scholarly journals Direct Quantitative Detection and Identification of Lactococcal Bacteriophages from Milk and Whey by Real-Time PCR: Application for the Detection of Lactococcal Bacteriophages in Goat's Raw Milk Whey in France

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Mai Huong Ly-Chatain ◽  
Loïc Durand ◽  
Véronique Rigobello ◽  
Annabelle Vera ◽  
Yann Demarigny
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Direct Detection ◽  
Raw Milk ◽  
Quantitative Detection ◽  
Amplification Efficiency ◽  
The Real ◽  
Milk Whey ◽  
Detection And Identification ◽  
Milk Fermentation

The presence ofLactococcusbacteriophages in milk can partly or completely inhibit milk fermentation. To prevent the problems associated with the bacteriophages, the real-time PCR was developed in this study for direct detection from whey and milk of three main groups ofLactococcusbacteriophages, c2, 936, and P335. The optimization of DNA extraction protocol from complex matrices such as whey and milk was optimized allowed the amplification of PCR without any matrix and nontarget contaminant interference. The real-time PCR program was specific and with the detection limit of 102PFU/mL. The curve slopes were −3.49, −3.69, and −3.45 with the amplification efficiency estimated at 94%, 94%, and 98% and the correlation coefficient () of 0.999, 0.999, and 0.998 for c2, 936 and P335 group, respectively. This method was then used to detect the bacteriophages in whey and goat's raw milk coming from three farms located in the Rhône-Alpes region (France).

scholarly journals An improved rapid quantitative detection and identification method for a wide range of fungi

2009 ◽  
Vol 58 (8) ◽  
pp. 1037-1044 ◽  
Author(s):  
Nobutoshi Soeta ◽  
Masanori Terashima ◽  
Mitsukazu Gotoh ◽  
Shuichi Mori ◽  
Kyoko Nishiyama ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Diagnostic Technique ◽  
Fungal Species ◽  
Quantitative Detection ◽  
Its2 Region ◽  
The Real ◽  
Detection And Identification ◽  
Wide Range ◽  
Pcr Products

To develop a rapid and quantitative diagnostic technique for the detection and identification of a wide range of fungi, an improved molecular method based on real-time PCR and the analysis of its products that targets the internal transcribed spacer (ITS) 2 region was established. The real-time PCR could quantitatively and specifically detect the ITS2 region from all 24 tested pathogenic fungal species at between 101 and 107 copies per test without amplification of bacterial or human DNA. The sequences of the primer-binding sites are conserved in the registered sequences of 34 other pathogenic fungal species, suggesting that the PCR would also detect these species. The hyperpolymorphic nature of the ITS2 region between fungal species in terms of length and nucleotide sequence provided valuable information for the determination of species. By labelling the 5′ end of the reverse primer with NED fluorescent dye, the fragment lengths of the real-time PCR products and their 3′-terminal fragments, derived using restriction enzyme ScrFI digestion, were easily evaluated by capillary electrophoresis. Using this analysis, the number and species of fungi present in samples could be estimated. Moreover, sequence analysis of the real-time PCR products could accurately determine species in samples containing a single species. This diagnostic technique can estimate a wide range of fungi from various clinical samples within 1 day and accurately identify them in 2 days. Quantitative results for fungal titre in samples can also provide useful information for understanding the progression of disease and the efficacy of antifungal chemotherapy.

scholarly journals Application of 5′-Nuclease PCR for Quantitative Detection of Listeria monocytogenes in Pure Cultures, Water, Skim Milk, and Unpasteurized Whole Milk

2000 ◽  
Vol 66 (10) ◽  
pp. 4266-4271 ◽  
Author(s):  
Hege Karin Nogva ◽  
Knut Rudi ◽  
Kristine Naterstad ◽  
Askild Holck ◽  
Dag Lillehaug
Keyword(s):  
Listeria Monocytogenes ◽  
Real Time ◽  
Real Time Pcr ◽  
Direct Detection ◽  
Skim Milk ◽  
Raw Milk ◽  
Magnetic Bead ◽  
Quantitative Detection ◽  
Listeriolysin O ◽  
Pure Cultures

