Fast and Sensitive Real-time PCR-based Detection of Porcine DNA in Food Samples by Using EvaGreen Dye

Abstract

Accidental and/or incidental adulterations of foods by porcine ingredients are common in the globalized food processing industry. Food mislabelling and fraudulent substitutions of non-porcine ingredients with porcine ones are objectionable to those who abstain from porcine derived products due to habitual (e.g., vegans and vegetarians), medical (e.g., porcine allergies), legal (fraudulent labelling), economic (e.g., substitution of expensive meat with cheaper pork meat) and cultural or religious grounds (e.g., Islamic and Jewish dietary restrictions). Thus, a strong demand exists for a fast and sensitive method for quantitative sensing of porcine DNA in food. In this study, we are reporting the development of probe-free real-time PCR assay with new primer sets targeting the cytochrome b gene for the fast and sensitive detection of porcine DNA in real food samples. Standard curve was developed with six tenfold dilutions of the DNA standard and the assay successfully detected up to 0.00001 ng/μL of porcine DNA and as low as 0.001 % porcine adulteration in raw pork-chicken binary mixture. The standard curve indicated a linear regression of R 2 value of 0.990 and an efficiency of 92.5 %. The Ct value range for the detected pork DNA from the 35 food samples tested was 16.03-28.76. We confirmed the assay's specificity to porcine DNA against nine non-porcine animal species and 6 vegetables species.

Sensitivitas Multiplex-Polymerase Chain Reaction Gen 12S rRNA dalam Mendeteksi Pemalsuan Daging Sapi dengan Daging Babi, Anjing dan Tikus

“…Sensitivitas primer gen 12S rRNA pada spesies sapi memiliki limit deteksi 0,5 ng (Yin et al, 2009). Pengujian sensitivitas pada spesies babi menggunakan primer gen Cytochrome Oxidase Subunit 1 (COI) mampu mendeteksi hingga 0,001 ng dan primer gen cyt b dengan metode Eva Green real-time PCR hingga 0,00001 ng/ µL (Lubis et al, 2018). Pengujian sensitivitas oleh Matsunaga et al (1999) menggunakan primer gen cyt b pada spesies sapi dan babi dinyatakan sensitif hingga konsentrasi 0,25 ng.…”

“…Penelitian ini apabila dibandingkan dengan penelitian terdahulu, primer gen 12S rRNA lebih sensitif (Matsunaga et al, 1999;Dalmasso et al, 2004;Yin et al, 2009;Ali et al, 2015;Hossain et al, 2017;Iwobi et al, 2017;Prusakova et al, 2018;Liu et al, 2019;Li et al, 2019). Primer gen 12S rRNA untuk spesies babi sama sensitif dengan primer gen COI yaitu 0,001 ng (Wang et al, 2019), kurang sensitif untuk spesies babi apabila dibandingkan dengan primer cyt b oleh Lubis et al (2018), dan kurang sensitif untuk spesies tikus oleh Chen et al (2020).…”

An Eva Green Real-Time PCR Assay for Porcine DNA Analysis in Raw and Processed Foods

“…The EvaGreen dye was also utilized by Raharjo et al [25] for detecting porcine DNA in meatballs. Using a qPCR technique based on EvaGreen, Lubis et al [26] successfully quantified pork DNA in raw and processed foods. SYBR ® Green I, the most common intercalating dye employed, fluoresce up to a thousand-fold upon binding to dsDNA [17].…”

“…Nevertheless, SYBR ® Green I has some downsides, including low sensitivity, stability, and reproducibility of results [27]. EvaGreen dye, on the other hand, offers better stability, excellent sensitivity, and higher fluorescence signal with less inhibitory effect on PCR even at saturating dye concentration [16,26,27].…”

Detection of animal DNA in vegan food by multiplex qPCR system

“…A first motivation for the detection of animals in vegetarian food is based on cultural preferences such as halal or kosher. Work has been done on the development of PCR systems for the detection of pig residues such as DNA in meat [1,2]. In addition, quality control laboratories need systems for the detection of specific animals in food and developed such systems [3][4][5][6].…”