Porcine DNA Research Articles

PENGGUNAAN PRIMER GEN CYTOCHROME OXIDASE 1 DALAM REAKSI POLYMERASE CHAI REACTION (PCR) UNTUK IDENTIFIKASI KANDUNGAN BABI PADA MAKANAN

Muslims make up the majority of the population in Indonesia so halal food safety is of paramount importance. From a religious and health perspective, adulteration and contamination of Sus scrofa meat is considered a violation of halal norms. Sus scrofa is often used to adulterate beef to take advantage of the physical resemblance and price difference. The need to identify Sus scrofa meat contamination in food products was highlighted in this study. After a successful isolation process, the Polymerase Chain Reaction (PCR) method was used to validate the presence of porcine DNA. PCR utilizes primers specifically made to replicate specific DNA sequences associated with pigs. PCR testing provides precise results for pig DNA, ensuring the accuracy of detection of pork contamination in foodstuffs. This research contributes to efforts to maintain halal food safety in Indonesia by emphasizing the important role of PCR in the detection process of Sus scrofa meat.

PCR-free and minute-scale electrochemical analysis of porcine DNA adulteration via molecularly amplified DNA sandwich assay

The increasing incidence of meat adulteration and mislabeling poses significant challenges in terms of food safety and consumer trust. This study proposes an electrochemical DNA biosensor for detecting porcine mitochondrial DNA in tainted meat products, offering a novel approach to address the above challenges. Unlike conventional nucleic acid amplification tests that rely on polymerase chain reactions (PCRs), the proposed biosensor employs a molecularly amplified DNA strategy with DNA tracers that bind to two regions of the target DNA, creating an elongated hybridization structure with multiple redox-tagging molecules. This design catalyzes detection signals autonomously, eliminating the need for PCR amplification. One-step DNA probe immobilization using poly-adenine (poly-A) oligonucleotides significantly improves hybridization efficiency and reduces the necessity for extensive sample purification, thereby simplifying the detection process. The proposed biosensor exhibits a linear detection range of 101–106 pM and a limit of detection (LOD) of 2.2 pM in controlled settings. Furthermore, the proposed biosensor distinguishes pork from beef in adulterated samples with a LOD of 1 % w/w. With its stability exceeding 9 weeks and a cost of less than 0.5 USD per test, the proposed biosensor offers a highly sensitive, economically viable solution with significant potential for widespread use in the meat industry and by end-users, effectively combating porcine adulteration.

A novel recombinase polymerase amplification approach with propidium iodide reporter for rapid detection of porcine adulteration in food

The adulteration of food products with porcine components is among the most widespread concerns in the global food industry. Apart from health and religious reasons, being able to detect porcine adulteration is crucial to warrant fair trading and legal compliance. Current detection methods are often constrained by their low sensitivity and expensive equipment. In this work, a recombinase polymerase amplification (RPA) assay for the rapid and sensitive detection of porcine is constructed, with propidium iodide (PI) dye as the reporter molecule. Under the optimized conditions, the RPA-PI assay achieved a wide linear range of 0.005–100 ng/μL and a detection limit of 0.005 ng/μL for porcine DNA with no cross-reactivity. Furthermore, the application of the assay to actual food samples demonstrated a 100 % consistency with conventional PCR results. This developed assay not only provides a novel porcine adulteration detection platform with simpler and faster protocols, but it also holds the potential to be developed further for point-of-care authenticity testing.

