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.

Quantitative Detection of Campylobacter jejuni on Fresh Chicken Carcasses by Real-Time PCR

2007 ◽  
Vol 70 (6) ◽  
pp. 1373-1378 ◽  
Author(s):  
ANNA-CLARA RÖNNER ◽  
HANS LINDMARK
Keyword(s):  
Detection Limit ◽  
Real Time ◽  
Dna Extraction ◽  
Campylobacter Jejuni ◽  
Real Time Pcr ◽  
Close Correlation ◽  
Quantitative Detection ◽  
Quantitative Real Time Pcr ◽  
Direct Plating ◽  
Pcr Method

Campylobacter jejuni infection is a significant cause of foodborne gastroenteritis worldwide. Consumption and handling of poultry products is believed to be the primary risk factor for campylobacteriosis. Risk assessments require quantitative data, and C. jejuni is enumerated usually by direct plating, which sometimes allows growth of non-Campylobacter bacteria. The objective of the present study was to develop a quantitative real-time PCR method (q-PCR) for enumerating C. jejuni in chicken rinse without a culturing step. The procedure to obtain the template for the PCR assay involved (i) filtration of 10 ml of chicken rinse, (ii) centrifugation of the sample, and (iii) total DNA extraction from the pellet obtained using a commercial DNA extraction kit. The detection limit of the method was comparable to that for plating 100 μl of chicken rinse on modified charcoal cefoperazone deoxycholate agar, and the detection limit could be further improved 10-fold by concentrating the DNA eluate by ethanol precipitation. A close correlation for spiked chicken rinse was obtained for the results of the quantitative real-time PCR method and direct plating (r = 0.99). The coefficient of correlation for the methods was 0.87 when samples from chicken carcasses on the slaughter line were analyzed, whereas a lower correlation (r = 0.76) was obtained when samples from retail carcasses were analyzed. Greater variation in the proportion of dead and/or viable but not culturable Campylobacter types in the retail samples may explain the decreased correlation between the methods. Overall, the new method is simple and fast and the results obtained are closely correlated with those for direct plating for samples containing a low proportion of dead Campylobacter cells.

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 Comparison of laboratory methods for diagnosis of Trichomonas vaginalis

2014 ◽  
Vol 63 (1) ◽  
pp. 5-9
Author(s):  
Zinaida Mikhaylovna Martikaynen ◽  
Aleksey Nikolayevich Grigoryev ◽  
Olga Sergeyevna Ryzhkova ◽  
Yelena Vasilyevna Shipitsyna ◽  
Alevtina Mikhaylovna Savicheva ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Trichomonas Vaginalis ◽  
Culture Media ◽  
High Sensitivity ◽  
Laboratory Methods ◽  
Pcr Method

The paper presents data on comparison of microscopy, culture and molecular (PCR) methods for diagnosis of trichomoniasis, as well as on evaluation of the quality of culture media for isolation of trichomonads produced by Russian and international manufacturers. The highest sensitivity was shown for the molecular (real-time PCR) method. Microscopy of stained and wet smears demonstrated relatively high sensitivity. The culture media that are widely used in our country for Trichomonas vaginalis isolation need optimization.

scholarly journals Development of ISSR-derived SCAR Marker and SYBR Green I Real-time PCR Method for Detection of Teliospores of Tilletia laevis Kühn

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Zhaoqun Yao ◽  
Dandan Qin ◽  
Delai Chen ◽  
Changzhong Liu ◽  
Wanquan Chen ◽  
...  
Keyword(s):  
Detection Limit ◽  
Real Time ◽  
Real Time Pcr ◽  
Scar Marker ◽  
Sybr Green ◽  
Sybr Green I ◽  
Tilletia Laevis ◽  
Highly Sensitive ◽  
Pcr Method ◽  
First Time

