scholarly journals Detection of Peanut Allergen by Real Time PCR: Looking For the Suitable Detection Marker as Affected by Processing

2021 ◽  
Author(s):  
Africa Sanchiz ◽  
Paulina Sánchez-Enciso ◽  
Carmen Cuadrado ◽  
Rosario Linacero
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dna Isolation ◽  
Peanut Flour ◽  
Coding Region ◽  
Peanut Allergen ◽  
Assay Sensitivity ◽  
Organelle Dna ◽  
Chloroplast Markers ◽  
Specificity And Sensitivity

Peanut (Arachis hypogaea) contains allergenic proteins, which make it harmful to the sensitised population. The presence of peanut in foods must be indicated on label, to prevent accidental consumption by allergic population.. In this work, we use chloroplast markers for specifically detection of peanut by real-time PCR, in order to increase the assay sensitivity. Three different protocols of DNA isolation were evaluated, for total and organelle-DNA extraction. Binary mixtures of raw and processed peanut flour in wheat were performed at concentrations ranging from 100000 to 0.1 mg/kg. DNA isolation from peanut, mixtures and other legumes was carried out following three protocols for obtaining genomic and chloroplast-enrich DNA. Quantity and quality of DNA was evaluated, obtaining better results for protocol 2. Specificity and sensitivity of the method has been assayed with specific primers for three chloroplast markers (mat k, rpl16 and trnH-psbA) and Ara h 6 peanut allergen-coding region was selected as nuclear low-copy target and TaqMan probes. Efficiency and linear correlation of calibration curves were within the adequate ranges. Moreover, the influence of pressure and thermal processing on the peanut detectability was analyzed.

scholarly journals Detection of Peanut Allergen by Real Time PCR: Looking For the Suitable Detection Marker and Dna Isolation Method

2021 ◽  
Author(s):  
Africa Sanchiz ◽  
Paulina Sánchez-Enciso ◽  
Carmen Cuadrado ◽  
Rosario Linacero
Keyword(s):  
Real Time ◽  
Conformational Changes ◽  
Real Time Pcr ◽  
Dna Isolation ◽  
Interspecific Variation ◽  
Peanut Flour ◽  
Coding Region ◽  
Peanut Allergen ◽  
Chloroplast Markers ◽  
Specificity And Sensitivity

Peanut (Arachis hypogaea) contains allergenic proteins, which make it harmful to the sensitive population. The presence of peanut in foods must be indicated on label, to prevent accidental consumption by allergenic population. The development of suitable analytical methodologies to detect this allergen in processed foods is advisable. Real Time PCR allowed a specific and accurate amplification of allergen sequences. The optimal genome targets for specific and sensitive detection are those that show interspecific variation and high copy number. Some food processing methods could induce structural and/or conformational changes in proteins and produce the fragmentation and/or degradation of genomic DNA. In this work, we use chloroplast markers for specifically detection of peanut by Real Time PCR, in order to increase the sensitivity. Three different protocols of DNA isolation were evaluated, for total and organelle-DNA extraction. Binary mixtures of raw and processed peanut flour in wheat were performed at concentrations ranging from 100000 to 0.1 ppm. DNA isolation from peanut, mixtures and other legumes was carried out following three protocols for obtaining genomic and chloroplast-enrich DNA. Quantity and quality of DNA was evaluated, obtaining better results for protocol 2. Specificity and sensitivity of the method has been assayed with specific primers for three chloroplast markers (mat k, rpl16 and trnH-psbA) and Ara h 6 peanut allergen-coding region was selected as nuclear low-copy target and TaqMan probes. Efficiency and linear correlation of calibration curves were within the adequate ranges. Moreover, the influence of pressure and thermal processing on the peanut detectability was analyzed.

scholarly journals Detection of Peanut Allergen by Real-Time PCR: Looking for a Suitable Detection Marker as Affected by Processing

Foods ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1421
Author(s):  
Africa Sanchiz ◽  
Paulina Sánchez-Enciso ◽  
Carmen Cuadrado ◽  
Rosario Linacero
Keyword(s):  
Real Time ◽  
Binary Mixtures ◽  
Linear Correlation ◽  
Real Time Pcr ◽  
Peanut Flour ◽  
Peanut Allergen ◽  
Assay Sensitivity ◽  
Chloroplast Markers ◽  
Efficient Detection ◽  
Specificity And Sensitivity

Peanut (Arachis hypogaea) contains allergenic proteins, which make it harmful to the sensitised population. The presence of peanut in foods must be indicated on label, to prevent accidental consumption by allergic population. In this work, we use chloroplast markers for specific detection of peanut by real-time PCR (Polymerase Chain Reaction), in order to increase the assay sensitivity. Binary mixtures of raw and processed peanut flour in wheat were performed at concentrations ranging from 100,000 to 0.1 mg/kg. DNA isolation from peanut, mixtures, and other legumes was carried out following three protocols for obtaining genomic and chloroplast-enrich DNA. Quantity and quality of DNA were evaluated, obtaining better results for protocol 2. Specificity and sensitivity of the method has been assayed with specific primers for three chloroplast markers (mat k, rpl16, and trnH-psbA) and Ara h 6 peanut allergen-coding region was selected as nuclear low-copy target and TaqMan probes. Efficiency and linear correlation of calibration curves were within the adequate ranges. Mat k chloroplast marker yielded the most sensitive and efficient detection for peanut. Moreover, detection of mat K in binary mixtures of processed samples was possible for up to 10 mg/kg even after boiling, and autoclave 121 °C 15 min, with acceptable efficiency and linear correlation. Applicability of the method has been assayed in several commercial food products.

