Porcine DNA Research Articles

Short communication. PCR detection of DNA of bovine, ovine-caprine and porcine origin in feed as part of a bovine spongiform encephalopathy control program

The rapid identification of residues of mammalian materials in animal feedstuffs is important for the effective control of feed as a potential source of transmission of bovine spongiform encephalopathy (BSE). It is included in the monitoring program developed to avoid the appearance of this disease. In the present work a PCR analysis was tested for the detection of mammalian residues (bovine, caprine, ovine and porcine). The DNA extraction method utilised guanidium thiocianate, and primers flanking a conserved region of mitochondrial DNA were synthesized for each species. PCR tests were specific for each species and allowed detection in feedstuffs of levels as low as 1% of bovine DNA and 1.5% of ovine, caprine and porcine DNA. These PCR tests may allow the rapid detection of residues of these species constituting a powerful tool against BSE

Development of a Porcine cDNA Microarray: Analysis of Clenbuterol Responding Genes in Pig (Sus scrofa) Internal Organs

Pig (Sus scrofa) fat accumulation can be reduced by feeding with high dosages of clenbuterol, but the molecular mechanism has not yet been explained. In our study, a porcine cDNA microarray representing 3 358 pig genes was successfully developed. This microarray is the first porcine DNA microarray in China and its false positive rate is 0.98%, which means the microarray platform is reliable. The microarray can be used to study gene expression profiles in multiple pig tissues because the present genes percentage of adipose, skeletal muscle, heart, liver, lung, kidney, and spleen were all more than 60%. This microarray was used to identify the genes responding to clenbuterol stimulation in pig internal organs, including heart, liver, lung, spleen, and kidney. Many genes were identified including enzymes involved in lipids metabolism (lipoprotein lipase up-regulated in liver, heart and lung, ATP-citrate lyase and carnitine palmitoyltransferase II precursor up-regulated in liver, succinyl-CoA up-regulated in lung, mitochondrial malate dehydrogenase down-regulated in spleen), and signaling pathway genes (cAMP-protein kinase A signaling pathway was found up-regulated in liver, heart, lung, and kidney as reported previously, while transforming growth factor was found down-regulated in heart and lung). However, no common gene responding to clenbuterol administration was found in all tissues. The expression levels of 14 genes were analyzed using real-time PCR with 82.1% of them induced to express similar magnitudes as in the microarray analyses. This work offers some understanding of how clenbuterol so effectively reduces pig adipose accumulation on the molecular level.

Novel approach for deriving genome wide SNP analysis data from archived blood spots

BackgroundThe ability to transport and store DNA at room temperature in low volumes has the advantage of optimising cost, time and storage space. Blood spots on adapted filter papers are popular for this, with FTA (Flinders Technology Associates) Whatman™TM technology being one of the most recent. Plant material, plasmids, viral particles, bacteria and animal blood have been stored and transported successfully using this technology, however the method of porcine DNA extraction from FTA Whatman™TM cards is a relatively new approach, allowing nucleic acids to be ready for downstream applications such as PCR, whole genome amplification, sequencing and subsequent application to single nucleotide polymorphism microarrays has hitherto been under-explored.FindingsDNA was extracted from FTA Whatman™TM cards (following adaptations of the manufacturer’s instructions), whole genome amplified and subsequently analysed to validate the integrity of the DNA for downstream SNP analysis. DNA was successfully extracted from 288/288 samples and amplified by WGA. Allele dropout post WGA, was observed in less than 2% of samples and there was no clear evidence of amplification bias nor contamination. Acceptable call rates on porcine SNP chips were also achieved using DNA extracted and amplified in this way.ConclusionsDNA extracted from FTA Whatman cards is of a high enough quality and quantity following whole genomic amplification to perform meaningful SNP chip studies.

