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Molecular Detection of Goat and Sheep Meat Origin Using Mitochondrial Cytochrome B Gene

Jahura FT
Department of Animal Breeding and Genetics, Bangladesh Agricultural University, Mymensingh-2202

Munira S
Department of Animal Breeding and Genetics, Bangladesh Agricultural University, Mymensingh-2202

Bhuiyan AKFH
Department of Animal Breeding and Genetics, Bangladesh Agricultural University, Mymensingh-2202

Hoque MR
Genet Bio Inc., Yuseong-gu, Daejeon 305-500, Republic of Korea

Bhuiyan MSA*
Department of Animal Breeding and Genetics, Bangladesh Agricultural University, Mymensingh-2202

Abstract/ Executive Summary:

The present study was conducted to discriminate between sheep and goat species meat origin utilizing mitochondrial cytochrome b (Cyt b) gene fragment. A total of 46 ear tissue and meat samples were collected from different slaughterhouses and farms of Mymensingh and Rangpur districts. Genomic DNA was extracted using GeNet Bio DNA isolation kit and DNA concentration and purity was quantified by NanoDrop spectrophotometer.Two pairs species specific primer were used to amplify Cyt b gene fragments. Selected primers were highly conserved across the breed within a species and worked well with the species of indigenous goat and sheep resulting similar size of the amplicons 330 and 585 bp respectively. The duplex PCR condition would enable to detect adulteration from goat and sheep mixed samples which revealed by two precise bands (330 and 585 bp) in a single reaction. This study suggests an accurate molecular technique for identification of sheep and goat meat species origin and differentiates species present in adulterate meat samples. In conclusion, this DNA based marker could be used for prevention of fraudulent practice in slaughterhouse and chain shops in Bangladesh.

Keywords: Goat, Sheep, Cytochrome b gene, PCR, Agarose gel

Location: Animal Genetics Lab, Bangladesh Agricultural University, Mymensingh

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Goat, Sheep

Objectives:

The objectives of this study were to adopt PCR based technique and to develop DNA marker from Cyt b gene for identification of goat and sheep species meat origin and detection of adulteration from mixed samples.

Methodology:

Sample collection, transportation and processing Forty-six (46) ear tissue or meat samples of goat and sheep were collected from different regions of Mymensingh and Rangpur districts. Samples collected for this study purpose were submersed into 96% alcohol immediately after collection and transferred to the Animal Genetics Lab, Bangladesh Agricultural University, Mymensingh and stored at room temperature into a small plastic tube. DNA extraction and quantification Samples were trimmed to remove extra hair root and exogenous fat before DNA extraction. DNA was extracted according to the instructions followed by the GeNet Bio genomic DNA isolation kit.DNA concentration was assessed by 0.8% gel electrophoresis as well as NanoDrop spectrophotometer (Model ND1000). Purity of extracted DNA was assessed by calculating the OD260/OD280 nm ratios using Nano Drop (Model ND1000) spectrophotometer. Selected DNA samples were homogenised with a concentration of ~ 50ng/µl by adding ddH2O and stored at - 20°C for further studies. Cyt b gene amplification by single and duplex PCR The primer sequence information was obtained from a published paper by Zarringhabaie et al. (2011). The Cytb gene sequence of Black Bengal goat and indigenous sheep were retrieved from NCBI gene bank data base (accession no. AB110597.1 and AB006800.1) and were aligned by CLUSTAL OMEGA software (EMBL lab) to check the suitability of selected primers for indigenous sheep and goat species of Bangladesh. Two sets of primer pairs which were used for PCR amplification. PCR amplification was performed by using gradient thermocycler (Biometra). Two types of PCR reaction was carried out which are given below:

i) Cyt b gene amplification by single PCR, PCR reaction was carried out in 20 µl volume comprising of 1X buffer, 1.5 mM MgCl2, 0.2 mM dNTP, 1.0 µM of each primer, 2 µl (~ 50ng/µl)of genomic DNA and 0.3 U Taq DNA polymerase.

ii) Cyt b gene amplification by duplex PCR

Duplex PCR means both forward and reverse primers were added in mixed samples in one PCR reaction. PCR reaction was carried out in 20 µl volume comprising of 1X buffer, 1.5 mM MgCl₂, 0.2 mM dNTP, 1.5 µM of each primer, 3 (1.5+1.5) µl (~ 50ng/µl)of mixed genomic DNA and 0.5 U Taq DNA polymerase. The PCR amplification was performed using Biometra thermo-cycler and cyclic conditions comprised of initial denaturation for 10 min at 94°C, followed by 35 cycles of denaturation for 30 s at 94°C, annealing at 62°C for 60 s, extension at 72°C for 45 s, and a final extension at 72°C for 10 min. The PCR products were electrophoresed at 120 V for 25 min in 2.0 % agarose gels. For this, 0.5x TBE buffer solution was used to prepare 2.0 % gel which stained with 5 μl (10 μg/μl)of ethidiumbromide (BioNEER, SouthKorea). The gel images were documented by UVsolo TS imaging system (Biometra, Germany).

Source of Information: Bang. J. Anim. Sci. 2016. 45 (2): 41-45

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Our DNA based study showed an accurate analytical technique for identification of goat and sheep species meat origin by conventional PCR and meat adulteration between these two species through duplex PCR analysis without further using any enzymatic digestion. Therefore, it can be suggested as a useful laboratory tool especially for meat traceability and has also commercial impact for prevention of malpractice in slaughterhouse, meat products factory and chain shops of Bangladesh.

Knowledge Management: Journal