COMPARATIVE STUDY OF SPECIFICITY AND SENSITIVITY FOR IHHNV DETECTION BETWEEN REAL-TIME PCR AND LAMP METHODS

2010 ◽  
Vol 36 (5) ◽  
pp. 984-989
Author(s):  
Zhe ZHAO ◽  
Chun-Hua REN ◽  
Xiao JIANG ◽  
Lü-Ping ZHANG ◽  
Juan FENG ◽  
...  
Keyword(s):  
Comparative Study ◽  
Real Time ◽  
Real Time Pcr ◽  
Specificity And Sensitivity

scholarly journals Detection of Peanut Allergen Traces with a Real Time PCR Assay - The Challenge to Protect Food-Allergic Consumers

2017 ◽  
Vol 7 (1) ◽  
pp. 32 ◽  
Author(s):  
Dimitra Houhoula ◽  
Stamatios Koussissis ◽  
Vladimiros Lougovois ◽  
John Tsaknis ◽  
Dimitra Kassavita ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Food Products ◽  
Molecular Techniques ◽  
Food Allergens ◽  
Pcr Assay ◽  
Peanut Allergen ◽  
Pcr Method ◽  
Detection And Quantification

The aim of the present study was the implementation of molecular techniques in the detection and quantification of allergic substances of peanut in various kinds of food products, e.g., breakfast cereals, chocolates and biscuits that are frequently related to allergies. In some cases, the presence of peanuts can be due to contamination during production and are not declared on the label. A total of 152 samples were collected from supermarkets and were analysed by a Real Time PCR method. The results indicated that 125 samples (83,3%) were found positive in peanut traces but the most important finding is that from the 84 samples that had no allergen declaration for peanuts, 48 (57,1%) of them were found positive. In conclusion, Real Time PCR can be a very important tool for the rapid detection and quantification of food allergens.

scholarly journals A New Highly Sensitive and Specific Real-Time PCR Assay Targeting the Malate Dehydrogenase Gene ofKingella kingaeand Application to 201 Pediatric Clinical Specimens

2018 ◽  
Vol 56 (8) ◽  
Author(s):  
Nawal El Houmami ◽  
Guillaume André Durand ◽  
Janek Bzdrenga ◽  
Anne Darmon ◽  
Philippe Minodier ◽  
...  
Keyword(s):  
Real Time ◽  
Malate Dehydrogenase ◽  
Real Time Pcr ◽  
Detection Sensitivity ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Content Type ◽  
Clinical Specimens ◽  
Specificity And Sensitivity ◽  
Pcr Assays

ABSTRACTKingella kingaeis a significant pediatric pathogen responsible for bone and joint infections, occult bacteremia, and endocarditis in early childhood. Past efforts to detect this bacterium using culture and broad-range 16S rRNA gene PCR assays from clinical specimens have proven unsatisfactory; therefore, by the late 2000s, these were gradually phased out to explore the benefits of specific real-time PCR tests targeting thegroELgene and the RTX locus ofK. kingae. However, recent studies showed that real-time PCR (RT-PCR) assays targeting theKingellasp. RTX locus that are currently available for the diagnosis ofK. kingaeinfection lack specificity because they could not distinguish betweenK. kingaeand the recently describedKingella negevensisspecies. Furthermore,in silicoanalysis of thegroELgene from a large collection of 45K. kingaestrains showed that primers and probes fromK. kingaegroEL-based RT-PCR assays display a few mismatches withK. kingae groELvariations that may result in decreased detection sensitivity, especially in paucibacillary clinical specimens. In order to provide an alternative togroEL- and RTX-targeting RT-PCR assays that may suffer from suboptimal specificity and sensitivity, aK. kingae-specific RT-PCR assay targeting the malate dehydrogenase (mdh) gene was developed for predicting no mismatch between primers and probe and 18 variants of theK. kingae mdhgene from 20 distinct sequence types ofK. kingae. This novelK. kingae-specific RT-PCR assay demonstrated high specificity and sensitivity and was successfully used to diagnoseK. kingaeinfections and carriage in 104 clinical specimens from children between 7 months and 7 years old.

scholarly journals Detection and identification of Acanthamoeba and other non-viral causes of infectious keratitis in corneal scrapings by real-time PCR and next-generation sequencing-based 16S-18S gene analysis

2020 ◽  
Author(s):  
Dennis Back Holmgaard ◽  
Celine Barnadas ◽  
Seyed Hossein Mirbarati ◽  
Lee O’Brien Andersen ◽  
Henrik Vedel Nielsen ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Relevance ◽  
Bacterial Species ◽  
Screening Method ◽  
Infectious Keratitis ◽  
Next Generation ◽  
Specificity And Sensitivity ◽  
Generation Sequencing