Sensitive detection of porcine DNA in processed animal proteins using a TaqMan real-time PCR assay

A TaqMan real-time PCR method was developed for specific detection of porcine-prohibited material in industrial feeds. The assay combines the use of a porcine-specific primer pair, which amplifies a 79 bp fragment of the mitochondrial (mt) 12 S rRNA gene, and a locked nucleic acid (LNA) TaqMan probe complementary to a target sequence lying between the porcine-specific primers. The nuclear 18 S rRNA gene system, yielding a 77 bp amplicon, was employed as a positive amplification control to monitor the total content of amplifiable DNA in the samples. The specificity of the porcine primers-probe system was verified against different animal and plant species, including mammals, birds and fish. The applicability of the real-time PCR protocol to detect the presence of porcine mt DNA in feeds was determined through the analysis of 190 industrial feeds (19 known reference and 171 blind samples) subjected to stringent processing treatments. The performance of the method allows qualitative and highly sensitive detection of short fragments from porcine DNA in all the industrial feeds declared to contain porcine material. Although the method has quantitative potential, the real quantitative capability of the assay is limited by the existing variability in terms of composition and processing conditions of the feeds, which affect the amount and quality of amplifiable DNA.

Porcine Bioengineered Scaffolds as New Frontiers in Regenerative Medicine

Porcine organs are attractive for xenotransplantation, if severe immunologic concerns can be overcome. Recently, reengineered organs, with heterologous cellular materials removed but preserved organ architecture and vasculature have been created using small rodents in an effort to produce customized bioengineered organs. However, few studies have been performed to generate bioengineered organs from porcine sources. The aim of this work was to produce 3-D bioengineered scaffolds from major porcine organs, preserving the native morphology and vascular structures with complete removal of cellular and nuclear materials. We decellularized porcine heart, liver, and kidney using a peristaltic pump system with 1% sodium dodecyl sulfate. The preservation of major architecture and vasculature was confirmed by gross findings, ultrasonography, and angiography. Hematoxylin and eosin staining revealed no evidence of nuclear or cytoplasmic residues. Quantitative DNA analysis demonstrated a substantial reduction (0%–8%) of porcine DNA in the scaffolds. These results suggested that 3-D bioengineered scaffolds of porcine organs may have tremendous potential to produce non-immunogenic transplantable organs as well as beneficial tools for biomedical studies on organ re-engineering and repair.

Porcine-Specific Polymerase Chain Reaction Assay Based on Mitochondrial D-Loop Gene for Identification of Pork in Raw Meat

The porcine-specific polymerase chain reaction assay was developed to detect the presence of pork in raw meat either in pure or in a mixture with different meat species. A porcine-specific DNA fragment could be observed by using a porcine-specific primer designed based on a porcine specific sequence of mitochondrial D-loop gene. Amplification of porcine DNA produced 174 bp porcine-specific DNA fragment and no polymerase chain reaction products were detected when assay applied to DNA of other meat species confirming the specificity of the primers. The assay was able to detect as low as 0.1% (v/v) porcine DNA spiked on DNA of cattle, sheep, goat, chicken, and deer. Furthermore, a detection limit of 0.001 ng/μL porcine DNA showed the high sensitivity conferred by the developed porcine-specific polymerase chain reaction assay.

Species-specific identification of ruminant components contaminating industrial crude porcine heparin using real-time fluorescent qualitative and quantitative PCR

Ever since the emergence of bovine spongiform encephalopathy, the source of pharmaceutical heparin has been restricted to porcine intestinal mucosa. In this project, two real-time fluorescent PCR methods were developed to assist with quality control analysis. The first is a qualitative method which relies on SYBR Green I chemistry to confirm the porcine origin of industrial crude porcine heparin (ICPH), identify any ruminant contaminants, and generally control purity. The second is based on TaqMan chemistry and is able to quantitatively identify porcine, bovine, caprine, and ovine components and contaminants in ICPH. By targeting mitochondrial DNA, both PCR systems showed a detection limit of 1 pg DNA and amplification efficiencies ranging between 96% and 102%. Moreover, quantitative PCR showed a detection limit of 0.02 ppm in samples comprising porcine, bovine, caprine, and ovine DNA. The results of qualitative PCR over 27 ICPH samples showed that all samples were porcine in origin and that 17 had ruminant contaminants. The results of quantitative PCR further showed that out of all 17 samples with ruminant contaminants, seven samples had bovine, ovine, and caprine contaminants, two samples had bovine and ovine contaminants, and eight samples had only ovine contaminants. In conclusion, the qualitative PCR system was found to be a relatively inexpensive, rapid, and flexible method of identifying the porcine origin of and ruminant contaminants in ICPH, while the quantitative PCR was found suitable to accurately analyze the components and contaminants in detail. Both methods are suitable for routine control assays for the evaluation of ICPH purity and origins of contaminants.

