I: Bacteroides spp./Porphyromonas spp./Prevotella spp., Peptostreptococcus spp./Parvimonas spp. or Eubacterium spp. In samples with asthenozoospermia Cluster II was characterized by the prevalence of Lactobacillus spp., while in samples with normospermia other bacteria groups were present along with lactobacilli, mainly obligate anaerobes. In samples with normospermia Corynebacterium spp. and Streptococcus spp., typical of normal microbiota of male UGT, were prevalent in Cluster III. In samples with asthenozoospermia Cluster III were characterized by the prevalence of Staphylococcus spp. In samples with asthenozoospermia Lactobacillus spp was present in Cluster IV along with Enterobacteriaceae/Enterococcus spp., which was not typical of the samples with normospermia. Limitations, reasons for caution Cluster analysis was not conducted for the samples with TBL lower than 103 GE/ml, since their results were incompatible with the data received for the negative control samples. Wider implications of the findings Further research could determine the detection rate of the described bacterial clusters in semen with other pathologies. Establishing the relationship between the characteristics of semen microbiota and infertility in men might allow the development of new algorithms for treating patients with reproductive disorders, depending on the composition of semen microbiota. Trial registration number not applicable

scholarly journals A Real-Time PCR Method for the Authentication of Common Cuttlefish (Sepia officinalis) in Food Products

Foods ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 286 ◽  
Author(s):  
Amaya Velasco ◽  
Graciela Ramilo-Fernández ◽  
Carmen G. Sotelo
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Food Products ◽  
Polymerase Chain Reaction Method ◽  
Cross Reactivity ◽  
Sepia Officinalis ◽  
Base Pairs ◽  
Mitochondrial Coi ◽  
Fresh Frozen ◽  
Pcr Method

Cephalopods are very relevant food resources. The common cuttlefish (Sepia officinalis) is highly appreciated by consumers and there is a lack of rapid methods for its authentication in food products. We introduce a new minor groove binding (MGB) TaqMan real-time PCR (Polymerase Chain Reaction) method for the authentication of S. officinalis in food products to amplify a 122 base pairs (bp) fragment of the mitochondrial COI (Cytochrome Oxidase I) region. Reference and commercial samples of S. officinalis showed a threshold cycle (Ct) mean of 14.40, while the rest of the species examined did not amplify, or showed a significantly different Ct (p < 0.001). The calculated efficiency of the system was 101%, and the minimum DNA quantity detected was 10−4 ng. No cross-reactivity was detected with any other species, thus, the designed method differentiates S. officinalis from other species of the genus Sepia and other cephalopod species and works for fresh, frozen, grilled, cooked and canned samples of Sepia spp. The method has proved to be reliable and rapid, and it may prove to be a useful tool for the control of fraud in cuttlefish products.

Sign in / Sign up

Export Citation Format

Share Document