Comparison of DNA Extraction Methods Between Conventional, Kit, Alkali and Buffer-Only for PCR Amplification on Raw and Boiled Bovine and Porcine Meat

Abstract

Detection of porcine contamination in food material by employing PCR techniques is integral in halal food confirmation. However, PCR is both costly and laborious, particularly in DNA isolation method. This study explores several different methods in DNA extraction for PCR amplification in bovine and porcine raw and boiled meat samples. Four methods for DNA extraction (conventional PCI method, DNA isolation kit, alkaline-based method, and a DNA lysis buffer-only from the same kit) was employed followed by PCR using primers from previous studies and compared for DNA quality and quantity (in six replicates) and PCR amplification on the best three DNA samples. This study shows that in all samples, the conventional method had the best DNA yield based on nanodrop measurement, followed by an alkali-based method, buffer-only method, and DNA isolation kit. Each method except lysis-buffer only had at least one sample with good DNA quality. Conventional and isolation kit showed reliable positive PCR detection for all porcine and bovine samples (92% positive). Using the alkaline-lysis method, DNA was amplified reliably on boiled meat samples (83% positive). Lysis-buffer-only method did not show consistent PCR amplification on the samples used (50% positive). The conclusion was that conventional PCI method and DNA isolation kit showed high reliability in PCR amplification of bovine and porcine meats, both raw and boiled. While high DNA yield was obtained using the alkaline-lysis method, PCR amplification was only successful on boiled samples. Lysis-buffer only method yielded in poor DNA quality and was not able to result in reliable DNA amplification.

Specificity of Various Mitochondrial DNA (<i>mt</i>DNA), <i>ND5</i>, <i>D-Loop</i>, and <i>Cty-b</i> DNA Primers in Detecting Pig (<i>Sus scrofa</i>) DNA Fragments

“…Meat samples (beef, goat, lamb and chicken) and pork as a positive control obtained from supermarkets/markets/supermarkets in the city of Malang, East Java, Indonesia. 0.5 M NaOH; 0.01 M EDTA pH 8; 2 M NH 4 CH 3 COOH; Isopropanol; Ethanol 70%; TE Buffer pH 7.6 and aquadest [10] [14]. Reaction mixture consists of Go Taq Green Master Mix (PROMEGA); DNA ladder 1 kb; BSA (Bovine Serum Albumin) 10 mg/ml and mt-DNA primers 10 pmol/μl (forward & reverse) [12].…”

“…The Polymerase Chain Reaction (PCR) technique is widely applied for the analysis of meat-based processed food products because it is fast, simple/specific, and sensitive [1] [8] [9] [10]. To support the process of detection of adulteration, many PCR-based methods have been developed using primers designed based on mitochondrial DNA [2] [11].…”

“…Another alternative method of DNA isolation is the Alkaline-lysis method. This method is quite simple, using temperature heating and alkaline treatment for the stages of cell lysis and DNA isolation [10]. In this research, the application of DNA isolation method uses Alkaline-lysis modification and tests the primer specificity of mt-DNA (ND5, D-Loop and Cyt-b) in detecting pig DNA fragments using conventional PCR techniques.…”

Genetic Diversity Analysis of Exon-2 Growth Hormone (GH) Gene in Crossbred Chicken (Sentul X Arab) using PCR-RFLP Technique

“…Some contaminants that can be identified using UV light spectroscopy are proteins, polysaccharides, and RNA [13]. [18], Biodrop spectrophotometer quantity results with values below 1.8 indicate contamination from protein and phenol solutions, while values above 2.0 indicate RNA contamination. The principle of the spectrophotometer is to calculate the difference in UV light where the double band of DNA can absorb UV light at 260 nm.…”

An Automated and Miniaturized Rotating-Disk Device for Rapid Nucleic Acid Extraction

“…Numerous well-known biological reagent companies have developed various nucleic acid extraction kits based on these conventional nucleic acid extraction methods for the separation and extraction of DNA and RNA from a wide variety of tissue samples. The conventional methods of nucleic acid extraction often include precipitation and centrifugation, which require an extensive number of steps, and thus are complicated, time-consuming (requiring up to 3 h, and much longer if incubated overnight) [6], and difficult to achieve miniaturized automation. Most of the methods require operators to be in direct contact with toxic chemical reagents.…”