Detection of porcine DNA in gelatine and gelatine-containing processed food products—Halal/Kosher authentication

A commercially available real-time PCR, based on a multi-copy target cytochrome b (cyt b) using porcine specific primers, has been validated for the Halal/Kosher authentication of gelatine. Extraction and purification of DNA from gelatine were successfully achieved using the SureFood® PREP Animal system, and real-time PCR was carried out using SureFood® Animal ID Pork Sens kit. The minimum level of adulteration that could be detected was 1.0% w/w for marshmallows and gum drops. A small survey was undertaken of processed food products such as gum drops, marshmallows and Turkish delight, believed to contain gelatine. Of fourteen food products from Germany, two samples were found to contain porcine gelatine, whereas of twenty-nine samples from Turkey twenty-eight were negative. However, one product from Turkey contained porcine DNA and thus was not Halal, and neither was the use of porcine gelatine indicated on the product label.

Swine-Specific PCR-RFLP Assay Targeting Mitochondrial Cytochrome B Gene for Semiquantitative Detection of Pork in Commercial Meat Products

Verification of pork adulteration in commercial meat products is increasingly important for the authentication of Halal labels in processed foods. Here, we documented a PCR–restriction fragment length polymorphism (RFLP) assay with high precision and reproducibility for the tracing of porcine DNA in commercial meat products. The assay combined the species-specific primers to selectively amplify a short fragment (109 bp) of porcine cytochrome b gene from a heterogeneous background of genomic DNAs followed by RFLP analysis to authenticate real amplicon. The analysis of PCR products and restriction digests was automated in a chip-based capillary electrophoresis incorporated in Agilent 2100 bioanalyzer. The swine specificity of the assay was checked with 11 different meat-providing animal and fish species. Model experiments, mimicking the processed foods, were performed in binary and ternary mixtures after mechanical grinding and prolonged autoclaving. Finally, four types of the most popular finished meat products (meatball, streaky beacon, frankfurter, and burger) which are prevalent in the Malaysian food market were analyzed in order to verify the assay performance. The assay was sensitive enough to detect 0.0001 ng of swine DNA in pure formats and 0.01% (w/w) spiked pork in extensively processed ternary mixture of pork, beef, and wheat flour.

Real-time PCR assays for detection and quantitation of porcine and bovine DNA in gelatin mixtures and gelatin capsules

Detection and quantitation of material from porcine and bovine species in gelatin and gelatin capsules is required for health safety concerns and for some religious practices. In this study, two species-specific qPCR assays were developed based upon repetitive elements. They allowed sensitive detection of porcine and bovine DNA at as low as 1pg/mL. The lack of cross-reactivity when the sets were used to amplify DNA from the other species indicates high specificity of the assay. When binary gelatin blends containing various amounts of porcine and bovine gelatin were prepared and analyzed by the qPCR assays, the determined ratios of porcine material to bovine material were very close to their theoretical values, and a contamination level as low as 1% of the other species in the gelatin blends could be determined. When evaluated in gelatin capsules, although significantly less DNA was detected, determination of porcine and bovine species identities and estimation of the relative abundance of each species was possible. Therefore, the porcine and bovine species-specific qPCR assays described here represent simple, reliable and sensitive DNA-based tests for determination and quantitation of the species of origin from highly processed products.

