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.

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).

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.

scholarly journals Deteksi Plasmodium falciparum dengan menggunakan metode real-time polymerase chain reaction di daerah Likupang dan Bitung

2016 ◽  
Vol 4 (1) ◽  
Author(s):  
David C. Tooy ◽  
Janno B. Bernadus ◽  
Angle Sorisi
Keyword(s):  
Polymerase Chain Reaction ◽  
Plasmodium Falciparum ◽  
Real Time ◽  
Real Time Pcr ◽  
18S Rrna ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Negative Results ◽  
Pcr Method ◽  
Polymerase Chain

Abstract: Malaria is one of the most important parasitic disease which is caused by Plasmodium spp. There are approximately 1,2 billion people in the world with high risk of getting malaria. Plasmodium falciparum (P. falciparum) is the cause of tropical malaria or falciparum malaria, and is responsible for most of the mortality rate. Currently, real-time polymerase chain reaction (RT-PCR) is being studied as an alterative of conventional malarian examination. Mangold et al reported that RT-PCR have 94.1% sensitivity and 100% specificity compared to microscopic examination in detecting P. falciparum. The aim of this research is to detect the presence of P. falciparum using RT-PCR in Likupang and Bitung region. This research were using descriptive design to find out the capability of real-time PCR method to detect P. falciparum in Likupang dan Bitung region. The researcher have examined 71 samples which are fulfill the research sample’s criteria. Postive results of P. falciparum found in 18 samples (25,3%) and negative results in 53 samples (74,6%) of total 71 samples with using RT-PCR. No positive results were found in samples from Likupang. There are positive result of P. falciparum in samples from Bitung. It is concluded that RT-PCR method can detect the presence of P. falciparum from the samples obtained from Likupang and Bitung based on the presence of its DNA. This detection efford is done by using 18S rRNA as target gene and ajust specific temperature on the RT-PCR instrument.Keywords: Plasmodium falciparum, Real-time Polymerase Chain Reaction (PCR), DetectionAbstrak: Malaria merupakan salah satu penyakit penting yang disebabkan oleh parasit Plasmodium spp. Kira-kira 1,2 miliar penduduk dunia memiliki risiko tinggi untuk mendapat malaria. Di Indonesia sendiri, terdapat 343.527 kasus terkonfirmasi dan 45 kematian karena malaria. Plasmodium falciparum (P. Falciparum) merupakan penyebab dari malaria tropika atau malaria falsiparum, dan bertanggung jawab atas sebagian besar angka mortalitas. Saat ini Real-Time Polymerase Chain Reaction (RT-PCR) telah banyak diteliti sebagai alternatif dari pemeriksaan malaria. Mangold dkk melaporkan bahwa real-time PCR memiliki nilai sensitivitas 94,1% dan nilai spesifisitas 100% terhadap pemeriksaan mikroskopis dalam mendeteksi P. falciparum. Penelitian bertujuan untuk mendeteksi P. falciparum dengan menggunakan RT-PCR di daerah Likupang dan Bitung. Penelitian ini menggunakan rancangan penelitian deskriptif untuk mengetahui kemampuan metode real-time PCR dalam mendeteksi P. falciparum di daerah Likupang dan Bitung. Tujuan penelitian ini ialah untuk mendeteksi keberadaan P. falciparum dengan menggunakan metode real-time PCR di daerah Likupang dan Bitung. Peneliti memeriksa 71 sampel darah yang memenuhi kriteria sampel penelitian. Hasil positif P. falciparum ditemukan pada 18 sampel (25,3 %) dan hasil negatif pada 53 sampel (74,6 %) dari total 71 sampel dengan menggunakan RT-PCR. Tidak ditemukannya hasil positif P. falciparum pada sampel dari Likupang. Ditemukan hasil positif P. falciparum pada sampel dari Bitung. Simpulan: Metode RT-PCR dapat mendeteksi P. falciparum berdasarkan keberadaan DNA-nya pada sampel yang diperoleh dari daerah Likupang dan Bitung. Deteksi ini berhasil dilakukan dengan menggunakan 18S rRNA sebagai gen target dan pengaturan suhu tertentu pada instrument RT-PCR.Kata kunci: P. falciparum, Real-time Polymerase Chain Reaction (PCR), Detection

scholarly journals Usefulness of real-time PCR assay targeting lipL32 gene for diagnosis of human leptospirosis in Uruguay

2013 ◽  
Vol 7 (12) ◽  
pp. 941-945 ◽  
Author(s):  
Sabina González ◽  
Juan Pablo Geymonat ◽  
Elba Hernández ◽  
Juan Martín Marqués ◽  
Felipe Schelotto ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dna Amplification ◽  
Diagnostic Sensitivity ◽  
Analytical Sensitivity ◽  
Serum Samples ◽  
Initial Management ◽  
Negative Results ◽  
Acute Infections ◽  
Positive Results