Assessment of Commercial DNA Extraction Kits for Porcine Gelatin Detection Using RT-PCR and ddPCR

Gelatin is extensively applied in various industries, including food, beverages, cosmetics, and pharmaceuticals. Although the determination of gelatin species is essential for religious, health, and consumer preference reasons, a standardized analysis method is absent. The challenge in identifying gelatin through DNA-based methods arises from the low DNA content and extensive DNA denaturation in the gelatin matrix. This study assessed the efficacy of two commercial DNA extraction kits, namely the Processed Food DNA Extraction (PF kit) and the DNeasy Mericon Food Kit (DM kit), for extracting DNA from porcine gelatin powder and commercial products derived from gelatin. Additionally, we evaluate the amplification of the extracted porcine DNA using real time polymerase chain reaction (RT-PCR) and droplet digital polymerase chain reaction (ddPCR) techniques. The PF extraction kit demonstrated successful DNA extraction from porcine gelatin powder and commercial samples of porcine gelatin-based candies with a higher concentration (32.24-286.07 ng/uL) and purity (A260/A280 ratio of 1.82-2.33) compared to the DM kit (3.95-7.30 ng/uL and an A260/A280 ratio of 1.29-2.45). RT-PCR and ddPCR analyses yielded positive results for porcine DNA from gelatin powder for both PF and DM kits, albeit with differing Cq values and copy numbers. The choice of DNA extraction kit significantly impacted the amplification results when analyzing commercial samples of porcine gelatin-based candies. Using RT-PCR, all samples yielded negative results with the DM kit, while the PF kit detected one positive result for porcine DNA. Improved outcomes were observed with more sensitive analysis methods such as ddPCR, where the DM kit identified one positive result for porcine DNA while the PF kit detected positive results for all tested candies.

CTAB DNA Extraction Method Optimization for Quantification of Lard Content in Cheese and Butter Using Real-Time PCR

ABSTRACT Porcine adulteration in dairy products for economic purposes has been tackled mostly by protein- and lipid-based porcine detection methods. In this study, a DNA-based approach was used to determine the presence of porcine DNA in dairy products, especially butter and cheese. DNA was extracted by optimizing the conventional CTAB extraction method followed by an assessment of DNA fragmentation by real-time PCR assays targeting different sizes of amplicon. Butter and cheese DNA samples were analyzed using both endogenous and porcine-specific real-time PCR assays and the lard content was estimated based on the linear regression analysis of a series of different percentages of lard-adulterated butter and cheese DNA samples. Even though the optimization steps did not improve the DNA yield, the successful amplification of the 200 bp amplicon depicted high DNA quality. Approximately 1% lard was able to be quantified exhibiting high potential of the assay in quantifying porcine content in lard-adulterated highly processed food products.

Porcine detection in commercial edible bird nest beverages using real time PCR targeting chromosomally encoded multiple porcine repetitive elements

Popular consumption of edible bird nest beverage (EBNB) is driven by its claimed anti-aging properties. Incorporation of other anti-aging and protein-rich compounds such as collagen and gelatin increase the adulteration potential of EBNB especially those from economical feasible porcine sources. In this study, fifteen commercial EBNB products were examined for the presence of porcine DNA using real time PCR targeting multiple porcine repetitive element (MPRE42). DNA extracted using DNeasy Mericon Food Kit (Qiagen) obtained 0.08–17 ng/µl DNA that is ample for downstream qPCR. Universal qPCR assay successfully amplified conserved 18 S rRNA genes in all samples except for B7 which might dictate presence of PCR inhibitors such as phenolic compounds. Porcine-specific MPRE42 assay detected the presence of porcine DNA in 40% of the EBNB products (B5, B10, B11, B16, B17, B20) with Cq value ranging from 33.85±0.33–38.55±1.23. Analysis of EBNB product (B3) being spiked with different amounts of porcine gelatin shows that porcine-specific MPRE42 assay can detect up to 0.5% (w/w) porcine gelatin in B3. A linear correlation between Cq values of the DNA extracted from spiked samples and serial diluted porcine gelatin were obtained which demonstrates potential quantitative application of this assay. In overall, upon strict validation according to ISO/IEC 17025, the method described in this study has a huge potential to be applied by enforcement and authorities for porcine gelatin detection in EBNB products.

