scholarly journals Development of a triplex real-time PCR for simultaneous detection of allergenic ingredients in processed food

2018 ◽  
Vol 36 (No. 1) ◽  
pp. 22-27 ◽  
Author(s):  
Wenju Zhang ◽  
Yulei Zhao ◽  
Qingjin Xu ◽  
Qin Chen
Keyword(s):  
Real Time ◽  
Simultaneous Detection ◽  
High Specificity ◽  
Processed Food ◽  
Specific Pcr ◽  
Specificity And Sensitivity ◽  
Pcr Method ◽  
Primer Sets ◽  
Specific Pcr Primer ◽  
Suitable System

SYBR Green real-time or quantitative PCR (Q-PCR) is a suitable system in which to establish a multiplex method to detect allergenic ingredients in food. In this study, a triplex Q-PCR method was developed to detect trace amounts of peanut, soybean and sesame in processed food. Specific PCR primer sets were designed and the concentration of the primers used in the triplex PCR was optimised. The triplex method showed high specificity and sensitivity which were similar to those of the simplex method, and it was applied for the detection of allergenic ingredients in commercially available processed food. The results demonstrate that the developed triplex Q-PCR is a quick, reliable and efficient method for the detection of allergenic ingredients in processed food.

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.

Method for Identifying Heat-Resistant Fungi of the Genus Neosartorya

2012 ◽  
Vol 75 (10) ◽  
pp. 1806-1813 ◽  
Author(s):  
TAKASHI YAGUCHI ◽  
YUMI IMANISHI ◽  
TETSUHIRO MATSUZAWA ◽  
KOUICHI HOSOYA ◽  
JUN HITOMI ◽  
...  
Keyword(s):  
Food Industry ◽  
High Specificity ◽  
Species Level ◽  
Processed Food ◽  
Food Spoilage ◽  
Specific Primer ◽  
Identification Methods ◽  
Heat Resistant ◽  
Pcr Method ◽  
Primer Sets

Species of the genus Neosartorya are heat-resistant fungi that cause the spoilage of heat-processed acidic foods due to the formation of heat-resistant ascospores, and they produce mycotoxins, such as fumitremorgins and gliotoxin. Their anamorphs are phylogenetically and morphologically very close to Aspergillus fumigatus, which has never been reported as a spoilage agent in heat-processed food products. Therefore it is important to discriminate between the species of Neosartorya and A. fumigatus in the food industry. In the present study, we examined β-tubulin and calmodulin genes to identify Neosartorya and A. fumigatus at the species level and found a region for specifically detecting these species. We succeeded in developing the PCR method of differentiating and identifying Neosartorya and A. fumigatus using specific primer sets. Moreover, we developed specific primer sets to identify Neosartorya species, N. fischeri, N. glabra, N. hiratsukae, N. pseudofischeri, and N. spinosa–complex, which are important in food spoilage; these fungi vary in heat resistance and productivity of mycotoxins, depending on the species. PCR using these primer sets did not detect other fungi involved in food spoilage and environmental contamination. These identification methods are rapid and simple with extremely high specificity.

Development of a real-time PCR method for the simultaneous detection of soya and lupin mitochondrial DNA as markers for the presence of allergens in processed food

2011 ◽  
Vol 127 (2) ◽  
pp. 834-841 ◽  
Author(s):  
Antonio M. Gomez Galan ◽  
Marcel Brohée ◽  
Eugénia de Andrade Silva ◽  
Arjon J. van Hengel ◽  
Hubert Chassaigne
Keyword(s):  
Mitochondrial Dna ◽  
Real Time ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Processed Food ◽  
Pcr Method

scholarly journals An Optimized Real-Time PCR to Avoid Species-/Tissue-Associated Inhibition for H5N1 Detection in Ferret and Monkey Tissues

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
LingJun Zhan ◽  
LinLin Bao ◽  
FengDi Li ◽  
Qi Lv ◽  
LiLi Xu ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rna Isolation ◽  
High Specificity ◽  
Sybr Green ◽  
Pcr Inhibitors ◽  
Specificity And Sensitivity ◽  
Avian Influenza Virus H5n1 ◽  
Pcr Method ◽  
Tissue Specimens

The real-time PCR diagnostics for avian influenza virus H5N1 in tissue specimens are often suboptimal, since naturally occurring PCR inhibitors present in samples, or unanticipated match of primer to unsequenced species’ genome. With the principal aim of optimizing the SYBR Green real-time PCR method for detecting H5N1 in ferret and monkey (Chinese rhesus macaque) tissue specimens, we screened various H5N1 gene-specific primer pairs and tested their ability to sensitively and specifically detect H5N1 transcripts in the infected animal tissues, then we assessed RNA yield and quality by comparing Ct values obtained from the standard Trizol method, and four commonly used RNA isolation kits with small modifications, including Roche High Pure, Ambion RNAqueous, BioMIGA EZgene, and Qiagen RNeasy. The results indicated that a single primer pair exhibited high specificity and sensitivity for H5N1 transcripts in ferret and monkey tissues. Each of the four kits and Trizol reagent produced high-quality RNA and removed all or nearly all PCR inhibitors. No statistically significant differences were found between the Ct values from the isolation methods. So the optimized SYBR Green real-time PCR could avoid species- or tissue-associated PCR inhibition in detecting H5N1 in ferret and monkey tissues, including lung and small intestine.

scholarly journals Identification of Novel Sensitive and Reliable Serovar-Specific Targets for PCR Detection of Salmonella Serovars Hadar and Albany by Pan-Genome Analysis

