A Novel Direct PCR Lysis Buffer Can Improve PCR from Meat Matrices

Comparison of different DNA preparatory methods for development of a universal direct PCR-RFLP workflow for identification of meat origin in food products

“…Among such attempts, the direct PCR approach allows PCR amplification without prior DNA extraction. This method efficiently reduces the time and cost of a developed assay for confirmation of meat origin (Guan et al, 2019). The said method has been successfully exploited for medical diagnostics, forensic purposes, meat origin identification, and DNA barcoding of insects, microbial and fungal fauna as well as for certain invertebrates (Kitpipit et al, 2014a;Tjhie et al, 1994;Thanakiatkrai et al, 2019;Werblow et al, 2016;Wu et al, 2020).…”

“…The developed procedure has several special aspects that make it desirable for species identification especially for confirmation of meat origin. First, the lengthy 2-3 days DNA extraction protocol is shortened to only 10 min of boiling the sample in a buffer and then directly proceeding it to PCR, hence the term 'direct PCR' (Guan et al, 2019). Second, as no purification steps are included in the extraction procedure through boiling methods, therefore inhibitors can hamper the PCR reaction (Schrader et al, 2012).…”

“…Within the mitochondrial genes, 16S rRNA has been used for broad range of mammalian and birds' species because of its evolutionary stability (Vences et al, 2016;Ha et al, 2017;Lalitha & Chandavar, 2017). Furthermore, the developed direct PCR method is coupled with RFLP assay which is much more desirable for molecular based meat identification, especially, in cases where large number of samples have to be processed as it does not require DNA sequencing and/or specialized equipment and reduces the cost and time for post-PCR processing of samples (Guan et al, 2019;Gargouri et al, 2021;Vaithiyanathan et al, 2021;Taha et al, 2021). It has been mostly applied for species discrimination in processed and unprocessed meat products because of its simplicity, quicker detection and reduced cost (Al et al, 2020;Asghar et al, 2022;Farag et al, 2022).…”

“…Direct PCR is rapid, eliminates the need for purification steps, and proves more sensitive than conventional PCR (Linacre et al, 2010;Swaran & Welch, 2012). Although previously reported, these methods either require special and expensive polymerases and extraction kits or have limited efficiency for amplification (Ben-Amar et al, 2017;Guan et al, 2019;Thanakiatkrai et al, 2019).…”