AbstractCommon bunt, caused by Tilletia laevis Kühn [syn. T. foetida (Wallr) Liro] and Tilletia tritici (Bjerk.) Wint. [syn. T. caries (DC) Tul.], is an important wheat disease worldwide. To quickly differentiate the closely related fungi T. laevis, T. tritici and Tilletia controversa (a pathogen that causes dwarf bunt of wheat and has been requested as a quarantined pathogen in many countries), a rapid diagnostic and detection method for an ISSR molecular marker was developed for the first time in this study. Based on the T. laevis-specific band (1300 bp) amplified by the primer ISSR860, a pair of SCAR primers (L60F/L60R) was designed to amplify a specific 660-bp DNA fragment from the isolates of T. laevis but not other related pathogens. The detection limit of the SCAR marker was 0.4 ng/μl of DNA from T. laevis; moreover, a SYBR Green I real-time PCR method was also successfully developed based on the SCAR marker with the detection limit of 10 fg/μl T. laevis DNA. This is the first report of a rapid, specific and highly sensitive SCAR marker and SYBR Green I real-time PCR method for detection of the teliospores of T. laevis based on ISSR technology. This method allows highly efficient, rapid and accurate differentiation of the pathogen from related pathogens, especially from the very similar pathogens T. tritici and T. controversa.

scholarly journals The Allele-Specific Probe and Primer Amplification Assay, a New Real-Time PCR Method for Fine Quantification of Single-Nucleotide Polymorphisms in Pooled DNA

2011 ◽  
Vol 78 (4) ◽  
pp. 1063-1068 ◽  
Author(s):  
A. Billard ◽  
V. Laval ◽  
S. Fillinger ◽  
P. Leroux ◽  
H. Lachaise ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Plant Pathogens ◽  
Amplification Refractory Mutation System ◽  
Specific Probe ◽  
Monitoring Methods ◽  
Single Nucleotide ◽  
Allele Specific ◽  
Amplification Assay ◽  
Pcr Method

ABSTRACTThe evolution of fungicide resistance within populations of plant pathogens must be monitored to develop management strategies. Such monitoring often is based on microbiological tests, such as microtiter plate assays. Molecular monitoring methods can be considered if the mutations responsible for resistance have been identified. Allele-specific real-time PCR approaches, such as amplification refractory mutation system (ARMS) PCR and mismatch amplification mutation assay (MAMA) PCR, are, despite their moderate efficacy, among the most precise methods for refining SNP quantification. We describe here a new real-time PCR method, the allele-specific probe and primer amplification assay (ASPPAA PCR). This method makes use of mixtures of allele-specific minor groove binder (MGB) TaqMan probes and allele-specific primers for the fine quantification of SNPs from a pool of DNA extracted from a mixture of conidia. It was developed for a single-nucleotide polymorphism (SNP) that is responsible for resistance to the sterol biosynthesis inhibitor fungicide fenhexamid, resulting in the replacement of the phenylalanine residue (encoded by the TTC codon) in position 412 of the enzymatic target (3-ketoreductase) by a serine (TCC), valine (GTC), or isoleucine (ATC) residue. The levels of nonspecific amplification with the ASPPAA PCR were reduced at least four times below the level of currently available allele-specific real-time PCR approaches due to strong allele specificity in amplification cycles, including two allele selectors. This new method can be used to quantify a complex quadriallelic SNP in a DNA pool with a false discovery rate of less than 1%.

scholarly journals Development of droplet digital PCR for the detection of Tilletia laevis, which causes common bunt of wheat, based on the SCAR marker derived from ISSR and real-time PCR

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Tongshuo Xu ◽  
Zhaoqun Yao ◽  
Jianjian Liu ◽  
Han Zhang ◽  
Ghulam Muhae Ud Din ◽  
...  
Keyword(s):  
Detection Limit ◽  
Real Time ◽  
Real Time Pcr ◽  
Scar Marker ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Common Bunt ◽  
Tilletia Laevis ◽  
Pcr Method ◽  
Dna Fragment

