scholarly journals Isıl İşlem Görmüş Sütlerde Salmonella Typhimurium Canlı Hücrelerinin PMA/Real-Time PCR ile Belirlenmesi

2017 ◽  
Vol 5 (5) ◽  
pp. 518
Author(s):  
Zülal Kesmen ◽  
Hakiye Aslan
Keyword(s):  
Salmonella Typhimurium ◽  
Real Time ◽  
Real Time Pcr ◽  
False Positive ◽  
Live Cells ◽  
Dead Cell ◽  
Milk Samples ◽  
Heat Treated ◽  
Pcr Method ◽  
Positive Results

Applying different technological processes during the production of food has a lethal effect on the bacteria but DNA of these bacterial strains may cause false positive results when detected by real time PCR technique because they preserve their existence for a certain period of time. To overcome this shortcoming of the real time PCR technique, a new method has been developed in recent years, based on the removal of dead cell DNA from the medium by treatment with Propodium Monoazide (PMA) before DNA extraction. In this study, real-time PCR method was combined with PMA application for the detection of live cells of Salmonella Typhimurium in heat treated milk samples. For this purpose, milk samples inoculated with S. Tyhimurium were heat treated at different temperatures (60, 65, 70 and 75°C) and times (15, 60, 300, 900 sec) and number of live bacteria was determined comparatively by direct real-time PCR, PMA/real-time PCR and conventional cultural method. As a result, unlike the direct real time PCR technique, PMA/real-time PCR method prevents to a certain extent of false positive results from dead cells at all tested temperatures and times but higher results were obtained from PMA/real-time PCR method when compared to conventional cultural results. Therefore, further studies should be carried out to optimize the conditions of the PMA application in order to eliminate the high positive results detected by the PMA / real-time PCR method

Detection of Allergen Walnut Component in Food by an Improved Real-Time PCR Method

2009 ◽  
Vol 72 (11) ◽  
pp. 2433-2435 ◽  
Author(s):  
HAIYAN WANG ◽  
FEI YUAN ◽  
YAJUN WU ◽  
HAIRONG YANG ◽  
BAOLIANG XU ◽  
...  
Keyword(s):  
Detection Limit ◽  
Real Time ◽  
Real Time Pcr ◽  
Seed Storage Protein ◽  
Seed Storage ◽  
Plant Materials ◽  
Agricultural Plant ◽  
Negative Results ◽  
Pcr Method ◽  
Positive Results

A real-time PCR method aimed at the gene sequence of the walnut vicilin-like seed storage protein was established for the detection of the allergen walnut in food. The primers and probe were designed based on published methods. The method provided positive results for walnut and negative results for other tested agricultural plant materials including pecan. The intrinsic detection limit of the method was 0.00125 ng of walnut DNA, and the practical detection limit was 0.001% (wt/wt) walnut content in wheat; both of these values are lower than that of previously published methods. Therefore, this real-time PCR method is sufficiently specific and sensitive for the detection of walnut component in food.

Comparative Studies of a Real-Time PCR Method and Three Enzyme-Linked Immunosorbent Assays for the Detection of Central Nervous System Tissues in Meat Products†

2008 ◽  
Vol 71 (10) ◽  
pp. 2059-2066 ◽  
Author(s):  
HOLGER SCHÖNENBRÜCHER ◽  
KATRIN ANNETTE GÖBEL ◽  
AMIR ABDULMAWJOOD ◽  
JÜRGEN A. RICHT ◽  
MICHAEL BÜLTE
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Real Time ◽  
Real Time Pcr ◽  
Meat Products ◽  
Spongiform Encephalopathy ◽  
Pcr Assay ◽  
Heat Treated ◽  
Pcr Method ◽  
Minced Meat

The removal of certain central nervous system (CNS) tissues (part of the bovine spongiform encephalopathy risk material) from the food chain is one of the highest priority tasks associated with avoiding contamination of the human food chain with the agent of bovine spongiform encephalopathy. A recently developed real-time PCR assay and three commercially available enzyme-linked immunosorbent assays (ELISAs) for the detection of CNS tissues in minced meat and three types of heat-treated sausages were evaluated. Bovine brain was used for spiking of internal reference material, and its detectability was examined during storage times of 12 months (for frozen minced meat and liver sausage) and 24 months (for sausages treated with medium and high heat). The real-time PCR method and both ELISA kits detected 0.1% CNS tissue in frozen minced meat and 0.1 or 1% CNS tissue in heat-treated meat products. The detectability of the amplified mRNA target region with the PCR assay was similar to the detectability of antigen by the ELISAs. Because the real-time PCR method also can be used to distinguish cattle, ovine, and caprine CNS tissues from porcine CNS tissues, it seems to be suitable as a routine diagnostic test for the sensitive and specific detection of CNS tissues in meat and meat products.

