Development and validation of TaqMan qPCR assay for porcine DNA detection in gelatine-based foods for halal authentication

Halal certification is vital for confirming the halal status of food products, particularly for Muslim consumers. Instances of halal fraud, including the use of counterfeit halal logos and the incorporation of non-halal ingredients in food products, negatively impact Muslim consumers. Gelatine, widely used in the food industry, varies in acceptability depending on its source, with porcine-derived gelatine strictly prohibited in Islam. Therefore, this research aims to identify the presence of porcine DNA in gelatine-containing food products by targeting mitochondrial cytochrome b (cytb) via a real-time polymerase chain reaction (qPCR) assay and to compare the qPCR findings with the sources of gelatine and the presence of a halal logo. Nine food products without a recognised JAKIM halal logo or bearing a foreign halal logo not recognised by JAKIM were purchased from a local supermarket in Sibu, Sarawak. DNA was extracted from the food products using the DNeasy Mericon Food kit, analysed with a spectrophotometer, and used as template DNA in the qPCR assay. Positive qPCR findings were validated through DNA sequencing and BLAST analysis. Of the nine products tested, eight contained detectable porcine DNA, including one product labelled with a halal logo. Sequencing data confirmed Sus scrofa as the predominant species, with BLAST identities ranging from 86.52% to 100% against the NCBI database. In summary, these results highlight the risks posed by mislabelling and fraudulent halal logos, and emphasise the importance of rigorous certification, transparent labelling, and routine molecular verification to protect Muslim consumers and maintain trust in halal markets.

DNA extraction procedures and validation parameters of a real-time PCR method to control milk containing only A2 β-casein

To develop a broad-range 28S ribosomal DNA quantitative PCR (qPCR) assay for detection of fungal DNA in equine endometrial samples. 12 fungal samples from a clinical diagnostic laboratory and 29 samples obtained from 17 mares. The qPCR assay was optimized with commercially acquired fungal organisms and validated with samples obtained from the clinical diagnostic laboratory. Subsequently, 29 samples from 17 mares suspected of having fungal endometritis were evaluated via the qPCR assay and via traditional fungal culture and endometrial cytology. Amplicons from the qPCR assay were subjected to genetic sequencing to identify the organisms. The qPCR assay theoretically had a detection threshold of 2 organisms of Candida albicans. Fungal DNA was amplified from all 12 fungal samples from the commercial diagnostic laboratory. Fungal identification by use of genetic sequencing was successful for 34 of 36 amplicons from the 12 samples assayed. A fungal agent was identified via qPCR assay and genetic sequencing in all 12 samples; in contrast, a fungal agent was identified in only 8 of 12 samples via standard fungal culture and biochemical analysis. The qPCR assay detected fungal DNA in samples from 12 of 17 mares suspected of having fungal endometritis. A rapid, sensitive, and repeatable qPCR assay was developed for detection of fungal DNA from equine endometrial samples. The qPCR may prove to be clinically useful as an adjunct to microbial culture and cytologic examination to provide identification of fungal organisms in a timely manner.

Development and evaluation of real-time PCR assays for the quantitative detection of Babesia caballi and Theileria equi infections in horses from South Africa

Fecal contamination in fresh produce is a public health concern because it may contain human pathogens. We introduced host-specific quantitative real-time polymerase chain reaction (qPCR) assays for the rapid detection and identification of fecal contamination sources from humans and farm animals (cow, pig, chicken) in fresh produce. Each composite fecal sample was spiked on lettuce at two contamination levels (0.2 mg or 2 mg feces g⁻¹), followed by qPCR assays for detecting each host-specific genetic marker: BoBac (cow); PF163 (pig); CP3-49 (chicken); and HF183 and gyrB (human). Two commercial DNA extraction kits were compared to evaluate DNA recovery yields and removal of PCR inhibition. Sketa2 assay was conducted to assess the presence of PCR inhibition in the contaminated lettuce. All the qPCR assays yielded reliable detection from contaminated lettuce (2 mg feces g⁻¹), where their target gene numbers were 1.5-5.0 × 10³ (HF183), 0.8-2.2 × 10³ (gyrB), 0.6-1.6 × 10³ (BoBac), 1.6-3.0 × 10³ (CP3-49) and 1.1-2.2 × 10³ (PF163) copies g⁻¹ of lettuce. Among the two extraction kits, QIAamp DNA Stool Kit resulted in 2-3 times higher sensitivity and 20% less PCR inhibition than the PowerFood™ kit. This study provides information on the optimized host-specific qPCR assay in identifying sources of fecal contamination in fresh produce and is useful for tracking the contamination source and improving agricultural practice.