Acanthamoeba is a free-living amoeba of extensive genetic diversity. It may cause infectious keratitis (IK), which can also be caused by bacteria, fungi, and viruses. High diagnostic sensitivity is essential to establish an early diagnosis of Acanthamoeba-associated keratitis. Here, we investigated the applicability of next-generation sequencing (NGS)-based ribosomal gene detection and differentiation (16S-18S) compared with specific real-time PCR for detection of Acanthamoeba. Two hundred DNAs extracted from corneal scrapings and screened by Acanthamoeba-specific real-time PCR were analyzed using an in-house 16S-18S NGS assay. Of these, 24 were positive using specific real-time PCR, 21 of which were positive using the NGS assay. Compared with real-time PCR; the specificity and sensitivity of the NGS assay were 100% and 88%, respectively. Genotypes identified by the NGS assay included T4 (n = 19) and T6 (n = 2). Fungal and bacterial species of potential clinical relevance were identified in 31 of the samples negative for Acanthamoeba, exemplified by Pseudomonas aeruginosa (n = 11), Moraxella spp. (n = 6), Staphylococcus aureus (n = 2), Fusarium spp. (n = 4), and Candida albicans (n = 1). Conclusively, the 16S-18S assay was slightly less sensitive than real-time PCR in detecting Acanthamoeba-specific DNA in corneal scrapings. Robust information on genotype was provided by the NGS assay, and other pathogens of potential clinical relevance were identified in 16% of the samples negative for Acanthamoeba. NGS-based detection of ribosomal genes in corneal scrapings could be an efficient screening method for detecting non-viral causes of IK, including Acanthamoeba.

Development of three real-time PCR assays to detect peanut allergen residue in processed food products

2007 ◽  
Vol 227 (3) ◽  
pp. 857-869 ◽  
Author(s):  
Elena Scaravelli ◽  
Marcel Brohée ◽  
Rosangela Marchelli ◽  
Arjon J. van Hengel
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Food Products ◽  
Processed Food ◽  
Peanut Allergen ◽  
Pcr Assays

Differentiation of infectious bronchitis virus vaccine strains Ma5 and 4/91 by TaqMan real-time PCR

2017 ◽  
Vol 20 (3) ◽  
pp. 599-601 ◽  
Author(s):  
T. Stenzel ◽  
D. Dziewulska ◽  
M. Śmiałek ◽  
B. Tykałowski ◽  
J. Kowalczyk ◽  
...  
Keyword(s):  
Real Time ◽  
Virus Vaccine ◽  
Infectious Bronchitis Virus ◽  
Real Time Pcr ◽  
Cross Reactivity ◽  
Specific Primers ◽  
Assay Sensitivity ◽  
Infectious Bronchitis ◽  
Molecular Assays ◽  
Rapid Molecular Assays

Abstract The aim of this study was to develop rapid molecular assays for differentiating vaccine strains Ma5 and 4/91 of the infectious bronchitis virus (IBV). Specific primers and probes for S1 and N genes were designed based on the nucleotide sequences of both vaccine strains. Cross-reactivity was not observed. Assay sensitivity was 2.373 × 103 copies of the Ma5 strain, and 3.852 x 103 copies of the 4/91 strain. Samples belonging to a known genotype demonstrated that the designed assays supported rapid and sensitive detection of Ma5 and 4/91 vaccine strains of IBV.

scholarly journals Canine distemper virus detection in asymptomatic and non vaccinated dogs

2010 ◽  
Vol 30 (2) ◽  
pp. 139-144 ◽  
Author(s):  
Helen L. Del Puerto ◽  
Anilton C. Vasconcelos ◽  
Luciana Moro ◽  
Fabiana Alves ◽  
Gissandra F. Braz ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Canine Distemper Virus ◽  
Early Stage ◽  
Virus Detection ◽  
Vero Cells ◽  
Canine Distemper ◽  
Coding Region ◽  
Protein Coding ◽  
Dissociation Curves

A quantitative real time polymerase chain reaction (PCR) revealed canine distemper virus presence in peripheral blood samples from asymptomatic and non vaccinated dogs. Samples from eleven domestic dogs with no signs of canine distemper and not vaccinated at the month of collection were used. Canine distemper virus vaccine samples in VERO cells were used as positive controls. RNA was isolated with Trizol®, and treated with a TURBO DNA-free kit. Primers were designed for canine distemper virus nucleocapsid protein coding region fragment amplification (84 bp). Canine b-actin (93 bp) was utilized as the endogenous control for normalization. Quantitative results of real time PCR generated by ABI Prism 7000 SDS Software showed that 54.5% of dogs with asymptomatic canine distemper were positive for canine distemper virus. Dissociation curves confirmed the specificity of the real time PCR fragments. This technique could detect even a few copies of viral RNA and identificate subclinically infected dogs providing accurate diagnosis of this disease at an early stage.

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