scholarly journals Tentacle Probes™: Differentiation of Difficult Single-Nucleotide Polymorphisms and Deletions by Presence or Absence of a Signal in Real-Time PCR

2007 ◽  
Vol 53 (12) ◽  
pp. 2042-2050 ◽  
Author(s):  
Brent C Satterfield ◽  
David A Kulesh ◽  
David A Norwood ◽  
Leonard P Wasieloski ◽  
Michael R Caplan ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
False Positive ◽  
Yersinia Pseudotuberculosis ◽  
Melting Curve ◽  
Melting Curve Analysis ◽  
Specific Cell ◽  
Single Nucleotide ◽  
Positive Results

AbstractBackground: False-positive results are a common problem in real-time PCR identification of DNA sequences that differ from near neighbors by a single-nucleotide polymorphism (SNP) or deletion. Because of a lack of sufficient probe specificity, post-PCR analysis, such as a melting curve, is often required for mutation differentiation.Methods: Tentacle Probes™, cooperative reagents with both a capture and a detection probe based on specific cell-targeting principles, were developed as a replacement for 2 chromosomal TaqMan–minor groove binder (MGB) assays previously developed for Yersinia pestis and Bacillus anthracis detection. We compared TaqMan-MGB probes to Tentacle Probes for SNP and deletion detection based on the presence or absence of a growth curve.Results: With the TaqMan-MGB Y. pestis yp48 assays, false-positive results for Yersinia pseudotuberculosis occurred at every concentration tested, and with the TaqMan-MGB B. anthracis gyrA assays, false-positive results occurred in 21 of 29 boil preps of environmental samples of near neighbors. With Tentacle Probes no false-positive results occurred.Conclusions: The high specificity exhibited by Tentacle Probes may eliminate melting curve analysis for SNP and deletion mutation detection, allowing the diagnostic use of previously difficult targets.

scholarly journals A multiplex real-time PCR method for differentiation of beef, buffalo meat and pork

2019 ◽  
Vol 18 (01) ◽  
pp. 63-71
Author(s):  
Tan M. Tran
Keyword(s):  
Detection Limit ◽  
Real Time ◽  
Real Time Pcr ◽  
High Sensitivity ◽  
Specific Probe ◽  
Food Authenticity ◽  
Processed Meats ◽  
Buffalo Meat ◽  
Heat Treated ◽  
Pcr Method

The objective of this study was to optimize a multiplex real-time PCR protocol for detection of DNA of beef, buffalo meat and pork, serving for food authenticity. The optimized concentrations were 200 nM primer and 100 nM specific probe for beef/buffalo meat, and 300 nM primer and 150 nM probe for pork. The amplification was performed using initial denaturation at 50oC for 2 min, 95oC for 2 min, followed by 45 cycles of denaturation at 95oC for 15 sec, and annealing and extension at 60oC for 40 sec. This protocol had high sensitivity and specificity. The detection limit of this method was found to be 0.1% in raw and heat-treated meat mix (80 – 121oC/15 min) or 0.005 ng DNA/reaction. The protocol of testing was applied for the commercial products both fresh and processed meats. The results demonstrated that 50% of raw beef sample (6/12) weren't found beef DNA. Eight of twelve beef sausage samples (66.67%) contained buffalo DNA. Beef DNA were found in all 12 samples of beef meatballs, but eight out of the 12 meatball samples were confirmed to have buffalo DNA (66.67%) and two out of the 12 meatball samples (16.67%) also contained porcine DNA.

scholarly journals Detection of Salmonella typhimurium ATCC 14028 in Powder Prepared Traditional Medicines Using Real-Time PCR

2021 ◽  
Vol 4 (3) ◽  
pp. 178-183
Author(s):  
Alfi Sophian ◽  
Ratna Purwaningsih ◽  
Muindar Muindar ◽  
Eka Putri Juniarti Igirisa ◽  
Muhammad Luthfi Amirullah
Keyword(s):  
Salmonella Typhimurium ◽  
Real Time ◽  
Real Time Pcr ◽  
Positive Control ◽  
Negative Control ◽  
Pcr Analysis ◽  
Direct Pcr ◽  
Traditional Medicines ◽  
Pcr Method ◽  
Alternative Testing

