Low cost and comprehensive pork detection in processed food products with a different food matrix

Fenny Aulia Sugiana, Henni Widyowati, M. Ali Warisman, Suryani, and Desriani (2018)

Indonesian Journal of Biotechnology, 23 (1): 21-27.ISSN 0853-8654

The adulteration of processed beef-based meat products with pork is a sensitive issue in Indonesia. Therefore a simple, low cost, and accurate method is required for the detection of pork, so as to protect consumers from accidental consumption of adulterated meat. In this study, we developed a detection method for the low cost identification of pork in processed meat products. We used the cost-efficient Taq DNA polymerase, DreamTaq Green PCR master mix (2x), and duplex PCR method to recognize pork simultaneously with 18S rRNA detection. A positive control containing a pork gene inserted into pGEM®-T easy was prepared, along with a negative control. The results of the duplex PCR were used to assess its specificity, detection limit, and its ability to recognize pork in processed meat products with a different food matrix. 18S rRNA detection was for confirming DNA integrity of DNA extracted from the processed food, while the positive control confirmed that the reagents were working well and the negative control confirmed a non-contamination problem. Following this, the duplex PCR was opধmized and the optimum concentration primer for duplex PCR detection was found to be 3 µM for pork and 0.2 µM for 18S rRNA. As liħle as 3.125 ng of the DNA template could be used to detect whether a sample contained pork. Of the nine commercial processed meat products tested, five were found to contain pork while four halal products showed no signs of pork. It can be concluded that duplex PCR is a simple, fast, sensitive, specific, and low cost method of detecting pork in processed meat products.

Keywords: 18S rRNA, duplex PCR, low cost, pork, processed meat

Katalog: http://perpus.biotek.lipi.go.id/index.php?p=fstream&fid=3480&bid=16089

Disusun oleh: Ludya AB/Pustakawan

Tandem Recombinant Plasmid Construction as Positive Control for PIK3CA H1047R Detection Based on SYBR Green I qPCR

Nahdaturrugaisiyah, Azamris, Bugi Ratno Budiarto, I Made Artika, Desriani (2018)

Pak. J. Biotechnol., Vol. 15 (3): 735-742.

PIK3CA H1047R mutation is found in breast cancer in high frequency and its detection could be applied as prognosis and predictive factor for trastuzumab therapy. qPCR is one of the simplest and robust method for PIK3CA H1047R detection. Provision of positive control for PIK3CA H1047R detection based on qPCR will support data analysis efficiently and avoid false negative result. In this research, we constructed a tandem recombinant plasmid (pGEM-tandPIK3CA) as positive control for Tm Shift SYBR Green I qPCR-based of PIK3CA H1047R detection system by ligating wild-type and PIK3CA H1047R fragments tandemly into pGEM-T Easy. The tandem plasmid was confirmed by restriction, DNA sequencing and qPCR. As a result, pGEMtandPIK3CA has been successfully constructed and confirmed. Statistical analysis shows high repeatability and reproducibility with % CV of <25%. The main advantage of this tandem positive control is its ability to serve as positive control for both wild-type PIK3CA and PIK3CA H1047R simultaneously, therefore improving the efficiency of the detection system.

Keywords: Breast cancer, PIK3CA H1047R, qPCR, positive control.

Katalog: http://perpus.biotek.lipi.go.id/index.php?p=fstream&fid=3475&bid=16075

Disusun oleh: Ludya AB/Pustakawan