2021 ◽  
Vol 12 ◽  
Author(s):  
Qinghua Ye ◽  
Yuting Shang ◽  
Moutong Chen ◽  
Rui Pang ◽  
Fan Li ◽  
...  
Keyword(s):  
Genome Analysis ◽  
High Specificity ◽  
Food Samples ◽  
Genomic Sequences ◽  
Specific Gene ◽  
Pan Genome ◽  
Specific Pcr ◽  
Pcr Method ◽  
Primer Sets ◽  
Pcr Targeting

The accurate and rapid classification of Salmonella serovars is an essential focus for the identification of isolates involved in disease in humans and animals. The purpose of current research was to identify novel sensitive and reliable serovar-specific targets and to develop PCR method for Salmonella C2 serogroups (O:8 epitopes) in food samples to facilitate timely treatment. A total of 575 genomic sequences of 16 target serovars belonging to serogroup C2 and 150 genomic sequences of non-target serovars were analysed by pan-genome analysis. As a result, four and three specific genes were found for serovars Albany and Hadar, respectively. Primer sets for PCR targeting these serovar-specific genes were designed and evaluated based on their specificity; the results showed high specificity (100%). The sensitivity of the specific PCR was 2.8 × 101–103 CFU/mL and 2.3 × 103–104 CFU/mL for serovars Albany and Hadar, respectively, and the detection limits were 1.04 × 103–104 CFU/g and 1.16 × 104–105 CFU/g in artificially contaminated raw pork samples. Furthermore, the potential functions of these serovar-specific genes were analysed; all of the genes were functionally unknown, except for one specific serovar Albany gene known to be a encoded secreted protein and one specific gene for serovars Hadar and Albany that is a encoded membrane protein. Thus, these findings demonstrate that pan-genome analysis is a precious method for mining new high-quality serovar-targets for PCR assays or other molecular methods that are highly sensitive and can be used for rapid detection of Salmonella serovars.

scholarly journals Development of a duplex real-time PCR method for the detection of influenza C and D viruses

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Letian Zhang ◽  
Meng Lu ◽  
Jiaxuan Lu ◽  
Ningning Wang ◽  
Zhongzhou Pan ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Detection Method ◽  
Phylogenetic Analyses ◽  
Genome Structure ◽  
Simultaneous Detection ◽  
Influenza Viruses ◽  
Clinical Test ◽  
Specificity And Sensitivity ◽  
Pcr Method

AbstractInfluenza viruses are major respiratory pathogens known to infect human and a variety of animals and are widely prevalent worldwide. Genome structure of influenza D virus (IDV) is identical to that of influenza C virus (ICV), and phylogenetic analyses suggest that IDV and ICV share a common ancestry and high homology. To date, the prevalence of ICV and IDV in China is unclear, but these viruses represent a potential threat to public health due to cross-species transmission and zoonotic potential. To efficiently monitor ICV and IDV, it is necessary to establish a dual detection method to understand their prevalence and conduct in-depth research. A duplex real-time PCR method for the simultaneous detection of ICV and IDV was developed. TaqMan fluorescent probes and specific primers targeting NP gene of ICV and PB1 gene of IDV were designed. This method exhibited good specificity and sensitivity, and the detection limit reached 1 × 101 copies/μL of plasmid standards of each pathogen. Thirty-one clinical swine samples and 10 clinical cattle samples were analyzed using this method. One positive sample of IDV was detected, and the accuracy of clinical test results was verified by conventional PCR and DNA sequencing. The duplex real-time PCR detection method represents a sensitive and specific tool to detect ICV and IDV. It provides technical support for virus research and clinical diagnosis of ICV and IDV. This information will benefit animal and human health.

scholarly journals An Editing-Site-Specific PCR Method for Detection and Quantification of CAO1-Edited Rice

Foods ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1209
Author(s):  
Hongwen Zhang ◽  
Jun Li ◽  
Shengbo Zhao ◽  
Xiaohong Yan ◽  
Nengwu Si ◽  
...  
Keyword(s):  
Genetically Modified Organisms ◽  
High Specificity ◽  
Editing Site ◽  
Locked Nucleic Acid ◽  
Molecular Characteristics ◽  
Site Specific ◽  
Specific Pcr ◽  
Specificity And Sensitivity ◽  
Pcr Method ◽  
Detection And Quantification

Genome-edited plants created by genome editing technology have been approved for commercialization. Due to molecular characteristics that differ from classic genetically modified organisms (GMOs), establishing regulation-compliant analytical methods for identification and quantification of genome-edited plants has always been regarded as a challenging task. An editing-site-specific PCR method was developed based on the unique edited sequence in CAO1-edited rice plants. Test results of seven primer/probe sets indicated that this method can identify specific CAO1-edited rice from other CAO1-edited rice and wild types of rice with high specificity and sensitivity. The use of LNA (locked nucleic acid) in a probe can efficiently increase the specificity of the editing-site-specific PCR method at increased annealing temperature which can eliminate non-specific amplification of the non-target. The genome-edited ingredient content in blinded samples at the level of 0.1% to 5.0% was accurately quantified by this method on the ddPCR platform with RSD of <15% and bias in the range of ±17%, meeting the performance requirements for GMO detection method. The developed editing-site-specific PCR method presents a promising detection and quantification technique for genome-edited plants with known edited sequence.

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.

Sign in / Sign up

Export Citation Format

Share Document