Page 124 - BBLP ejournal2018.docx

วารสารเทคโนโลยีชีวภาพการผลิตปศุสัตว์

Sequence analysis and identification of animal species

To obtain sequence information on cyt b gene of each animal species tested, the PCR

products were purified using PCR purification kit (Macherey-Nagel, Germany) and sequenced the

purified products using the automated dye-terminator cycle sequencing method with Ampli Taq DNA
polymerase in ABI 3130 DNA Analysis Sequencer (ABI, USA). The products, then, were blasted to

the sequencing information of GenBank (NCBI) using the BLAST Sequence Analysis program

(https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch#).

Quantification of multiplex PCR products

To quantify the mixed DNA from different animal spp., DNA samples of cattle, chicken and

pig were diluted to the concentration of 20 ng/µl. These DNAs were mixed for using as the templates

in 2 different PCR reactions. The first reaction was the mixture between cattle and pig’s DNA and

the second reaction was the mixture between chicken and pig’s DNA. Both reaction were mixed in

ratio of cattle or chicken’s DNA to pig’ s DNA as 1:0, 1:1, 1:0.5, 1:0.25, 1:0.1, 1:0.05, 1:0.025, 1:0.01,

and 0:1 (v/v) with 3replicationsfor each reaction in order to test for the efficiency of the tests.

Sampling food testing

Eleven meat-labeled products from food shelves and vendors including white pork sausage,

pork sausage, pork ball, chicken sausage, chicken ball, beef ball, and beef stew were used for

testing original species which were sampling randomly. DNA from all samples was extracted and

PCR reactions were evaluated as described previously. The PCR products were illustrated on
agarose gel compared with DNA from 6-known species.

Results

Nucleotide sequence analysis and prediction of primer specific of each spp. cyt b gene

To obtain sequence information on the cyt b gene of different potential animal species tested,

the data from NCBI database. Sequence comparison among cytb amplified regions of 6 animal

species: cattle (Accession no.NC006853), buffalo (Accession no.NC006295), sheep (Accession no.

FR873152), goat (Accession no. EU130780), chicken (Accession no. EU839454) and pig (Accession

no. AB015078) were used. Species-specific primers of mt cyt band expecting PCR amplicon sizes

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