Abstract Common bunt of wheat caused by Tilletia laevis and/or T. caries (syn. T. tritici), is a major disease in wheat-growing regions worldwide that could lead to 80% or even total loss of production. Even though T. laevis can be distinguished from T. caries on the bases of morphology of teliospores using microscopy technique. However, molecular methods could serve as an additional method to quantify the pathogen. To develop a rapid diagnostic and quantify method, we employed the ISSR molecular marker for T. laevis in this study. The primer ISSR857 generated a polymorphic pattern displaying a 1385 bp T. laevis-specific DNA fragment. A pair of specific primers (L57F/L57R) was designed to amplify a sequence-characterized amplified region (SCAR) (763 bp) for the PCR detection assay. The primers amplified the DNA fragment in the tested isolates of T. laevis but failed in the related species, including T. caries. The detection limit of the primer set (L57F/L57R) was 5 ng/µl of DNA extracted from T. laevis teliospores. A SYBR Green I real-time PCR method for detecting T. laevis with a 100 fg/µl detection limit and droplet digital PCR with a high sensitivity (30 fg/µl detection limit) were developed; this technique showed the most sensitive detection compared to the SCAR marker and SYBR Green I real-time PCR. Additionally, this is the first study related the detection of T. laevis with the droplet digital PCR method.

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

Duplex Real-Time PCR Method for the Differentiation of Cronobacter sakazakii and Cronobacter malonaticus

2016 ◽  
Vol 80 (1) ◽  
pp. 50-56 ◽  
Author(s):  
XIAOFANG LI ◽  
JINGHUA CUI ◽  
XIAOLI DU ◽  
ZHIGANG CUI ◽  
YIBING HUANG ◽  
...  
Keyword(s):  
Detection Limit ◽  
Real Time ◽  
Real Time Pcr ◽  
Reliable Method ◽  
Common Species ◽  
Powdered Infant Formula ◽  
Cronobacter Sakazakii ◽  
Pcr Assay ◽  
Surveillance Programs ◽  
Pcr Method

ABSTRACT Cronobacter sakazakii and Cronobacter malonaticus are the most common species of Cronobacter, so it is necessary to detect the two species as soon as possible in surveillance programs. We developed a real-time PCR method for identifying C. sakazakii and C. malonaticus from the genus Cronobacter. In this study, the two pairs of primers and probes were designed, targeting 16S rRNA and fusA, respectively. The specificity of the real-time PCR assay was validated with 112 strains of Cronobacter, including 56 C. sakazakii, 32 C. malonaticus, 16 Cronobacter dublinensis, 6 Cronobacter turicensis, and 2 Cronobacter muytjensii. The results showed that C. sakazakii and C. malonaticus were all correctly identified, consistent with the results of another method by analyzing the clustering of the fusA sequence. The detection limit for pure culture was 102 CFU/ml and 103 CFU/g for artificially contaminated rehydrated powdered infant formula. Therefore, the developed real-time PCR was a rapid, sensitive, and reliable method for the identification of C. sakazakii and C. malonaticus.

Detection of Streptococcus pneumoniae in Sputum Samples by Real- Time PCR.

2020 ◽  
Vol 18 ◽  
Author(s):  
Pegah Shakib ◽  
Mohammad Reza Zolfaghari
Keyword(s):  
Streptococcus Pneumoniae ◽  
Real Time ◽  
Real Time Pcr ◽  
False Negative ◽  
Cross Sectional Study ◽  
Laboratory Culture ◽  
Cross Sectional ◽  
Negative Results ◽  
False Negative Results ◽  
Pcr Method

Background: Conventional laboratory culture-based methods for diagnosis of Streptococcus pneumoniae are time-consuming and yield false negative results. Molecular methods including real-time (RT)-PCR rapid methods and conventional PCR due to higher sensitivity and accuracy have been replaced instead traditional culture assay. The aim of the current study was to evaluate lytA gene for detection of Streptococcus pneumoniae in the cerebrospinal fluid of human patients with meningitis using real-time PCR assay. Material and Methods: In this cross-sectional study, a total of 30 clinical specimens were collected from patients in a period from September to December 2018. In order to evaluate the presence of lytA gene, conventional and real-time PCR methods were used without culture. Results: From 30 sputum samples five (16.66%) isolates were identified as S. pneumoniae by lytA PCR and sequencing. Discussion: In this research, an accurate and rapid real-time PCR method was used, which is based on lytA gene for diagnosis of bacteria so that it can be diagnosed. Based on the sequencing results, the sensitivity for detection of lytA gene was 100% (5/5).

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