ABSTRACT PCR techniques have significantly improved the detection and identification of bacterial pathogens. Countless adaptations and applications have been described, including quantitative PCR and the latest innovation, real-time PCR. In real-time PCR, e.g., the 5′-nuclease chemistry renders the automated and direct detection and quantification of PCR products possible (P. M. Holland et al., Proc. Natl. Acad. Sci. USA 88:7276–7280, 1991). We present an assay for the quantitative detection of Listeria monocytogenesbased on the 5′-nuclease PCR using a 113-bp amplicon from the listeriolysin O gene (hlyA) as the target. The assay was positive for all isolates of L. monocytogenes tested (65 isolates including the type strain) and negative for all otherListeria strains (16 isolates from five species tested) and several other bacteria (18 species tested). The application of 5′-nuclease PCR in diagnostics requires a quantitative sample preparation step. Several magnetic bead-based strategies were evaluated, since these systems are simple and relatively easy to automate. The combination of nonspecific binding of bacteria to paramagnetic beads, with subsequent DNA purification by use of the same beads, gave the most satisfactory result. The detection limit was approximately 6 to 60 CFU, quantification was linear over at least 7 log units, and the method could be completed within 3 h. In conclusion, a complete quantitative method for L. monocytogenes in water and in skimmed and raw milk was developed.

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.

scholarly journals Real-time quantitative PCR for enteric adenovirus serotype 40 in environmental waters

2005 ◽  
Vol 51 (5) ◽  
pp. 393-398 ◽  
Author(s):  
Sunny Jiang ◽  
Hojabr Dezfulian ◽  
Weiping Chu
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
Amplification Efficiency ◽  
Sewage Effluent ◽  
Hexon Gene ◽  
Pcr Assay ◽  
Environmental Waters ◽  
Cycle Number ◽  
The Real

Adenoviruses 40 and 41 have been recognized as important etiological agents of gastroenteritis in children. A real-time PCR method (TaqMan® assay) was developed for rapid quantification of adenovirus 40 (Ad40) by amplifying an 88 bp sequence from the hexon gene. To establish a quantification standard curve, a 1090 bp hexon region of Ad40 was amplified and cloned into the pGEM®-T Vector. A direct correlation was observed between the fluorescence threshold cycle number (Ct) and the starting quantity of Ad40 hexon gene. The quantification was linear over 6-log units and the amplification efficiency averaged greater than 95%. Seeding studies using various environmental matrices (including sterile water, creek water, brackish estuarine water, ocean water, and secondary sewage effluent) suggest that this method is applicable to environmental samples. However, real-time PCR was sensitive to inhibitors present in the environmental samples. Lower efficiency of PCR amplification was found in secondary sewage effluent and creek waters. Application of the method to fecal contaminated waters successfully quantified the presence of Ad40. The sensitivity of the real-time PCR is comparable to the traditional nested PCR assay for environmental samples. In addition, the real-time PCR assay offers the advantage of speed and insensitivity to contamination during PCR set up. The real-time PCR assay developed in this study is suitable for quantitative determination of Ad40 in environmental samples and represents a considerable advancement in pathogen quantification in aquatic environments.Key words: adenovirus, real-time PCR, environmental waters, serotype 40.

scholarly journals Conventional and real-time pcr assays for detection and identification of rhizoctonia solani AG-2-2, the causal agent of root rot of sugar beet

2019 ◽  
Vol 34 (1) ◽  
pp. 19-29
Author(s):  
Mira Vojvodic ◽  
Dejan Lazic ◽  
Petar Mitrovic ◽  
Brankica Tanovic ◽  
Ivana Vico ◽  
...  
Keyword(s):  
Sugar Beet ◽  
Real Time ◽  
Real Time Pcr ◽  
Root Rot ◽  
Direct Detection ◽  
Causal Agent ◽  
Future Research ◽  
Rhizoctonia Root Rot ◽  
Detection And Identification ◽  
High Level

