Page 123 - BBLP ejournal2018.docx

Journal of Biotechnology in Livestock Production

Materials and Methods

Primer designation

Primers specific to each animal spp. were designed from published mitochondrial cyt b gene

sequences of the National Center of Biotechnology Information (NCBI) (Anonymous)

(http://www.ncbi.nlm.nih.gov). The expecting PCR primers were calculated using free Primer design

program available from the internet, Primer3plus (Anonymous) (http://www.bioinformatics.nl/cgi-
bin/primer3plus/primer3plus.cgi).

Samples

In order to select primers specific for each animal species and to develop an appropriate
multiplex PCR condition, 10 meat samples of each species of animal (cattle, buffalo, goat, sheep,

chicken, and pig) were collected from the slaughter houses to ensure the species of animals as well

as to quantify of multiplex PCR products (mixed DNA species). Also 11 samples from animal

products or food including white pork sausage, pork sausage, pork ball, chicken sausage, chicken

ball, beef ball, and beef stew were collected randomly from food shelves and vendors in the purpose

o
of testing the adulteration of meat-labeled products. All samples were kept in -200 C until testing in
the laboratory.

DNA extraction

DNA from all samples was extracted using QIAGEN Genomics DNA Extraction Kit (QIAGEN,

USA) according to manufacturer. Microvolume Spectrophotometer (Nanodrop, DeNovix. USA) was
used for checking the quality and quantity of each DNA sample based on an optical density (OD)

ratio of 260/280 nm between 1.6 and 1.8.

PCR reactions
The PCR amplification was performed in a total of 20 µl with 20 ng of genomic DNA, 0.2 µM

of each primer, 0.2 mM of each dNTP, 1.5 mM of MgCl 2, 1x PCR reaction buffer, and 1U of Platinum

Taq DNA polymerase. Amplification conditions included an initial denaturation at 950 C for 2 min,
o
followed by 40 cycles at 950 C for 45 sec, 620 C for 60 sec, and 720 C for 45 sec, then followed
o
o
o
by a final extension at 72 C for 10 min. The PCR products were mixed with GelRed Loading Buffer
o
(Biotium, USA) and separated by 2% (w/v) agarose gel electrophoresis. The gel was visualized and

documented on Gel-Doc system (Alpha image 3400, USA).

113