DNA Extraction from Beef Harboring Sarcocystis spp.: Comparison of Three Different Analytical Methods

Background: DNA extraction is one the most important steps for molecular analysis of food-borne pathogens. In this research, three methods of DNA extractions from beef harboring Sarcocystis spp. were compared for the quality, quantity, safety, as well as cost-effectiveness.Methods: About 100 mg intersostal and diaphragm were collected from 10 slaughtered cattle. After ensuring their contamination with Sarcocyst using Polymerase Chain Reaction (PCR) with the specific primer pair, three methods of salting out, Phenol- Chloroform-Isoamylalcohol (PCI), and commercial kit were performed. Quantification, qualification, and amplification analysis of the extracted DNA was done using spectrophotometer,agarose gel electrophoresis, and PCR, respectively. Statistical analysis was performed using ANOVA test, by SPSS, Inc, Chicago, IL software (v.16.0).Results: Qualification in all methods was appropriate but the ones related to salting out and PCI methods were the best in comparison with the ones from commercial kit. Quantification analysis indicated the mean concentration of 249.3±3.94, 67.8±5.1, and 31±2.7 ng/μl for PCI, salting out, and commercial kit, respectively. The purification analysis represented the mean ratios of A (260)/(280), 1.7±0.3, 1.63±0.2, and 1.81±0.6 for PCI,salting out, and commercial kit, respectively. No significant difference (p> 0.05) was found between the yielded concentration and purification among three methods. Conclusion: The commercial kit is expensive, but salting out and PCI methods are cost effectiveness, however the last is considered as a toxic method. Because, amplification in all methods was appropriate, we introduced salting out for molecular detection ofSarcocystis in beef.

Keywords:

Sarcocystis , Meat , Molecular Diagnostic Techniques