Introduction: Assays based on DNA amplification can provide information that contributes to the initial management of patients with leptospirosis. However, these have not been adopted in Uruguay. Our aim was to evaluate the performance of the lipL32 real-time PCR (qPCR) for diagnosis of leptospirosis. Methodology: We analyzed by microscopic agglutination test (MAT) and lipL32 qPCR serum samples from 183 patients with suspected leptospirosis. To establish the analytical sensitivity of the qPCR, experimentally spiked samples with known amounts of Leptospira interrogans were analyzed. Results: The analytical sensitivity of the qPCR was 102 leptospires/mL. In 98 patients MAT results were negative meanwhile 85 showed positive reactions, revealing acute infections. Twenty six acute-phase sera of these 85 patients showed a positive signal by qPCR (diagnostic sensitivity 30%). In these patients the average time between onset of symptoms and collection of the first sample was 8 days. In patients with negative results for qPCR and positive MAT results (n=59) the average interval between onset of symptoms and collection of the first sample was 13 days. The qPCR did not yield false positive results. Conclusions: The qPCR had a lower diagnostic sensitivity than MAT and a higher cost. However, it allowed to make an early diagnosis in 26 patients. In patients with confirmed acute infections and negative results by qPCR, more than 8 days had elapsed between the onset of the illness and extraction of the first serum sample. Our data support that the qPCR from sera have clinical utility within the first week of illness.

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 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 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 Quantitation of Viral DNA by Real-Time PCR Applying Duplex Amplification, Internal Standardization, and Two-Color Fluorescence Detection

2001 ◽  
Vol 67 (6) ◽  
pp. 2837-2839 ◽  
Author(s):  
Franz Gruber ◽  
Falko G. Falkner ◽  
Friedrich Dorner ◽  
Thomas Hämmerle
Keyword(s):  
Real Time ◽  
Fluorescence Detection ◽  
Real Time Pcr ◽  
Parvovirus B19 ◽  
False Negative ◽  
Viral Dna ◽  
Negative Results ◽  
False Negative Results ◽  
Pcr Method ◽  
Internal Standardization

ABSTRACT A real-time PCR method was developed to quantitate viral DNA that includes duplex amplification, internal standardization, and two-color fluorescence detection without the need to generate an external standardization curve. Applied to human parvovirus B19 DNA, the linear range was from 102 to at least 5 × 106 copies per ml of sample. The coefficient of variation was 0.29 using a run control of 2,876 copies per ml. The method reduces the risk of false-negative results, yields high precision, and is applicable for other DNA targets.

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.

Direct Real-Time PCR with Ethidium Monoazide: A Method for the Rapid Detection of Viable Cronobacter sakazakii in Powdered Infant Formula

2012 ◽  
Vol 75 (9) ◽  
pp. 1572-1579 ◽  
Author(s):  
JUN-ICHI MINAMI ◽  
TAKASHI SOEJIMA ◽  
TOMOKO YAESHIMA ◽  
KEIJI IWATSUKI
Keyword(s):  
Real Time ◽  
Infant Formula ◽  
Real Time Pcr ◽  
Powdered Infant Formula ◽  
Cronobacter Sakazakii ◽  
Ethidium Monoazide ◽  
Rapid Assay ◽  
Negative Results ◽  
Current Detection ◽  
Positive Results

The goal of this study was to establish a rapid assay for the specific detection of viable Cronobacter sakazakii in powdered infant formula (PIF). Samples were subjected to treatment multiple times with ethidium monoazide with a concentration gradient (gEMA) prior to PCR to discriminate viable from dead C. sakazakii cells. To improve the current detection limits, we developed a new buffer for direct quantitative real-time PCR (DqPCR) without DNA isolation. Using 17 PIF samples, our rapid assay was compared with the new U.S. Food and Drug Administration (FDA) method published in the Bacteriological Analytical Manual in 2012. Although both the new FDA method and our rapid assay, which consists of DqPCR combined with gEMA (gEMADqPCR), produced negative results for all 17 PIF samples, 5 of the 17 PIFs were positive by DqPCR when they were not treated with EMA. Furthermore, for PIF samples artificially contaminated with viable C. sakazakii, gEMA-DqPCR successfully detected between 1 and 9 CFU of viable C. sakazakii in 300 g of PIF within 9 h, including a 6-h preincubation. Our results indicate that multiple EMA treatments are required to avoid false-positive results due to the contamination of commercial PIF with dead or injured C. sakazakii cells. Our rapid assay may also improve the sensitivity of the screening portion required by the new FDA method published in the Bacteriological Analytical Manual in 2012.

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