In-house validation and calibration of pork detection using duplex SYBR-Green I real-time PCR approach
2019
Rachmadhani | Warisman, M. A. | Suryani | Desriani
An effective detection method for identifying pork in processed food is urgently needed as there is a possibility that the food is adulterated with other materials. The aim of the present work was to validate duplex detection and quantification of pork and 18S rRNA using real-time PCR SYBR Green I approach and its quality control development. The validation parameters which consisted of specificity, detection limit (sensitivity), precision, linearity, PCR efficiency, and robustness were investigated. All these activities were done after obtaining the best formulation for the duplex. Quality control for pork detection was developed to avoid dubious things. The optimum primer concentration by duplex method of pork and 18S rRNA were 0.75 pmol (pork) and 0.1 pmol (18S rRNA), respectively. The detection limit of DNA template concentration was 0.78125 ng, which could be more sensitive since PCR result still obtained thick bands. The high precision and linearity marked by the % CV and R² were between 0.145 – 0.898 and 0.9813, respectively. In addition, the efficiency of PCR was 95.584%. All processed food products containing pork were amplified and showed bands as targeted for pork and 19S rRNA genes, while non-pork processed food products did not show amplification of pork gene (18S rRNA only). Standard curve as calibration for pork quantification was developed successfully with good efficiency (E = 90%).
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