Abstract
This study aimed to develop a TaqMan Real-Time Polymerase Chain Reaction method, using a novel primer for detection of pork adulteration in meatballs. The study is important as it described a TaqMan method for product adulteration analysis. TaqMan is known to have a more specific result compared to SYBR green analytical method. Assay in the study combined species-specific primers and TaqMan probes to targeting 153 bp fragment of D-loop mitochondrial region of pork. A specificity test was conducted on fresh tissues of pork, beef, chicken, wild boar, dog, and mouse. Meatballs as samples were prepared from a mixture of pork-beef and wild boar-beef with concentrations as follows: 5%, 10%, 25%, 75%, 90%, and 100%. The linearity and sensitivity of the method were performed by measuring the amplification curve from the dilution series, namely 1000, 200, 100, 10, 5, 1, and 0.5 pg/μL of DNA, extracted from 100% pork meatballs. A repeatability test was conducted as many as six repetitions on 100% pork and 100% wild boar meatballs. This study showed that mitochondrial D-loop species-specific primers and TaqMan probes could identify the DNA of pork and wild boar on the fresh tissues. Additionally, it also resulted in a threshold cycle (Ct) of 17.02 and 17.95 for pork, 22.22 for wild boar, while the negative result for others. The detection limit has shown 5 pg in the meatball formulation. The Relative Standard Deviation (RSD) of repeatability was 1.936% for pork, while 2.140% for wild boar. The developed method was also applied to analyzing commercial meatballs. A TaqMan real-time PCR analytical method using specific primer targeting on 153 bp fragment of the D-loop mitochondrial region could be applied as a standard method for identifying pork and wild boar in food samples intended for halal authentication studies
Highlights
Some methods based on DNA identification including Polymerase Chain Reaction (PCR) using specific primers (Aida et al, 2005; Sahilah et al, 2011), duplex-PCR (Soares et al, 2010), multiplex -PCR (Koppel et al, 2011), real-time-PCR (Eugster et al, 2008; Tjondro and Sismindari, 2012), PCR with electrophoresis, PCR-RFLP (Restriction Fragment Length Polymorphism), real-time PCR with TaqMan probe (Dooley et al, 2004; Kesmen et al, 2009; Ali et al, 2012), real-time PCR molecular beacon, SYBR green real-time PCR were reported
This study aimed to develop a TaqMan Real-Time Polymerase Chain Reaction method, using a novel primer for detecting pork adulteration in meatballs
A pair of primers (F: AAA TCG CCC ACT CTTT TCC C and R: TGT GTT AGG GCC TTT GAC G) targeting 153 bp fragment of the D-loop mitochondrial and a 25-nt porcine TaqMan probe (6-FAM/AGC CCA TGC/ZEN/TCA CAC ATA ACT GAG G /3IABkFQ) were specific to be used for identification of pig and wild boar DNA using a Taqman real-time PCR with the optimum annealing temperature was 53.3°C
Summary
A verification of declared components in commercial meat products does help consumers to make a definite purchase decision, and protect their health, religious belief, and endangered wildlife. A lot of methods have been developed to identify the meats including capillary gel electrophoresis (Hernandes-Chavez, 2011), Enzyme-Linked Immunosorbent Assay (ELISA) (Chen and Hsieh, 2000), chromatographic based techniques (Chou et al, 2007), electronic nose (Nurjuliana et al, 2011), and Fourier Transform Infrared (FTIR). Some methods based on DNA identification including Polymerase Chain Reaction (PCR) using specific primers (Aida et al, 2005; Sahilah et al, 2011), duplex-PCR (Soares et al, 2010), multiplex -PCR (Koppel et al, 2011), real-time-PCR (Eugster et al, 2008; Tjondro and Sismindari, 2012), PCR with electrophoresis, PCR-RFLP (Restriction Fragment Length Polymorphism), real-time PCR with TaqMan probe (Dooley et al, 2004; Kesmen et al, 2009; Ali et al, 2012), real-time PCR molecular beacon, SYBR green real-time PCR were reported.
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