The detection of Salmonella typhimurium ATCC 14028 using real-time PCR on powdered traditional medicinal products was carried out in the microbiology and molecular biology testing laboratory of the Food and Drug Administration in Gorontalo. This research aims to provide a reference for alternative testing methods in testing the products of traditional powder preparations on the market. The sample consisted of 10 traditional powder preparations spiked with positive control of S. typhimurium ATCC 14028 phase 2. The method used in the study was real-time PCR analysis using the SYBR® Green method, while DNA isolation using the direct PCR method. Data analysis was performed by analyzing the sample's melting temperature (Tm) curve and comparing it with positive control. The results showed that S. typhimurium ATCC 14028 was detected in samples at an average Tm value of 84.18°C, with ranges of 84.0-84.5°C. For positive control, the Tm value was at 85.2°C, while for the negative control, the Tm value was not detected. Based on these data, it can be concluded that S. typhimurium ATCC 14028 in traditional medicine products powder preparations can be detected using real-time PCR.

Real-Time PCR Method for Quantification of Staphylococcus aureus in Milk

2007 ◽  
Vol 70 (1) ◽  
pp. 90-96 ◽  
Author(s):  
M. GOTO ◽  
H. TAKAHASHI ◽  
Y. SEGAWA ◽  
H. HAYASHIDANI ◽  
K. TAKATORI ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Real Time ◽  
Real Time Pcr ◽  
Pure Culture ◽  
High Specificity ◽  
Pcr Assay ◽  
Milk Samples ◽  
High Temperature Short Time ◽  
Specificity And Sensitivity ◽  
Pcr Method

A reproducible real-time PCR method that targets the putative transcriptional regulator gene of Staphylococcus aureus was developed to quantify this microorganism in milk samples. On the basis of partial sequences of this gene determined from S. aureus strains, we designed the specific primers and probe for use in a quantitative PCR assay. These specificities were confirmed with 25 strains of S. aureus and 35 strains of other bacteria. A real-time PCR assay with serial 10-fold dilutions of purified DNA and pure culture was conducted. It was possible to construct standard curves with a high correlation coefficient (r2 = 0.99) in the range of 50 ng to 50 fg for purified DNA and 107 to 101 CFU/ml for a pure culture. The constructed standard curve for milk samples was similar to that for the pure culture, and the quantification of S. aureus in the range of 107 to 101 CFU/ml was possible. Moreover, to determine how our real-time PCR method would perform under actual analytical conditions, we quantified the DNA from S. aureus after two types of heat treatments were used for the pasteurization of milk. The amount of DNA found was affected after heat treatment at 63°C for 30 min (low-temperature long-time method) but not at 72°C for 15 s (high-temperature short-time method). The results indicate that the real-time PCR method developed in this study is effective for monitoring S. aureus contamination in milk because of its high specificity and sensitivity.

scholarly journals Comparison of Two Commercially Available qPCR Kits for the Detection of Candida auris

2021 ◽  
Vol 7 (2) ◽  
pp. 154
Author(s):  
Janko Sattler ◽  
Janina Noster ◽  
Anne Brunke ◽  
Georg Plum ◽  
Pia Wiegel ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
False Positive ◽  
Limit Of Detection ◽  
Detection Methods ◽  
Limited Data ◽  
Candida Auris ◽  
Human Blood Samples ◽  
Newcastle Upon Tyne ◽  
Positive Results

Candida auris is an emerging pathogen with resistance to many commonly used antifungal agents. Infections with C. auris require rapid and reliable detection methods to initiate successful medical treatment and contain hospital outbreaks. Conventional identification methods are prone to errors and can lead to misidentifications. PCR-based assays, in turn, can provide reliable results with low turnaround times. However, only limited data are available on the performance of commercially available assays for C. auris detection. In the present study, the two commercially available PCR assays AurisID (OLM, Newcastle Upon Tyne, UK) and Fungiplex Candida Auris RUO Real-Time PCR (Bruker, Bremen, Germany) were challenged with 29 C. auris isolates from all five clades and eight other Candida species as controls. AurisID reliably detected C. auris with a limit of detection (LoD) of 1 genome copies/reaction. However, false positive results were obtained with high DNA amounts of the closely related species C. haemulonii, C. duobushaemulonii and C. pseudohaemulonii. The Fungiplex Candida Auris RUO Real-Time PCR kit detected C. auris with an LoD of 9 copies/reaction. No false positive results were obtained with this assay. In addition, C. auris could also be detected in human blood samples spiked with pure fungal cultures by both kits. In summary, both kits could detect C. auris-DNA at low DNA concentrations but differed slightly in their limits of detection and specificity.

