Poultry DNA Research Articles

Investigation of meat species adulteration in beef-based meat products via real-time PCR in Türkiye

The addition of poultry and other meats to products made from 100% beef is prohibited in Türkiye. In addition, in Türkiye, where the majority of the population is Muslim, the use of pork and single-hoofed meat is a matter of concern due to religious reasons. This study utilized a sensitive and specific real-time polymerase chain reaction (PCR) method to identify the different meat species in meat products marketed as 100% beef and sold in the eastern Türkiye provinces of Erzurum, Erzincan, Kars, Ağrı, and Bingöl. The real-time PCR method was used to investigate the presence of DNA specific to six animal species (chicken, turkey, pork, horse, donkey, and camel). The analysis revealed no traces of horse, donkey, camel, or pork meat in any of the 100 samples of Turkish fermented sausage (sucuk), salami, and sausage. Contrary to the product labels claiming 100% beef content, poultry DNA residues were identified in a salami sample from Erzincan, while turkey DNA residues were found in sucuk samples from both Ağrı and Kars. The study’s findings reveal that contrary to label information, various types of meat can be present in meat products sold by both trademarked and local butchers. Consequently, this paper emphasizes the need for routine checks on market-sold products to ensure they comply with legislation and avoid consumer deception. Furthermore, the Real-Time PCR method, with its rapid results and high sensitivity, is deemed beneficial for food safety, consumer rights, and the prevention of unfair competition.

Hydroxymethylbilane synthase (HMBS) gene-based endogenous internal control for avian species

With PCR becoming one of the most important and widely-used diagnostic tools for infectious diseases of poultry, an urgent need has developed for an endogenous internal control (EIC) that monitors the quality and quantity of poultry DNA in test samples. In this study we developed a SYBR-qPCR to target the poultry homolog of the hydroxymethylbilane synthase (HMBS) gene as an EIC for avian species. The avian HMBS-based qPCR was very sensitive, detecting one HMBS gene copy in a 20 µL reaction, and is highly specific for avian species. It amplified DNA from 11 organs and tissues of chickens showing it can be used as an EIC on a large variety of samples. The application of the established EIC on clinically and experimentally infected samples demonstrated that false negativity and result variations could result from samples being collected using different operators, techniques, preservatives, and storage times. The high sensitivity and specificity of the avian HMBS-based qPCR, its ability to quantify DNAs extracted from a wide range of tissues and poultry species along with its usefulness in reducing false negativity in PCR results associated with inadequate sampling and storage degradation makes it an ideal EIC for poultry DNA and RNA PCR diagnostics. The study also highlights the importance of appropriate sampling and storage of samples in ensuring accuracy of molecular diagnostic testing.

A novel quantitative real-time PCR method for the detection of mammalian and poultry species based on a shared single-copy nuclear DNA sequence

Accurate quantification of species fractions is critical to determine meat adulteration. This study aimed to develop a novel quantitative real-time PCR (qRT-PCR) method for detection of mammalian and poultry DNA. A shared single-copy nuclear DNA sequence derived from the first exon of the LcoR gene was identified as a multi-species universal reference for a qRT-PCR assay. The conservation and copy number of the LcoR gene were evaluated among different species. The limit of detection was 0.01 ng DNA or 0.01% meat ingredient, and the limit of quantification was 0.01 ng DNA or 0.05% meat ingredient. Both the relative error (R.E.) and relative standard deviation (R.S.D.) were ≤ 25%. Moreover, modified coefficient k was introduced into this quantitative system to improve the accuracy and reliability of results, with maximum R.E. improved from 19.43% to 16.16%. The quantitative method would contribute to fighting against meat adulteration and maintaining a fair market.

Applicability of the poultry qPCR method to detect DNA of poultry processed animal protein materials

After the Bovine Spongiform Encephalitis (BSE) crisis most processed animal proteins (PAPs) were banned from use in animal feed. For the foreseen reintroduction of pork PAPs in poultry feed, and poultry PAPs in pork feed and to comply with the species-to-species ban that prohibits cannibalism, a sensitive and specific TaqMan PCR detection method for poultry DNA has been designed and published. This poultry method is able to detect DNA of chicken, turkey, duck and geese in one PCR reaction. PAPs however, are a difficult and variable matrix. Therefore, the usability of the poultry method was investigated on a range of different poultry PAPs. It was shown that the poultry detection method is capable of detecting poultry DNA in eight out of nine different poultry PAPs mixed at a 0.1% level in chicken feed. The method can also detect at least 0.1% poultry PAPs mixed in pork PAPs. These results show that the poultry method fulfils the 0.1% detection limit requirement in the EU legislation.

Specificity of a novel TaqMan PCR method for detection of poultry DNA

After the Bovine Spongiform Encephalopathy (BSE) crisis emerged in 1985/1986, all processed animal proteins (PAPs) were finally banned for use in animal feed in the European Union. To partially lift this feed ban, paths for re-introduction of PAPs from species other than ruminants e.g. pig and poultry, are described in the Transmissible Spongiform Encephalopathies (TSE) Roadmap 2. Cannibalism, however, is still not allowed. Specific detection methods for pig and poultry meal and PAPs are prerequisites for reintroduction of pig and poultry processed animal proteins into animal feed. Developing a sensitive PCR method that specifically detects the taxonomically diverse and therefore artificial group ‘poultry’ and that does not detect other birds at the same time is a challenge. Here, a novel TaqMan PCR method for poultry detection is presented. The specificity of the poultry method against target and non-target species has been extensively investigated. The efficiency, linearity and sensitivity was tested using dilution series of chicken, turkey, duck and goose DNA isolated from meat and autoclaved meat as a model system for PAPs.