β-D-glucuronidase activity triggered monitoring of fecal contamination using microbial and chemical source tracking markers at drinking water intakes

Intense rainfall and snowmelt events may affect the safety of drinking water, as large quantities of fecal material can be discharged from storm or sewage overflows or washed from the catchment into drinking water sources. This study used β-d-glucuronidase activity (GLUC) with microbial source tracking (MST) markers: human, bovine, porcine mitochondrial DNA markers (mtDNA) and human-associated Bacteroidales HF183 and chemical source tracking (CST) markers including caffeine, carbamazepine, theophylline and acetaminophen, pathogens (Giardia, Cryptosporidium, adenovirus, rotavirus and enterovirus), water quality indicators (Escherichia coli, turbidity) and hydrometeorological data (flowrate, precipitation) to assess the vulnerability of 3 drinking water intakes (DWIs) and identify sources of fecal contamination. Water samples were collected under baseline, snow and rain events conditions in urban and agricultural catchments (Québec, Canada). Dynamics of E. coli, HF183 and WWMPs were similar during contamination events, and concentrations generally varied over 1 order of magnitude during each event. Elevated human-associated marker levels during events demonstrated that urban DWIs were impacted by recent contamination from an upstream municipal water resource recovery facility (WRRF). In the agricultural catchment, mixed fecal pollution was observed with the occurrences and increases of enteric viruses, human bovine and porcine mtDNA during peak contaminating events. Bovine mtDNA qPCR concentrations were indicative of runoff of cattle-derived fecal pollutants to the DWI from diffuse sources following rain events. This study demonstrated that the suitability of a given MST or CST indicator depend on river and catchment characteristics. The sampling strategy using continuous online GLUC activity coupled with MST and CST markers analysis was a more reliable source indicator than turbidity to identify peak events at drinking water intakes.

Amplification-Based CRISPR/Cas12a Biosensor Targeting the COX1 Gene for Specific Detection of Porcine DNA.

We propose a CRISPR/Cas12a-mediated recombinase polymerase amplification (RPA) detection method that combines RPA with Cas12a cleavage for the detection of halal food adulteration, which is of global concern, particularly for Muslim consumers. We optimized the reagent concentrations for the Cas12a cleavage steps and designed and screened gRNA targeting a conserved area of the mitochondrial cytochrome C oxidase subunit I (COX1) gene. This procedure successfully detected the presence of porcine components as low as 5 pg/μL in the linear range of 5-1000 pg/μL. The assay's detection limit was 500 times lower than CRISPR-based approaches that exclude a preamplification step, allowing the detection of trace porcine DNA in food samples. The assay additionally showed no cross-reaction with nontarget species. Therefore, this detection platform shows tremendous potential as a method for the quick, sensitive, and specific detection of porcine-derived components.

Validating duplex-PCR targeting ND2 for bovine and porcine detection in meat products

Food authentication is a mandatory effort to assure the fair-trade. This study developed a duplex polymerase chain reaction (PCR) from the NADH dehydrogenase subunit 2 (ND2) gene to amplify specific segments of a cattle and porcine DNA. A universal forward primer composed of nineteen base pairs (bp) (3′-CCAAACACAACTCCGAAAA-5′) and species-specific reverse primers composed of twenty (3′-CCAAACACAACTCCGAAAA-5′) and twenty-one (3′-TGGCAAGAATTAGGACGGTTA-5′) bp were used to limit the amplified DNA segment for porcine and cattle. The PCR reaction would generate a product with a profile of 168 and 227 bp, respectively. To investigate the accuracy and limit of detection, an in vitro experiment was conducted using simplex and duplex PCR on commercial meatballs randomly purchased from a commercial market in Surakarta, Indonesia. The findings of this study indicated that ND2 could be used as an alternative genetic marker for the identification of porcine and beef species in meat-derived products.