Detection of Raw Pork Targeting Porcine-Specific Mitochondrial Cytochrome B Gene by Molecular Beacon Probe Real-Time Polymerase Chain Reaction

Pork identification in raw meat using real-time polymerase chain reaction (PCR) was developed. Total DNA from meat samples were successfully extracted and found to be of high quality and produced clear PCR products. Porcine-specific molecular beacon probe and primers that amplifies 119 bp of the cytochrome b gene fragment of swine (Sus scrofa domestica) was used. Analysis of data showed that the Cq (quantification cycle) from 10 ng/μl porcine DNA is (18.70 ± 0.12 to 19.08 ± 0.06). Meanwhile, the other samples exhibited negative result, which confirmed the specificity of the primers. The method also showed that the limit of detection of pork was 0.0001 ng. Based on the regression analysis of the standard curve, the 96% efficiency of real-time PCR was achieved with high correlation coefficient (r2 = 0.9989). Sensitivity of the assay in discriminating pork as low as 0.1% (w/w) pork in pork–beef mixtures was also obtained. Reproducibility of the assay was successfully validated by applying sample and experimental replicates in every assay being conducted. Thus, this methodology could serve as a fast and sensitive method for detection of pork for meat species verification.

Identification of pork genome in commercial meat extracts for Halal authentication by SYBR green I real‐time PCR

SummaryThe identification of pork DNA in meat extracts is very important for Halal authentication and Muslim consumers demand protection from falsely labelled meat products. A pig‐specific SYBR green I real‐time PCR assay has been developed to address this issue. Using specific primers for pig mitochondrial DNA, successful amplification has been obtained by DNA extracted from control meat samples. With SYBR green I real‐time PCR, the specificity of the amplification was showed by Tm value. Detection limit of the real‐time PCR was down to 0.1 ng of porcine DNA. An appropriate linearity was obtained by construction of a standard curve based on Ct value and different concentrations of porcine DNA. By conventional PCR, no amplification was shown by porcine DNA less than 0.1 ng. The established method was conducted on commercially available meat extracts for detection and quantification of porcine DNA. The results showed the SYBR green I real‐time PCR could be considered a robust method for Halal authentication of meat extracts.

Discrimination of two alleles of the melanocortin receptor 1 gene to discern European wild boar (Sus scrofa scrofa) and domestic pig (Sus scrofa domestica) in meat products by real-time PCR

A duplex real-time polymerase chain reaction (PCR) technique for the discrimination of two subspecies of sus scrofa in meat products has been developed. Primers and probes target at a sequence in the melanocortin receptor 1 (MCR1) gene being associated in the expression of wild-type coat color. Both PCRs amplify a 56-bp product of DNA from wild boar (Sus scrofa scrofa) and domestic pig (Sus scrofa domestica) likewise. One of the TaqMan probes specifically anneals to the wild boar sequence and the other one to the domestic pig sequence, their base sequence differing only in a single nucleotide (single nucleotide polymorphism, SNP). This qualitative-method allows the detection of genomic DNA from wild boar and domestic pig as low as 25 pg in a 25-μl reaction volume. No cross reactivities were found with either genomic DNA from various other meat species, or with other ingredients of meat products (e.g. spices). The PCR efficiency is >95% for both targets. Although both PCRs are impaired by the presence of bovine and porcine DNA (wild boar detection is impaired by domestic pig DNA and vice versa), the method is applicable for the detection of low levels of wild boar and domestic pig meat simultaneously in commercial meat products. The limit of detection (LOD) in meat samples is 2% for wild boar and 5% for domestic pig.

A quantitative PCR method to quantify ruminant DNA in porcine crude heparin.

