Simultaneous identification of four meat species (cattle, chicken, fish, and pig) using next generation sequencing (NGS)

Abstract

Meat adulteration has become a serious problem in global which directly affects to food consumers and producers. Therefore, it requires a tool to authenticate meat species to ensure safety of food products. Next generation sequencing (NGS) coupled with ribosomal RNA mitochondrial DNA gene can be used to analyze mixture of meat species in multiple meat samples. Therefore, this study aims to utilize NGS coupled with rRNA gene to identify 4 meat species (cattle, chicken, fish, and pig). Three primer sets (12S-Ki, 16S-KH, and 16S-Ki) were used to amplify DNA from the four meat species. All primer sets could be successfully amplified DNA fragments which corresponded to their size expectation. 16S-KH showed better detection effect in all species comparing with others. While the 12S-Ki and 16S-Ki could not be used to amplify in fish and chicken species. This may occur due to mismatches between sequences of primers and annealed regions of these species. Library construction of all PCR amplicons were prepared and sequenced by NGS. Amplicons amplified by 12S-Ki (fish) and 16SKi (chicken and fish) could not be mapped to the database because no PCR amplicons could not be amplified. NGS coupled with 16S-KH was then evaluated for precision test. The experimental precision was directly investigated comparing the results obtained from libraries that derives from DNA of four meat species which separately amplified for 3 different runs. As expected, the number and proportion of mapped reads between three different runs were also concordant. The percentage of mapped reads ranged from 14.05% to 31.04%, 15.14% to 31.98%, and 14.21% to 33.05% (1st, 2nd, and 3rd run, respectively). This demonstrated that NGS coupled with rRNA mtDNA gene could be reliably implemented as a routine testing. This developed technique can be applied to control and monitor meat adulterations in halal meat production and industry.