A simplex and multiplex PCR assay for simultaneous detection of beef, pork, and chicken meat in sausages based on mitochondrial DNA Cytochrome oxidase sub-unit I

Adulteration in the processed beef product industry has long been an issue affecting consumers’ rights and legal protection as beef is an expensive raw material. The intense market competition opens up opportunities for manufacturers of processed beef products to counterfeit and mixes their products with other cheap meat ingredients such as chicken and pork. In addition, the use of pork to substitute beef in the production of sausages without proper labelling on the final products is both an adulteration and halal assurance issue in the Muslim-majority market. Multiplex PCR is a sensitive assay used for the identification of raw materials used in the production of processed food and it reduces the cost and time of analyzing a large number of samples. The purpose of this research was to apply the multiplex PCR assay targeting mitochondrial DNA Cytochrome Oxidase SubUnit I (COI) sequence and to determine the detection limits for DNA concentrations in beef, pork, and chicken. The DNA genomes that were extracted from each meat were diluted to concentrations of 25; 10; 1; 0.1; 0.01; and 0.001 ng/µL, and then used as a DNA template for the simplex- and multiplex-PCR. The results of simplex- and multiplex-PCR showed that the COI primers used were able to amplify the DNA of bovine, porcine, and chicken accurately as indicated by amplicons of 263 bp, 168 bp, and 596 bp, respectively. It was concluded that the simplex- and multiplex-PCR methods using COI primers can be used to identify bovine, porcine, and chicken DNA isolated from sausages with a sensitivity of 0.001 ng/μL.

Development of novel isothermal-based DNA amplification assay for detection of pig tissues in adulterated meat.

For the first time, we describe an innovative polymerase spiral reaction (PSR) assay for the rapid, simple, and accurate detection of pig tissues or pork in adulterated meat including heat-treated and processed ones. The PSR assay specifically targeting the mitochondrial cytochrome b (cyt-b) gene of the pig was successfully optimized permitting assay results in 65min time. The developed detection method was 100% specific amplifying only the cyt-b gene and displaying negative results with all the tested non-pork meats. The sensitivity of the developed PSR (760fg porcine DNA) was tenfold better than the end-point PCR and able to detect heat-treated (121°C) and adulterated (0.5% pork in beef) meat and processed pork products such as sausages, salami, meatball, soup, curry, etc. The developed PSR-based method can be used for point-of-care detection with minimum instrumentation and technical expertise to guarantee instant clearance of exported and imported meat products. This is the first time that PSR has been adapted for food authenticity purposes.

Analysis of soft gelatin capsule with real-time polymerase chain reaction for halal autenthication

Halal medicine is an interesting topic to always discuss because it is a priority choice for Muslim consumers, one of which is halal capsules. Currently, molecular biology techniques such as real-time polymerase chain reactions are rapidly developing, including for the analysis of non-halal components based on DNA sequences. This study aimed to validate the quantitative PCR method for identifying DNA in gelatin-based products and to apply the confirmation method designed for capsule samples on the market circulating in Yogyakarta to prove the halalness of these samples. Validation of the porcine DNA detection analysis method on standard extraction of porcine gelatin using primer pairs obtained in previous studies. Validated methods are used for testing market capsule shells. The qPCR method using D-loop primers is specifically capable of amplifying porcine gelatin DNA up to a concentration of 0.5 pg/µL, with a CV value in the amplification response of porcine gelatin DNA isolates (1000 pg/µL) of 0.85% which meets the test criteria using the PCR. Three samples of commercial soft capsules tested gave a positive amplification response, meaning that the samples tested contained porcine DNA, and one negative sample, which probably had non-porcine gelatin. The application of this method is also very useful for ensuring the authenticity of the capsule shell, especially from cross-contamination and counterfeiting.

CRISPR/Cas12a-mediated Enzymatic recombinase amplification for rapid visual quantitative authentication of halal food

Economically motivated adulteration (EMA) has become a concern in food safety. We propose a CRISPR/Cas12a Mediated Enzymatic Recombinase Amplification detection system (CAMERA) that integrates Enzymatic Recombinase Amplification (ERA) and Cas12a cleavage to detect halal food adulteration. We designed and screened crRNA targeting CLEC, a porcine-specific nuclear single-copy gene, and optimized the reagent concentrations and incubation times for the ERA and Cas12a cleavage steps. CAMERA was highly specific for pork ingredients detection. The DNA concentration and fluorescence signal intensity relationship was linear at DNA concentrations of 20–0.032 ng/μL. CAMERA detected as few as two CLEC copies and quantified samples with porcine DNA content as low as 5% within 25 min. The system could be operated in a miniaturized working mode that requires no technical expertise or professional equipment, making CAMERA a valuable tool in resource-limited areas for the qualitative and quantitative detection of pork ingredients in halal food.