BackgroundIn the pharmaceutical industry, hard- and soft-shelled capsules are typically made from gelatin, commonly derived from bovine and porcine sources. To ensure that pharmaceutical products comply with halal regulations in Muslim countries (no porcine products allowed), development of a valid, reliable, quick, and most importantly, cost-effective tests are of utmost importance.MethodsWe developed a species-specific duplex polymerase chain reaction (PCR) assay targeting 149 bp porcine and 271 bp bovine mitochondrial DNA (mtDNA) to simultaneously detect both porcine and bovine DNA (in one reaction at the same time) in gelatin. Some additional simplex PCR tests (targeting 126 bp bovine and 212 bp porcine mtDNA) and real-time PCR using a commercially available kit (for identification of porcine DNA) were used to verify the selectivity and sensitivity of our duplex PCR. After optimization of DNA extraction and PCR methods, hard/soft pharmaceutical gelatin capsules (containing drug) were tested for the presence of porcine and/or bovine DNA.ResultsDuplex PCR detected the presence of as little as 0.1% porcine DNA, which was more accurate than the commercially available kit. Of all gelatin capsules tested (n = 24), 50% contained porcine DNA (pure porcine gelatin alone or in combination with bovine gelatin).ConclusionsDuplex PCR presents an easy-to-follow, quick, low-cost and reliable method to simultaneously detect porcine and bovine DNAs (>100 bp) in minute amounts in highly processed gelatin-containing pharmaceutical products (with a 0.1% sensitivity for porcine DNA) which may be used for halal authentication.Graphical abstractSimultaneous detection of porcine and bovine DNA in gelatin capsules by duplex PCR

Melioidosis, caused by category B bioterrorism agent Burkholderia pseudomallei, is a seasonal disease of tropical and subtropical regions with a high mortality rate. An early and culture-independent detection ofB. pseudomalleiis required for the appropriate disease management and prevention. The present study is designed to identify novel and unique sequences ofB. pseudomalleiand development of quantitative polymerase chain reaction (qPCR) assay. A novel B. pseudomallei-specific target sequence was identified by in silico analysis for the qPCR assay development. The specificity of the developed assay was assessed using purified DNA of 65 different bacterial cultures, and the sensitivity was estimated using a cloned target gene. Further, a type III secretion protein HrpB1 (HrpB1) gene-based duplex qPCR assay incorporating suitable extraction and amplification control was developed, and its viability was assessed in different clinical and environmental matrices for the detection of B. pseudomallei. In this study, an 80-nucleotide-longB. pseudomallei-specific region within the geneHrpB1was identified by computational analysis. The developedHrpB1-based qPCR assay was highly specific forB. pseudomalleidetection when evaluated with 65 different bacterial cultures. The sensitivity of the qPCR assay with theHrpB1-recombinant plasmid was found to be five copies per qPCR reaction. The assay's detection limit was found to be 5 × 102 CFU/mL for human blood and urine, 5 × 101 CFU/mL in river water, and 2 × 103 CFU/gm in paddy field soil. The results of the study showed the applicability of a novelHrpB1-based qPCR assay for sensitive and specific detection ofB. pseudomalleiin diverse clinical and environmental samples.

Validation and Application of a Real-Time PCR Assay Based on the CRISPR Array for Serotype-Specific Detection and Quantification of Enterohemorrhagic Escherichia coli O157:H7 in Cattle Feces

Development and Comparison of Sampling Techniques for a Broad Range, Semiquantitative Polymerase Chain Reaction Assay for Detection of Bacterial DNA in the Equine Uterus