A molecular assay for quantification and simultaneous detection of soybean and poultry DNA in sausages following multi-extraction

A duplex PCR assay was designed for simultaneous identification of soybean and poultry in sausages. A Multi-Fast DNA Isolation Kit was used for simultaneous extraction of amplifiable soybean and poultry DNA. Primers generated specific DNA fragments of 173 and 183 bp in length for soybean and poultry, respectively. The duplex assay sensitivity was 0.35% for specific detection of poultry mixed with soybeans and other additive materials and was 0.05% for specific detection of soybeans mixed with poultry and other additive materials. The optimized duplex PCR assay was applied to 15 commercial sausages. Unintentional and intentional contaminants were analyzed using real-time PCR based Sakalar Quantification Table of DNA (SQT-DNA). Techniques developed here are suitable tools for qualitative and quantitative comparison of sausage contents.

Authenticity control of game meat products – A single method to detect and quantify adulteration of fallow deer (Dama dama), red deer (Cervus elaphus) and sika deer (Cervus nippon) by real-time PCR

This contribution presents a single real-time PCR assay allowing the determination of the deer content (the sum of fallow deer (Dama dama), red deer (Cervus elaphus) and sika deer (Cervus nippon)) in meat products to detect food adulteration. The PCR assay does not show cross-reactivity with 20 animal species and 43 botanical species potentially contained in game meat products. The limit of quantification is 0.5% for fallow deer and red deer and 0.1% for sika deer. The deer content in meat products is determined by relating the concentration obtained with the deer PCR assay to that obtained with a reference system which amplifies mammals and poultry DNA. The analysis of binary meat mixtures with pork, a meat mixture containing equal amounts of fallow deer, red deer and sika deer in pork and a model game sausage showed that the quantification approach is very accurate (systematic error generally <25%).

Development and Use of Quantitative Competitive PCR Assay for Detection of Poultry DNA in Sausage

This study focused on the development and evaluation of a quantitative competitive polymerase chain reaction (QC-PCR) for detection and quantification of poultry DNA in sausage. PCR is well known to be quantitative if internal DNA standards are co-amplified together with the target DNA. A DNA competitor differing by 83 bp in length from the poultry target sequence was constructed and used for PCR together with the target DNA. Specificity of the new primers was evaluated with DNA from cattle and sheep. The results of QC-PCR showed that the percentage of contamination was in the range of 23.87–52.06%.

Development and use of quantitative competitive PCR assay for detection of poultry DNA in fish meal

Development and use of quantitative competitive PCR assay for detection of poultry DNA in fish meal

Specific Discrimination of Chicken DNA from Other Poultry DNA in Processed Foods Using the Polymerase Chain Reaction

In the present study, specific discrimination of chicken DNA contamination in processed foods using the polymerase chain reaction was investigated. The primer pair was designed to amplify a 102-bp fragment of the chicken mitochondrial 16S ribosomal RNA gene. While the DNA from chicken meat was amplified, the DNA from other poultry meat, mammalian meat, fish, shellfish, and cereals was not amplified. The primer amplified DNA fragments derived from model processed and nonprocessed food samples containing 0.001, 0.01, 0.1, 1, 10, and 100% chicken.

Application of Quantitative Real-Time PCR in the Detection of Prion-Protein Gene Species-Specific DNA Sequences in Animal Meals and Feedstuffs

This study describes a method for quantitative and species-specific detection of animal DNA from different species (cattle, sheep, goat, swine, and chicken) in animal feed and feed ingredients, including fish meals. A quantitative real-time PCR approach was carried out to characterize species-specific sequences based on the amplification of prion-protein sequence. Prion-protein species-specific primers and TaqMan probes were designed, and amplification protocols were optimized in order to discriminate the different species with short PCR amplicons. The real-time quantitative PCR approach was also compared to conventional species-specific PCR assays. The real-time quantitative assay allowed the detection of 10 pg of ruminant, swine, and poultry DNA extracted from meat samples processed at 130°C for 40 min, 200 kPa. The origin of analyzed animal meals was characterized by the quantitative estimation of ruminant, swine, and poultry DNA. The TaqMan assay was used to quantify ruminant DNA in feedstuffs with 0.1% of meat and bone meal. In conclusion, the proposed molecular approach allowed the detection of species-specific DNA in animal meals and feedstuffs.

Polymerase Chain Reaction–Based Analysis To Detect Terrestrial Animal Protein in Fish Meal

The recent European bovine spongiform encephalopathy crisis has focused attention on the importance of adopting stringent control measures to avoid the risk of the diffusion of mad cow disease through meat meal–based animal feedstuffs. Potential adulteration of such feedstuffs with bone particles from terrestrial animals is determined by microscopic examination by law before the release of these feedstuffs for free circulation in the European Community. This study describes a DNA monitoring method to examine fish meal for contamination with mammalian and poultry products. A polymerase chain reaction (PCR) method based on the nucleotide sequence variation in the 12S ribosomal RNA gene of mitochondrial DNA was developed and evaluated. Three species-specific primer pairs were designed for the identification of ruminant, pig, and poultry DNA. The specificity of the primers used in the PCR was tested by comparison with DNA samples for several vertebrate species and confirmed. The PCR specifically detected mammalian and poultry adulteration in fish meals containing 0.125% beef, 0.125% sheep, 0.125% pig, 0.125% chicken, and 0.5% goat. A multiplex PCR assay for ruminant and pig adulteration was optimized and had a detection limit of 0.25%.