Soil-borne fungi belonging to the genus Rhizoctonia are considered to be among the most destructive sugar beet pathogens. Although multinucleate R. solani AG-2-2 is frequently detected as the main causal agent of root rot of sugar beet worldwide, several binucleate (AG-A, AG-E and AG-K) and multinucleate Rhizoctonia (R. solani AG-4, AG-5 and AG-8) have also been included in the disease complex. Due to their soil-borne nature and wide host range, the management of Rhizoctonia root rot of sugar beet is highly demanding. Identification of Rhizoctonia AG associated with root rot of sugar beet is the essential first step in determining a successful disease management strategy. In this paper we report a highly specific and sensitive real-time PCR protocol for detection of R. solani AG-2-2 which showed a high level of specificity after testing against 10 different anastomosis groups and subgroups, including AG-2-1 as the most closely related. Moreover, a similar conventional PCR assay showed the same specificity but proved to be at least a 100 times less sensitive. Future research will include further testing and adaptation of this protocol for direct detection and quantification of R. solani AG-2-2 in different substrates, including plant tissue and soil samples.

scholarly journals Multiplex Fast Real-Time PCR for Quantitative Detection and Identification of cos- and pac-Type Streptococcus thermophilus Bacteriophages

2008 ◽  
Vol 74 (15) ◽  
pp. 4779-4781 ◽  
Author(s):  
Beatriz del Rio ◽  
María Cruz Martín ◽  
Noelia Martínez ◽  
Alfonso H. Magadán ◽  
Miguel A. Alvarez
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Industrial Process ◽  
Streptococcus Thermophilus ◽  
Quantitative Detection ◽  
Detection And Identification ◽  
Pcr Method

ABSTRACT The fermentation of milk by Streptococcus thermophilus is a widespread industrial process that is susceptible to bacteriophage attack. In this work, a preventive fast real-time PCR method for the detection, quantification, and identification of types of S. thermophilus phages in 30 min is described.

scholarly journals Direct detection of Dengue viruses without extraction of RNA on the mobile real-time PCR device

2020 ◽  
Author(s):  
Masaaki Muraoka ◽  
Yukiko Tanoi ◽  
Tetsutaro Tada ◽  
Aya Tabata ◽  
Mikio Mizukoshi ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Direct Detection ◽  
Positive Control ◽  
Nucleic Acid Amplification ◽  
Severe Dengue ◽  
Mosquito Vectors ◽  
Rt Pcr ◽  
The Real ◽  
One Step

ABSTRACTDengue virus (DENV) is the cause of dengue / severe dengue and a virus of the Flaviviridae family, furthermore, dengue fever has rapidly spread in the world in recent decades. DENV is transmitted by female mosquitoes, mainly of the specie Aedes aegypti. The main method to control or prevent the transmission of DENV is to combat the mosquito vectors. Among these, one of important methods is to monitor the DENVs in the mosquito vectors.For the detection of DENV, nucleic acid amplification tests (NAAT) were recommended, of which criterion standard is real-time RT-PCR with highly sensitive and specific. However, it takes long time as to judge the result per a reaction, besides the necessity of the treatment of RNA in advance, example of extraction, concentration and purification.It was our object in this time to develop the method of real-time RT-PCR detecting DENVs in shorter time, moreover without especial treatment of RNA from the mosquito in advance. Besides, this work was performed with combing the mobile real-time PCR device with the one-step RT-PCR reagent.Firstly, we succeeded in shortening the time of real-time RT-PCR for the detection of DENV per one reaction, so that the judgement needed less than 20 minutes if genomic RNA treated in advance. Moreover, each value on the real-PCR device was quantitatively correlated with the positive control RNA from 1.0 × 10 ^ 3 copies to 1.0 × 10 ^ 0 copies per reaction (This correlation coefficient R2 > 0.95). Additionally, it made sure that this method could be applied to each DENV serotype.Secondly, we established the basis of procedure for the real-time RT-PCR without the treatment in advance so-called “direct”. As the result that the positive control RNA additive was utilized instead of the real DENV, spiked into the mosquito homogenized and sampled the supernatant without treatment, it was possible to detect on the real-time RT-PCR even if mosquitoes immediately after blood-feeding. For this reason, this method might be able to utilize in human sera, too.According to the results of this work, we could suggest the method is possible to detect DENV more quickly and more simply than heretofore. The Real-time “direct” RT-PCR, especially, could be performed with mobile real-time PCR PCR1100 device and one step RT-PCR reagent only. This method must help to detect some viruses other than DENV, too.

