Detection of pork adulteration in processed meat by species-specific PCR-QIAxcel procedure based on D-loop and cytb genes

Abstract

Detection of pork meat adulteration in "halal" meat products is a crucial issue in the fields of modern food inspection according to implementation of very strict procedures for halal food labelling. Present study aims at detecting and quantifying pork adulteration in both raw and cooked manufactured sausages. This is by applying an optimized species-specific PCR procedure followed by QIAxcel capillary electrophoresis system. Manufacturing experiment was designed by incorporating pork with beef meat at 0.01 to 10 % substitution levels beside beef and pork sausages as negative and positive controls, respectively. Subsequently, sausages were divided into raw and cooked sausages then subjected to DNA extraction. Results indicated that PCR amplifications of mitochondrial D-loop and cytochrome b (cytb) genes by porcine-specific primers produced 185 and 117 bp pork-specific DNA fragments in sausages, respectively. No DNA fragments were detected when PCR was applied on beef sausage DNA confirming primers specificity. For internal control, a 141-bp DNA fragment of eukaryotic 18S ribosomal RNA (rRNA) gene was amplified from pork and beef DNA templates. Although PCR followed by either QIAxcel or agarose techniques were efficient for targeted DNA fragments differentiation even as low as 0.01 % (pork/meat: w/w). For proficiency, adequacy, and performance, PCR-QIA procedure is highly sensitive, a time-saver, electronically documented, mutagenic-reagent free, of little manual errors, accurate in measuring PCR fragments length, and quantitative data supplier. In conclusion, it can be suggested that optimized PCR-QAI is considered as a rapid and sensitive method for routine pork detection and quantification in raw or processed meat.

Recent advances in the application of capillary electromigration methods for food analysis and Foodomics

“…In consequence, CE is still the method of choice in many food applications for DNA analysis. It is interesting to note that, in the period covered by the present review, most of de published CE methods for DNA analysis have been aimed at assessing food authenticity and traceability .…”

“…Using commercial QIAGEN QIAxcell CGE microfluidic system, Barakat et al. developed a procedure for detection of pork adulteration in sausages. Authors optimized various porcine‐ and pork‐specific PCR systems based on amplifications of mitochondrial D‐loop and cytochrome b genes, and used 18S ribosomal gene sequence as internal control for amplification.…”

Identification of Different Animal Species in Meat and Meat Products: Trends and Advances

“…Haunshi et al (2009) designed primers specific for pigeon identification based on cyt b and species-specific markers for chicken duck and pig D-loop mitochondrial genes that could strictly identify the mentioned species in fresh and processed meats. Barakat et al (2014) amplified the mitochondrial cyt b and D-loop genes using porcine-specific primers followed by QIAxcel capillary electrophoresis system to detect and quantify the pork meat in "halal" meat products using raw and cooked sausages as models. This method proved rapidity and sensitivity as it gives specific DNA fragments for pork meat only.…”

Simultaneous detection of fungal, bacterial, and viral pathogens in insects by multiplex PCR and capillary electrophoresis

“…In such cases, capillary electrophoresis can be used as a sensitive method to quantitatively analyze and compare the presence of each target pathogen gene (Barakat et al, 2014;Chung et al, 2012;Shin et al, 2014;Shin et al, 2010). Capillary electrophoresis can be successfully applied in cases of insect cross infection with several pathogens to quantitatively evaluate relative importance of infectious agents based on the ratio of target gene expression (Thomas et al, 2010b);…”