scholarly journals Detection of Sesame Allergen Traces with Two PCR Assays - The Challenge to Protect Food-Allergic Consumers

2015 ◽  
Vol 3 (4) ◽  
pp. 210
Author(s):  
Dimitra Panagiotis Houhoula ◽  
Vasilios Belsis ◽  
Leonidas Georgopoulos ◽  
Virginia Giannou ◽  
Vasiliki R. Kyrana ◽  
...  
Keyword(s):  
Food Allergy ◽  
Real Time ◽  
Real Time Pcr ◽  
Food Industry ◽  
Food Samples ◽  
Rt Pcr ◽  
Highly Sensitive ◽  
Pcr Method ◽  
Pcr Assays ◽  
Positive Results

The purpose of this study was to investigate the possible presence of sesame in commercial foods normally carrying no warning for the allergen, but which may have been subjected to contamination during processing. One hundred units of widely consumed goods with high potential to contain allergenic substances deriving from nuts were analyzed, using sensitive and capable PCR (C-PCR) and Real Time PCR (RT-PCR) methodologies. Of the products examined, 15 (15.0%) declared the presence of sesame, 36 (36.0%) carried no food allergy label, 44 (44.0%) were marked by the phrase “may contain traces of nuts” and 5 (5.0%) carried the indication “may contain sesame traces”. The sesame-positive products detected using the C-PCR method were 15 (100%), 12 (33.3%), 14 (31.8%) and 3 (60%), respectively. Using the RT-PCR technique, positive results were obtained for 15 (100%), 18 (50.0%), 18 (20.5%) and 5 (100%) samples, respectively. The results indicate that the PCR methods applied are highly sensitive and selective, which makes them suitable for the detection of sesame traces in food samples. In addition, they can be useful for monitoring the effectiveness of cleaning processes in the production units of the food industry.

scholarly journals Detection of Salmonella typhimurium ATCC 14028 in supplement health product liquid preparation using Real-Time PCR (qPCR)

2020 ◽  
Vol 18 (2) ◽  
Author(s):  
Alfi Sophian ◽  
RATNA PURWANINGSIH ◽  
BERTHA LOLO LUKITA ◽  
ENI CAHYA NINGSIH
Keyword(s):  
Melting Point ◽  
Salmonella Typhimurium ◽  
Real Time ◽  
Real Time Pcr ◽  
Positive Control ◽  
Health Product ◽  
Direct Pcr ◽  
Liquid Preparation ◽  
Pcr Method ◽  
Negative Controls

Abstract. Sophian A, Purwaningsih R, Lukita BL, Ningsih EC. 2018. Detection of Salmonella typhimurium ATCC 14028 in supplement health product liquid preparation using Real-Time PCR (qPCR). Biofarmasi J Nat Prod Biochem 18: 61-65. Detection of Salmonella typhimurium ATCC 14028 using Real-Time PCR (qPCR) on health supplement products was carried out in the microbiology and molecular biology testing laboratory of the Food and Drug Supervisory Office in Gorontalo. The purpose of this study was to provide an alternative testing method reference in the testing of liquid supplement health supplement products in the market. The sample consisted of 35 samples of liquid supplement health supplements spike with positive control of Salmonella typhimurium ATCC 14028 phase 2. The method used in the study was qPCR analysis using the SYBR Green method, whereas DNA isolation using the direct PCR method. Data analysis was performed based on 2 main criteria: (i) Ct (Cycle threshold) analysis, which is looking at the value of the sample Ct and comparing it with controls and (ii) analysis of melting temperature (Tm), which is the melting point at the temperature at which melt occurs and comparing the melting point to the positive control. The results showed that in the sample, Salmonella typhimurium ATCC 14028 was detected at an average Ct value of 14.43, and an average Tm value of 86.05, for the specificity, LOD and positive control tests were all amplified. For negative controls, Ct and Tm values ​​were not detected. Based on these data it can be concluded that real-time PCR (qPCR) can be used to detect Salmonella typhimurium ATCC 14028 in liquid supplement health supplement products.

Sign in / Sign up

Export Citation Format

Share Document