Direct-multiplex PCR assay for meat species identification in food products

Highlights

• Assay simultaneously amplifies six meat species with no prior DNA extraction.

• No cross-reactivity is observed with 13 domestic and wildlife meat species.

• Sensitivity of the assay is approximately five cells and varies between targets.

• Robustness of assay was tested with 115 commercial foods (97.4% successful).

• 29 of 115 commercial samples tested have meat species not listed as ingredient.

Abstract

This is the first time that direct PCR – DNA amplification without prior DNA extraction – was successfully developed and fully validated for rapid and economical simultaneous identification of six commonly consumed meat species. To achieve this, six species-specific primers were selected from previous reports and newly designed from the mitochondrial cytochrome b (cyt b), cytochrome oxidase I (COI), and 12s rRNA gene. The assay generated PCR products of 100, 119, 133, 155, 253, and 311 bp for pork, lamb/mutton, chicken, ostrich meat, horsemeat and beef, respectively. Validation showed that the assay is robust, rapid, economical, reproducible, specific, and sensitive down to 12,500 mitochondrial copy (equating to seven fg). It could be used with a variety of raw meats and products, including highly degraded and processed food samples. This proposed method will be greatly beneficial to the consumers, food industry, and law enforcement.

Introduction

Unlisted, mislabeled, or fraudulent ingredients in food products is a serious problem and impacts humans in several ways. Several manufacturers have exploited consumers by adding or substituting ingredients in food products for cheaper ones to earn more profits (Moore, Spink, & Lipp, 2012). Certain ingredients in high demand could boost the sales of food products, and this serves as an incentive for mislabeling, as evidenced in the case of crocodile meat (Unajak et al., 2011). These occurrences negatively impact the health and safety of the consumers and the economy, as well as breach religious laws. Both national and international regulations are therefore issued to protect consumer rights; thus, a reliable technique for meat identification is needed by regulatory agencies.

There are a multitude of options available for species identification in raw meats and food products: proteins, lipid, volatile organic compounds, and DNA analyses (Ali et al., 2012). Among these, DNA-based techniques are reliable, robust, and rapid. High variations in nucleotide compositions between individuals and species enable differentiation and DNA withstands environmental degradation better than the other compounds (Che Man et al., 2007, Che Man et al., 2012, Rojas et al., 2010). Both nuclear and mitochondrial DNA have been used, for example, the nuclear DNA’s porcine leptin gene, bovine beta-actin gene, turkey prolactin receptor (Köppel et al., 2010, Köppel et al., 2008, Köppel et al., 2009), and the mitochondrial D-loop region, cytochrome b, cytochrome oxidase subunit I (COI), 16s rRNA, 12s rRNA, and NADH dehydrogenase subunit 5/6 genes (Ali et al., 2011, Branicki et al., 2003, Che Man et al., 2007, Che Man et al., 2012, Dalmasso et al., 2004, Fajardo et al., 2010, Unajak et al., 2011). These markers are used with several techniques, such as species-specific amplification (Linacre, 2006, Linacre, 2009), real-time PCR (Köppel et al., 2008, Köppel et al., 2009, Köppel et al., 2010, Rojas et al., 2010), PCR–RFLP (Ali et al., 2011, Murugaiah et al., 2009), and sequencing (Ali et al., 2012). However, these methods are complicated and required purification of DNA from food products. DNA degradation from acidity regulation and heating process, as well as trace contamination, can also prevent successful detection (Köppel et al., 2008, Köppel et al., 2009).

Direct PCR, the amplification of DNA directly from samples with no prior DNA extraction, was first proposed in the 1990s (Mercier et al., 1990, Tjhie et al., 1994). With improved polymerase enzymes and PCR buffer in recent years (Hedman et al., 2009, Hedman et al., 2010, Hedman et al., 2011, Kermekchiev et al., 2009, Zhang et al., 2010), direct PCR has been touted as a sensitive and robust technique, as well as a time-saver. Direct PCR possesses several advantages. First, it is rapid as the extraction and quantification step are bypassed. Second, it is more sensitive than conventional PCR (Linacre et al., 2010, Swaran and Welch, 2012), as the process of DNA extraction has very low efficiency (16–30%) (Colussi et al., 2009, Kishore et al., 2006). Lastly, it improves DNA analysis results from several types of biological sample, including trace, degraded, aged, and difficult samples (Kitpipit et al., 2014, Linacre et al., 2010, Park et al., 2008, Swaran and Welch, 2012). Diluting the samples in phosphate-buffered saline (PBS) increases the amplification success rate for various sample types (Kitpipit et al., 2013b, Kitpipit et al., 2014). This dilution protocol has been used in a recently published direct-triplex PCR assay for pork, lamb, and chicken and it has been shown to overcome inhibitors found in food products (Kitpipit, Sittichan, & Thanakiatkrai, 2013a); however, a larger multiplex that includes other commonly consumed meat, including another haram meat (horsemeat), will shorten and simplify the source identification process, as well as brings additional benefit the halal food industry.

Thus, the aim of the study was to develop a direct-hexaplex PCR assay for simultaneous identification of six commonly consumed meats (pork, lamb/mutton, chicken, ostrich meat, horsemeat, and beef). Using the dilution protocol for sample preparation, the assay was then fully validated for its specificity, reproducibility, sensitivity, and robustness.