Heparin is a well-known glycosaminoglycan extracted from porcine intestines. Increased vigilance for transmissible spongiform encephalopathy in animal-derived pharmaceuticals requires methods to prevent the introduction of heparin from ruminants into the supply chain. The sensitivity, specificity, and precision of the quantitative polymerase chain reaction (PCR) make it a superior analytical platform for screening heparin raw material for bovine-, ovine-, and caprine-derived material. A quantitative PCR probe and primer set homologous to the ruminant Bov-A2 short interspersed nuclear element (SINE) locus (Mendoza-Romero et al. J. Food Prot. 67:550–554, 2004) demonstrated nearly equivalent affinities for bovine, ovine, and caprine DNA targets, while exhibiting no cross-reactivity with porcine DNA in the quantitative PCR method. A second PCR primer and probe set, specific for the porcine PRE1 SINE sequence, was also developed to quantify the background porcine DNA level. DNA extraction and purification was not necessary for analysis of the raw heparin samples, although digestion of the sample with heparinase was employed. The method exhibits a quantitation range of 0.3–3,000 ppm ruminant DNA in heparin. Validation parameters of the method included accuracy, repeatability, precision, specificity, range, quantitation limit, and linearity.

Sequence polymorphisms in porcine homologs of murine coat colour‐related genes

Herein, we report the variability among 57 porcine homologs of murine coat colour-related genes. We identified single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) within 44 expressed gene sequences by aligning eight pig complementary DNA (cDNA) samples. The sequence alignment revealed a total of 485 SNPs and 15 InDels. The polymorphisms were then validated by performing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with reference DNA samples obtained from 384 porcine individuals. Of the 384 individuals, three parents of the experimental F(2) family were included to detect polymorphisms between them for linkage mapping. We also genotyped previously reported polymorphisms of 12 genes, and one SNP each in three genes that were detected by performing a BLAST search of the Trace database. A total of 211 SNPs and three InDels were successfully genotyped from our porcine DNA panel. We detected SNPs in 33 of the 44 genes among the parents of an experimental F(2) family and then constructed a linkage map of the 33 genes for this family. The linkage assignment of each gene to the porcine chromosomes was consistent with the location of the BAC clone in the porcine genome and the corresponding gene sequence. We confirmed complete substitutions of EDNRB and MLPH in the Jinhua and Clawn miniature breeds, respectively. Furthermore, we identified polymorphic alleles exclusive to each pig group: 13 for Jinhua, two for Duroc, three for Meishan, four for the Japanese wild boar, one for the Clawn miniature pig and four for the Potbelly pig.

A suspected case of hunting accident. Case report

Nowadays, the use of molecular biology in forensics has made it possible to identify human victim and sometimes even the circumstances under which the death occurred through. In our case, a corpse of a 50-years old man with a gunshot wound was found in the woods. The suspected murderer declared that it had been a hunting accident while he shot a wild boar. During the autopsy, a bullet (Borra-bullet Gualandi, 32 gr) was found in the abdomen of the victim. The authors investigated the presence of boar and victim blood both on the bullet, in order to substantiate the thesis of the hunting accident. Laboratory investigations underscored the presence of human cellular material on the bullet, which corresponds to profile of the victim and human and porcine ribosomal DNA. Through this investigation, the authors showed that it was a hunting accident, because the bullet passed through the body of the boar before hitting the victim.

Shotgun metagenomics of biological stains using ultra-deep DNA sequencing

A detailed molecular analysis of blood or other biological stains at a crime scene is often hampered by the low quantity and quality of the extractable DNA. However, the determination of the origin and composition of a stain is in most cases a prerequisite for the final elucidation of a criminal case. Standard methodologies, e.g. amplification of DNA followed by microsatellite typing or mitochondrial DNA sequencing, are often not sensitive enough to result in sufficient and conclusive data. We have applied ultra-deep DNA sequencing using the 454 pyrosequencing technology on a whole genome amplified (WGA) environmental biological stain, which was analysed unsuccessfully with standard methodologies following WGA. With the combination of WGA and 454 pyrosequencing, however, we were able to generate 7242 single sequences with an average length of 195 bp. A total of 1,441,971 bp DNA sequences were generated and compared with public DNA sequence databases. Using RepeatMasker and basic logical alignment search tool (BLAST) searches against known microbial and mammalian genomes it was possible to determine the metagenomic composition of the stain, i.e. 4.2% bacterial DNA, 0.3% viral DNA, 2.7% fungal DNA, 10.3% mammalian repetitive DNA, 0.9% porcine DNA, 0.13% human DNA and 81.5% DNA of unknown origin. Our data demonstrate that 454 pyrosequencing has the potential to become a powerful tool not only in basic research but also in the metagenomic analysis of biological trace materials for forensic genetics.

