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      Mitochondrial Markers for the Detection of Duck Breeds Using Polymerase Chain Reaction

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          Abstract

          Species identification of the components of various duck breeds has revealed that the lowest identifiable number of components depends on the breed. The results (shown on the agarose gel) of a species-specific PCR reaction for Rouen ducks were less intense than the results for the same amount of components from other popular duck breeds, suggesting differences in the Rouen duck genome. Therefore, the present study aimed to identify part of the Rouen duck’s gene sequences and to develop two new primer pairs. The first pair enables breed-independent identification of duck DNA, and the second distinguishes Rouen ducks from Chinese and Indian Runner ducks. The sequencing reaction yielded sequences of 1386 bp in length, and the identified sequence differs by around 7% from the sequences of Chinese duck species. The detected sequence contributes to improving species identification methods for duck DNA. On its basis, two primers for the identification of duck DNA were designed. The first allows for DNA amplification with the same sensitivity regardless of duck breed. The second primer’s pair is breed specific, and it distinguishes Rouen ducks from Chinese and Indian Runner ducks. Both methods are very sensitive (0.05%).

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          Most cited references14

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          Identification of meat species by TaqMan-based real-time PCR assay.

          In this study, a convenient, sensitive and specific real-time PCR assay was described for the species identification and their quantification in raw and cooked meat products. Specific primers and TaqMan probes were designed on the mitochondrial ND2, ND5 and ATP 6-8 genes for donkey, pork and horse, respectively, and the performance of the method was tested. In the results, no cross-reaction was observed between the donkey and pork species specific primer-probe systems and non-target species (bovine, ovine, chicken and turkey). Only one cross reaction was observed between the horse species specific primer-probe set and 100ng pork DNA at the ct 33.01 level (corresponding to 0.01ng horse DNA). The real-time quantitative assay used in this study allowed the detection of as little as 0.0001ng template DNA from pure meat for each species investigated and experimental meat mixtures. In conclusion, it can be suggested that the TaqMan probe assay used in this research might be a rapid and sensitive method for the routine meat species identifications studies in raw or cooked meat products.
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            A multiplex PCR assay for the identification of animal species in feedstuffs.

            A multiplex Polymerase Chain Reaction (PCR) assay was applied to feedstuff analysis for the identification of the most used species in rendering plants (ruminant, poultry, fish and pork materials). Primers were designed in different regions of mitochondrial DNA (12S rRNA, tRNA Val and 16S rRNA) after alignment of the available sequences in the GenBank database. The primers generated specific fragments of 104-106, 183, 220-230 and 290 bp length for ruminants, poultry, fish and pork, respectively. The detection limit was 0.004% for fish primers and 0.002% for ruminants, poultry and pork primers. The multiplex PCR proposed in this study can be considered a valid alternative to the microscopic method for the detection of animal derived materials banned by a European Union Regulation as a preventive measure against the spread of Bovine Spongiform Encephalopathy.
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              Quantitative detection of poultry meat adulteration with pork by a duplex PCR assay.

              A species-specific duplex polymerase chain reaction (PCR) assay was developed for the simultaneous detection of pork and poultry meat species using the mitochondrial cytb and 12S rRNA as target genes for pork and poultry, respectively. By the amplification of binary reference meat mixtures, a linear normalised calibration curve was obtained using the fluorescence intensities of PCR products for pork (149 bp) and poultry (183 bp) species. The proposed method allowed the quantification of pork meat addition to poultry meat in the range of 1-75%, with a sensitivity of 0.1%. The in-house validation using samples with known amounts of pork meat (1.0%, 2.5%, 7.5%, 20.0% and 40%) evidenced a high reproducibility of the methodology (coefficient of variation from 4.1% to 7.6%). The successful application of the duplex PCR was also demonstrated by the high correlation (R(2)=0.99) obtained from regression analysis between the predicted and the actual values of pork meat addition in blind meat mixtures. The suggested methodology presents a low cost, fast, easy and reliable alternative to estimate the level of poultry meat adulteration by the addition of pork meat.
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                Author and article information

                Contributors
                Role: Academic Editor
                Role: Academic Editor
                Journal
                Genes (Basel)
                Genes (Basel)
                genes
                Genes
                MDPI
                2073-4425
                03 June 2021
                June 2021
                : 12
                : 6
                : 857
                Affiliations
                Department of Animal Molecular Biology, National Research Institute of Animal Production, Krakowska 1, 32-083 Balice, Poland; piotr.krzyscin@ 123456iz.edu.pl (P.K.); dominika.rubis@ 123456iz.edu.pl (D.R.)
                Author notes
                Article
                genes-12-00857
                10.3390/genes12060857
                8228690
                34204948
                24cdd1ea-6fa5-4f13-9999-b13d7f2b2670
                © 2021 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( https://creativecommons.org/licenses/by/4.0/).

                History
                : 21 April 2021
                : 01 June 2021
                Categories
                Case Report

                12s rrna,16s rrna,rouen,mh078252,identification of duck dna,mtdna of duck

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