Porcine Mx proteins inhibit pseudorabies virus replication through interfering with early gene synthesis

Pseudorabies virus (PRV) is an enveloped, linear double-stranded DNA herpesvirus that resulted in huge financial losses to the swine industry. In addition to vaccination, the development of antiviral molecules is also a beneficial supplement to the control of Pseudorabies (PR). Although our previous studies have shown that porcine Mx protein (poMx1/2) significantly inhibited the proliferation of RNA virus, it was unknown whether poMx1/2 could inhibit porcine DNA virus, such as PRV. In this study, it was investigated the inhibitory effect of porcine Mx1/2 protein on PRV multiplication. The results showed that both poMx1 and poMx2 had anti-PRV activities, which required GTPase ability and stable oligomerization. Interestingly, the two GTPase deficient mutants (G52Q and T148A) of poMx2 also had the antiviral ability against PRV, which was consistent with previous reports, indicating that these mutants recognized and blocked the viral targets. Mechanistically, the antiviral restriction of poMx1/2 came from their inhibition of the early gene synthesis of PRV. Our results for the first time shed light on the antiviral activities of two poMx proteins against DNA virus. The data from this study provide further insights to develop new strategies for preventing and controlling the diseases caused by PRV.

Impact of vegetable fat on the sensory and physicochemical quality characteristics of chevon sausage

Goat fat forms unstable emulsions in making sausages and pork fat may be added however, Muslims have restrictions on ingesting pork. This study attempts to investigate the effect of plant-based fat (corn oil, palm oil, and margarine) on the sensory and physicochemical properties of chevon sausage. The moisture content, crude fat, saturated fat, ash, crude protein, and cooking loss of the three different sausage formulations were evaluated. Fifty consumers evaluated the sensory acceptability in terms of color, aroma, taste, texture, and overall acceptability. The most preferred sausage was subjected to microbiological and porcine DNA test. Saturated fat levels in corn oil, palm oil, and margarine, were 3.68%, 6.76%, and 4.41%, respectively. Among the treatments, the chevon sausage prepared with corn oil exhibited the least cooking loss. Although all treatments were rated between like moderately and like very much, the taste of chevon sausage prepared with palm oil was judged to be less appealing than the sausage with corn oil. With regards to proximate composition and sensory acceptability in terms of color and aroma, the results for the three treatments were comparable. The microbiological results showed that the chevon sausage with corn oil is safe to eat within 54 days if stored at -18 °C and the sausage is free from porcine DNA. The fatty acid composition of chevon sausage formulated with pre-emulsified corn oil is characterized by its high content of palmitic acid (16.21%), stearic acid (8.51%), oleic acid (28.31%), and linoleic acid (43.55%).

Analysis of Porcine DNA in Gelatine Products using SYBR Green Real Time Polymerase Chain Reaction for Halal Verification

Real-time PCR was employed in this study to determine whether gelatine items from nearby marketplaces contained porcine DNA. The mitochondrial NADH dehydrogenase subunit 5 (MT-ND5) gene from pigs (Sus scrofa) was the subject of oligonucleotide primer design; the forward and the reverse primer sequences produced 161bp amplicon. SYBR Green fluorescent dye was used to detect swine DNA in samples of 22 different types of gelatine products, 12 of which had labels identifying their sources and indicating their certification as Halal, and the remaining 10 had neither labels nor certification information. The 40 cycles of positive amplication with a Ct value of 19.81 served as the criterion for determining the specificity of the primers. In this work, the detection threshold for porcine DNA is set at 0.001 µg/ml. The efficiency of the PCR in this study is 91.72%, and the R2 is 0.9985, which indicates good efficiency. The 12 labelled products exhibit positive amplifications for porcine gelatine products while exhibiting negative amplifications for both bovine and fish gelatine products in labelled samples. Nine out of the ten samples of gelatine products that were tested had positive amplifications without source or Halal labelling.