Background – Soy sauce is a common fermented condiment used especially in East and Southeast Asian cooking. Yet, food adulteration, or the inclusion of porcine ingredients, is still a point of concern for consumers following halal food regulations. Purpose – This study aims to analyze the quality and quantity of DNA extracted from soy sauce, to determine specificity, sensitivity, and efficiency of PCR assay for porcine DNA detection and to screen porcine DNA in commercial soy sauce in the market. Design/methodology/approach – Real-time PCR analysis was utilized for the rapidity, sensitivity, and specificity in detection and quantification of porcine DNA in soy sauce products. Specifically designed primers were constructed from the cytochrome b gene (S. scrofa domestica). DNA samples were extracted using Qiagen DNeasy Mericon Food. SYBR Green-based real- time PCR was conducted for DNA detection of 11 samples, two raw meat samples, one soybean sample, and eight soy sauce samples. PCR amplification was performed with an initial pre-denaturation at 95°C for 60 seconds, followed by 35 cycles of denaturation at 95°C for 15 seconds and combined annealing and extension at 60°C for 45 seconds. Findings – Primer specificity was confirmed since the positive amplification was found to be for porcine DNA alone with a Ct value of 19.78 at 35 cycles. The technique possessed a detection limit of 0.001 ng/µL of porcine DNA. The efficiency of the real-time PCR assay from the regression analysis of the standard curve was 79.8% with a high R² value of 0.9986. There was no amplification of the DNA in any of the soy sauce samples, whereas in the spiked sample, amplification occurred at 33.19 Ct value. Research limitations – Soy sauce sample is a highly process and fermented food which contains mixture of ingredients that can interfere with DNA extraction. Besides, presence of polyphenolic compounds in soy-derived products can inhibit PCR reactions and limit the quality DNA extracted from the sample. Thus, we suggest optimizing DNA extraction methods and using inhibitor removal steps to minimize PCR inhibition in future studies. Originality/value – This study highlights real-time PCR as a powerful, fast, and sensitive method for detecting porcine DNA in processed food for halal food law enforcement and verification of food label authenticity.

Development and field evaluation of a species-specific mt-COI targeted SYBR-Green Real Time PCR for detection and quantification of Haemonchus contortus in cattle in Turkey

A quantitative PCR assay for rapid detection of Shigella species in fresh produce.

BackgroundThe tick-borne apicomplexan bovine parasite Theileria annulata is endemic in many tropical and temperate areas, including Minorca (Balearic Islands, Spain). Real-time PCR is widely used for the detection of piroplasms but quantification is not commonly considered.ResultsWe developed a real-time quantitative PCR (qPCR) assay for the detection and quantification of T. annulata that included an internal amplification control (IAC) to monitor for the presence of potential inhibitors. Specificity, sensitivity, precision, linear range and PCR efficiency were calculated and different methods for transformation of quantification cycle (Cq) values into quantities (Q) were evaluated. The assay was able to detect (100% probability) and quantify (linear response) 100 gene copies, and clinical sensitivity was set at 10 T. annulata per μl of blood. The assay was then validated on 141 bovine blood samples analyzed in parallel by a Luminex® suspension array, showing the utility of the qPCR assay developed here for the detection and quantification of the parasite in field conditions. Once validated it was used to monitor T. annulata parasitaemia throughout a year in 8 carrier animals from a farm in Minorca.ConclusionsThe developed qPCR assay offers a reliable and simple way to quantify T. annulata infection loads, which could prove crucial in studying the role of carrier animals as a source of the infection, or assessing the efficacy of treatment and control measures.

Development and validation of a specific real-time PCR assay for the detection of the parasite Perkinsus olseni

Western redcedar (WRC; Thuja plicata) root- and butt-rot diseases are caused by a set of wood-decay fungal pathogens, which have posed a significant threat to forest health and resulted in substantial economic losses of WRC production. Traditional approaches for disease detection are labor-intensive and more suitable on mature trees at late infection stages. This study developed and validated internal transcribed spacer region next-generation sequencing (ITS-NGS) and quantitative polymerase chain reaction (qPCR) assays for detecting decay-disease infections in WRC seedlings with high sensitivity and specificity. The efficiencies of ITS-PCR amplification were in silico predicted and validated through ITS-NGS using a pure fungal DNA mixture. For diagnosis of decay pathogens in WRC seedlings, fine root and root collar samples were collected from greenhouse inoculation trials. ITS-NGS identified positive infection rates of 100% for Armillaria ostoyae, Heterobasidion occidentale, and Poriella subacida in diseased seedlings, but the diagnostic efficiency for Coniferiporia weirii was affected by the types of sampled tissues. Species-specific qPCR assays were developed for C. weirii and revealed positive infection rates up to 100% in inoculated seedlings. Relative fungal abundances measured by ITS-NGS and qPCR were highly comparable, with significant correlation, demonstrating the reliability of both molecular diagnostic approaches. Moreover, qPCR provided higher quantification accuracy for a trace amount of the pathogen in total DNA extracted from host tissues. These results provided evidence for the application of ITS-NGS and qPCR assays as robust and reliable molecular tools for detecting early and latent infections of fungal pathogens in complex tissue samples for enhancing WRC disease management. [Formula: see text] Copyright © 2026 His Majesty the King in Right of Canada, as represented by the Minister of Natural Resources Canada. This is an open access article distributed under the CC BY 4.0 International license .