Rapid and Simultaneous Quantitation of Escherichia coli O157:H7, Salmonella, and Shigella in Ground Beef by Multiplex Real-Time PCR and Immunomagnetic Separation†

2007 ◽  
Vol 70 (6) ◽  
pp. 1366-1372 ◽  
Author(s):  
LUXIN WANG ◽  
YONG LI ◽  
AZLIN MUSTAPHA
Keyword(s):  
Escherichia Coli ◽  
Detection Limit ◽  
Real Time ◽  
Real Time Pcr ◽  
Ground Beef ◽  
Immunomagnetic Separation ◽  
Quantitative Detection ◽  
Escherichia Coli O157 ◽  
The Real ◽  
E Coli

The objective of this study was to establish a multiplex real-time PCR for the simultaneous quantitation of Escherichia coli O157:H7, Salmonella, and Shigella. Genomic DNA for the real-time PCR was extracted by the boiling method. Three sets of primers and corresponding TaqMan probes were designed to target these three pathogenic bacteria. Multiplex real-time PCR was performed with TaqMan Universal PCR Master Mix in an ABI Prism 7700 Sequence Detection System. Final standard curves were calculated for each pathogen by plotting the threshold cycle value against the bacterial number (log CFU per milliliter) via linear regression. With optimized conditions, the quantitative detection range of the real-time multiplex PCR for pure cultures was 102 to 109 CFU/ml for E. coli O157:H7, 103 to 109 CFU/ml for Salmonella, and 101 to 108 CFU/ml for Shigella. When the established multiplex real-time PCR system was applied to artificially contaminated ground beef, the detection limit was 105 CFU/g for E. coli O157:H7, 103 CFU/g for Salmonella, and 104 CFU/g for Shigella. Immunomagnetic separation (IMS) was further used to separate E. coli O157:H7 and Salmonella from the beef samples. With the additional use of IMS, the detection limit was 103 CFU/g for both pathogens. Results from this study showed that TaqMan real-time PCR, combined with IMS, is potentially an effective method for the rapid and reliable quantitation of E. coli O157:H7, Salmonella, and Shigella in food.

scholarly journals Real-Time PCR Assay for the Detection and Quantification of Roe Deer to Detect Food Adulteration—Interlaboratory Validation Involving Laboratories in Austria, Germany, and Switzerland

Foods ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2645
Author(s):  
Barbara Druml ◽  
Steffen Uhlig ◽  
Kirsten Simon ◽  
Kirstin Frost ◽  
Karina Hettwer ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Roe Deer ◽  
Amplification Efficiency ◽  
Pcr Assay ◽  
Food Adulteration ◽  
The Real ◽  
Game Meat ◽  
Meat Samples ◽  
Detection And Quantification

Game meat products are particularly prone to be adulterated by replacing game meat with cheaper meat species. Recently, we have presented a real-time polymerase chain reaction (PCR) assay for the identification and quantification of roe deer in food. Quantification of the roe deer content in % (w/w) was achieved relatively by subjecting the DNA isolates to a reference real-time PCR assay in addition to the real-time PCR assay for roe deer. Aiming at harmonizing analytical methods for food authentication across EU Member States, the real-time PCR assay for roe deer has been tested in an interlaboratory ring trial including 14 laboratories from Austria, Germany, and Switzerland. Participating laboratories obtained aliquots of DNA isolates from a meat mixture containing 24.8% (w/w) roe deer in pork, roe deer meat, and 12 meat samples whose roe deer content was not disclosed. Performance characteristics included amplification efficiency, level of detection (LOD95%), repeatability, reproducibility, and accuracy of quantitative results. With a relative reproducibility standard deviation ranging from 13.35 to 25.08% (after outlier removal) and recoveries ranging from 84.4 to 114.3%, the real-time PCR assay was found to be applicable for the detection and quantification of roe deer in raw meat samples to detect food adulteration.

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