Comparative analysis of fatty acid-binding protein 4 promoters: Conservation of peroxisome proliferator-activated receptor binding sites1

The objective of this research was to further characterize the promoter regions of the bovine and porcine fatty acid-binding protein 4 (FABP4) genes relative to those of other mammals. The DNA sequences of FABP4 promoters for the human, mouse, cow, pig, and dog were obtained from the genomic database of the National Center for Biotechnology Information and also from the sequencing of bovine and porcine genomic DNA clones obtained by 5' PCR racing of genomic DNA. Sequence alignments of these FABP4 promoters using the basic local alignment search tool of the National Center for Biotechnology Information revealed 3 highly conserved promoter regions across the species. Two computational bioinformatics databases and the literature identified the conserved transcription factor-binding sites for C/EBP, adapter primer-1, and boxes of CAAT and TATA in the first conserved proximal promoter region, a direct repeat 1-type PPAR responsive element in the second distal conserved region, and another PPAR responsive element in the third distal conserved promoter region of FABP4 in all 5 mammals. Five new short interspersed repetitive elements (SINE) in the bovine FABP4 promoter and 2 new SINE in the porcine were found, but these SINE did not disrupt the 3 conserved regions, indicating that important regulatory elements are maintained regardless of evolutionary pressure. In conclusion, the conserved cis-acting elements, especially the 2 key adipocyte transcription factors C/EBP and PPAR, may contribute greatly to adipogenic regulation and adipose tissue-specific expression of FABP4 in these mammals. This helps to further characterize and decipher important cis-acting elements that are important for adipocyte development in adipose and muscle tissue.

Cytoplasmic localization of oocyte‐specific variant of porcine DNA methyltransferase‐1 during early development

DNA methyltransferase-1 (Dnmt1) is involved in the maintenance of genomic methylation patterns. Rather than full-length Dnmt1, mouse oocytes have a truncated variant called Dnmt1o. Immunofluorescence data showed that Dnmt1o localized to the cytoplasm, but this has not been confirmed using more direct methods. The cytoplasmic localization of Dnmt1o has been assigned to the main cause of global DNA demethylation in early mouse embryos. We studied localization of Dnmt1o in mouse and pig embryos. We identified pig Dnmt1o protein and its transcript with unique 5'-end sequence. Physically separating mouse and pig 2-cell embryos into their nuclear and cytoplasmic components demonstrated that Dnmt1o of both species localized to the cytoplasm. Cloned pig embryos had Dnmt1o as the main form, with no indication of somatic Dnmt1. These findings indicate that Dnmt1o is cytoplasmic during early development; its presence in both pig and mouse embryos further suggests that Dnmt1o is conserved in mammals.

Universal mitochondrial PCR combined with species-specific dot-blot assay as a source-tracking method of human, bovine, chicken, ovine, and porcine in fecal-contaminated surface water

Mitochondrial (mt) DNA has the potential to be used as an animal-specific genetic marker for source-tracking of fecal contamination in surface waters and groundwaters. In this report, we describe the development of a method to detect in a single assay human, bovine, ovine, porcine and chicken mitochondrial (mt) DNA in water. Consensus nucleic sequences were found between human, bovine, porcine, ovine and chicken mtDNA to design three sets of PCR universal primers. Upon polymerase chain reaction (PCR) with a digoxigenin-labeled nucleotide and the universal primers, species determination was carried out by dot-blotting membranes containing specific oligonucleotides for these five animals. Our method was carried out with three river samples and three wastewater samples, and the results were compared with those obtained by multiple nested PCR with specific primers for these five species. Our results showed that the dot-blot assays were as specific and sensitive as the nested-PCR approach. The proposed method has the advantage that it requires the use of only one PCR per sample and very little amounts of DNA. Finally, it is an alternative to multiplex PCR approach which is less sensitive, and shows the way for the development of DNA arrays for source-tracking of many more animal species in fecal-contaminated water.