Electrochemical Biosensor for Detection Of Porcine DNA Based on Tetraaza Complex as Sensing Material

Copper tetraaza complex was prepared and the structure was confirmed by FTIR, 1HNMR and 13CNMR. The investigation of the binding interaction of complex with calf thymus DNA (CT-DNA) was investigated using UV-vis absorption, luminescence titrations, viscosity measurements and molecular docking. The findings shows that the complex displayed the interaction ability towards CT-DNA. It is suggested that complex bind to DNA by electrostatic interaction and envisaged that the differences in binding strengths were caused by the complex dissimilar charge and counter anion. The tetraaza complex was further applied in developing the porcine sensor. The developed sensor exhibits a broad linear dynamic range, low detection limit, good selectivity, and reproducibility. Analysis of real samples showed that the biosensor had excellent selectivity towards the pork meat compared to chicken and beef meat.

Rapid Detection of Porcine DNA in Meatball Using Recombinase Polymerase Amplification Couple with Lateral Flow Immunoassay for Halal Authentication

Point-of-care diagnostic methods for animal species determination are critical for rapid, simple, and accurate enforcement of food labelling. PCR is the most common method for species identification. However, the requirement of using a thermal cycler created drawbacks for the PCR application, particularly in low-resource settings. Hence, in this study, a method for porcine DNA detection using recombinase polymerase amplification (RPA), coupled with nucleic acid lateral flow immunoassay (NALFIA), was developed. Porcine-specific primers targeting pig (Sus scrofa) cytochrome b gene fragments specifically amplify a 197 bp fragment of the mitochondrial gene as being visualized by 2% agarose gel and PCRD NALFIA. The reaction temperature and time were 39 °C and 20 min, respectively. Herein, the specificity of the primers to porcine was confirmed after being assayed against six animal species, namely cow, goat, chicken, duck, dog, and rabbit. The porcine-specific RPA assay shows a high limit of detection of 0.01 ng/µL pork DNA. Based on the preliminary performance data obtained from this study, the potential of this method as a rapid and sensitive tool for porcine DNA detection in meat-based products is foreseen.

Rapid Detection of Porcine DNA by Real-Time Polymerase Chain Reaction (qPCR) on Imported Processed Foods

A pair of porcine-specific primers defining a 145 bp region of the mitochondrial D-loop 443 were used to identify the presence of porcine DNA in ten samples of imported instant noodles and soft candies by Real-Time Quantitative Polymerase Chain Reaction (qPCR). This study was performed in three steps: sample preparation, DNA extraction, and DNA amplification using Real-Time PCR. The porcine DNA was amplified at an annealing temperature of 53oC for 39 cycles. The result confirmed that three samples of instant noodles and three soft candies were found to contain porcine D.N.A. This method could effectively authenticate Halal products in processed foods, verify food labelling, and provide consumer protection.

Taq man multiplex real-time pcr for identification of porcine and canine dna in halal food

The efficient and timely detection of prohibited animals in Halal food is very important. This study aimed to establish a rapid detection method for porcine and canine DNA in a food product. Porcine and Canine DNA were Contaminated in halal food products using multiplex Real-Time PCR shown positive specificity only in Porcine and Canine meat but shown negative results in chicken, cow, and fish. The results from porcine showed that the lowest level is at 0.001% and 0.1% in raw meat and cooked meat heated at 120 degrees celsius for 15 min, and 0.1% in diluted canine DNA respectively. The method for detection of the contamination of porcine and canine DNA in Halal food products with multiplex real-time PCR technique is suitable and high sensitivity for detecting the DNA of prohibited animals in meat products and raw materials, such as detecting contaminated porcine DNA in sweet jelly and soup cubes.