Dataset Information

Development of real-time PCR method for rapid and accurate detection of Centipedes (Scolopendra mutilans) in food.

ABSTRACT: Centipedes contain pharmacologically active compounds used as important medicinal material. However, the poisons produced by centipedes can cause human diseases; therefore, its use as a food ingredient is prohibited. This is the first report to develop a real-time PCR method for detection of centipedes. The primer and probe targeting the mitochondrial cytochrome c oxidase subunit 1 (COI) gene were newly designed. The specificity was verified using ten species and was confirmed to amplify only the centipede species. The real-time PCR method exhibited good linearity with a high-determination coefficient (R ² = 0.999) and a detection limit was 0.001 ng. The performance of our method was also verified using five real-time PCR platforms under Universal and Fast PCR conditions. Finally, its applicability to processed food was evaluated using binary insect mixtures, and at least 0.1% of centipedes was detected. Therefore, our method can specifically and sensitively detect centipedes in food, contributing to food safety.

SUBMITTER: Suh SM

PROVIDER: S-EPMC10130274 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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Development of real-time PCR method for rapid and accurate detection of Centipedes (<i>Scolopendra mutilans</i>) in food.

Suh Seung-Man SM Kim Eiseul E Kim Mi-Ju MJ Yang Seung-Min SM Kim Hae-Yeong HY

Food science and biotechnology 20230110 7

Centipedes contain pharmacologically active compounds used as important medicinal material. However, the poisons produced by centipedes can cause human diseases; therefore, its use as a food ingredient is prohibited. This is the first report to develop a real-time PCR method for detection of centipedes. The primer and probe targeting the mitochondrial cytochrome c oxidase subunit 1 (COI) gene were newly designed. The specificity was verified using ten species and was confirmed to amplify only th ...[more]

PMID: 37123063

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Project description:BackgroundQuantitative Polymerase Chain Reaction (qPCR) is a well-established method for quantifying levels of gene expression, but has not been routinely applied to the detection of constitutional copy number alterations of human genomic DNA. Microdeletions or microduplications of the human genome are associated with a variety of genetic disorders. Although, clinical laboratories routinely use fluorescence in situ hybridization (FISH) to identify such cryptic genomic alterations, there remains a significant number of individuals in which constitutional genomic imbalance is suspected, based on clinical parameters, but cannot be readily detected using current cytogenetic techniques.ResultsIn this study, a novel application for real-time qPCR is presented that can be used to reproducibly detect chromosomal microdeletions and microduplications. This approach was applied to DNA from a series of patient samples and controls to validate genomic copy number alteration at cytoband 22q11. The study group comprised 12 patients with clinical symptoms of chromosome 22q11 deletion syndrome (22q11DS), 1 patient trisomic for 22q11 and 4 normal controls. 6 of the patients (group 1) had known hemizygous deletions, as detected by standard diagnostic FISH, whilst the remaining 6 patients (group 2) were classified as 22q11DS negative using the clinical FISH assay. Screening of the patients and controls with a set of 10 real time qPCR primers, spanning the 22q11.2-deleted region and flanking sequence, confirmed the FISH assay results for all patients with 100% concordance. Moreover, this qPCR enabled a refinement of the region of deletion at 22q11. Analysis of DNA from chromosome 22 trisomic sample demonstrated genomic duplication within 22q11.ConclusionIn this paper we present a qPCR approach for the detection of chromosomal microdeletions and microduplications. The strategic use of in silico modelling for qPCR primer design to avoid regions of repetitive DNA, whilst providing a level of genomic resolution greater than standard cytogenetic assays. The implementation of qPCR detection in clinical laboratories will address the need to replace complex, expensive and time consuming FISH screening to detect genomic microdeletions or duplications of clinical importance.

Project description:BackgroundGenomic surveillance efforts for SARS-CoV-2 are needed to understand the epidemiology of the COVID-19 pandemic. Viral variants may impact routine diagnostic testing, increase viral transmissibility, cause differences in disease severity, have decreased susceptibility to therapeutics, and/or confer the ability to evade host immunity. While viral whole-genome sequencing (WGS) has played a leading role in surveillance programs, many laboratories lack the expertise and resources for performing WGS. This study describes the performance of multiplexed real-time reverse transcription-PCR (RT-PCR) assays for identification of SARS-CoV-2 variants.MethodsSARS-CoV-2 specimens were tested for spike-gene variants using a combination of allele-specific primer and allele-specific detection technology (PlexPrime® and PlexZyme®). Targeted detection of spike gene mutations by RT-PCR was compared to variant detection in positive specimens by WGS, including the recently emerged SARS-CoV-2 Omicron variant.ResultsA total of 398 SAR-CoV-2 RT-PCR positive and 39 negative specimens previously tested by WGS were re-tested by RT-PCR genotyping. PCR detection of spike gene mutations N501Y, E484K, and S982A correlated 100% with WGS for the 29 lineages represented, including Alpha (B.1.1.7), Beta (B.1.351), and Gamma (P.1). Incorporating the P681R spike gene mutation also allowed screening for the SARS-CoV-2 Delta variant (B.1.617.2 and AY sublineages). Further sampling of 664 specimens that were screened by WGS between June and August 2021 and then re-tested by RT-PCR showed strong agreement for Delta variant positivity: 34.5% for WGS vs 32.9% for RT-PCR in June; 100% vs 97.8% in August. In a blinded panel of 16 Omicron and 16 Delta specimens, results of RT-PCR were 100% concordant with WGS results.ConclusionsThese data demonstrate that multiplexed real-time RT-PCR genotyping has strong agreement with WGS and may provide additional SARS-CoV-2 variant screening capabilities when WGS is unavailable or cost-prohibitive. RT-PCR genotyping assays may also supplement existing sequencing efforts while providing rapid results at or near the time of diagnosis to help guide patient management.

Dataset Information

Development of real-time PCR method for rapid and accurate detection of Centipedes (Scolopendra mutilans) in food.

Publications

Development of real-time PCR method for rapid and accurate detection of Centipedes (<i>Scolopendra mutilans</i>) in food.

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