aica® Droplet Chip Digital PCR System Accurately Evaluates Korean Beef Molecular Markers to Assist Germplasm Identification
Introduction
Bos taurus coreanae is a domesticated mammal that is consumed as a food resource in the Korean consumer market, and its beef consumption far exceeds that of other breeds. This consumption pattern has led to the emergence of molecular studies to distinguish Bos taurus from other cattle breeds. Not only cattle, but also different breeds of other economic animals have large differences in economic value in the market, so accurate germplasm identification is imperative.
In a previous study, Bos taurus-specific SVs generated by TE-associated deletion events were identified using traditional PCR methods and Sanger sequencing validation. It can be used as a molecular marker to distinguish different cattle breeds (i.e., Bos taurus vs. Holstein cattle). However, PCR has defects, and each sample has various final copy quantifications. In order to overcome the limitations of traditional PCR and accurately evaluate the Bos taurus-specific SV loci identified in previous studies, the Department of Biomedical Sciences of Dankook University, in conjunction with the Institute of Animal Husbandry and the Dankook University College of Medicine, used the naica️® droplet chip digital PCR system to perform more precise detection of Bos taurus-specific SV loci, and published the results in the journal Genomics & Informatics.
Transposable elements (TEs) make up about half of the cattle genome. They can be a powerful species-specific marker without recurrent mutations of structural variations (SVs) during genome evolution. Therefore, the authors applied the naica️® droplet chip digital PCR system to accurately quantify Korean cattle-specific SVs. Although the allele frequency variation of samples in Korean cattle populations is low, the naica️® droplet chip digital PCR system can perform high-sensitivity detection through absolute quantification, which can achieve more accurate quantification than PCR. Therefore, the naica️® droplet chip digital PCR system platform is more suitable for the quantitative evaluation of molecular markers than traditional PCR.
Application highlights:
▶ Accurate quantitative detection of Korean cattle-specific SV using naica® droplet chip digital PCR system.
▶ dPCR assay can be quantified with high precision when counting single molecules and analyzing small copies of specific populations, which has higher accuracy compared with qPCR.
▶ After sanger sequencing, it was determined that the naica️® droplet chip digital PCR system is accurate in detection, and the operation and cost are lower than sequencing.
▶ The naica️® droplet chip digital PCR system is suitable for quantitative evaluation of molecular markers.
Experimental method:
Test sample information:
A total of five brown Korean cattle DNA and five Holstein DNA were extracted as experimental samples.
Detection method:
In order to more accurately detect Korean cattle-specific SV, the "Del_96" site was applied to the naica️® droplet chip digital PCR system (Stilla Technologies). Confirm the concentration quantification of Korean cattle and Holstein cattle DNA before performing the naica️® droplet chip digital PCR system. FAM primer set and FAM probe are used to detect Korean cattle and Holstein cattle genomes. The VIC primer set and VIC probe were designed to be specifically absent in Korean cattle (Figure 1B). Therefore, the FAM primer set and FAM probe (positive control) were designed to detect in all cattle DNA. The VIC primer set and VIC probe were designed to detect fluorescence only in Korean cattle.
Experimental results:
The FAM dye was detected in all bovine genomes, and the VIC dye showed significant detection only in Korean cattle samples. This indicates that all Korean cattle genomes contain a specific deleted sequence (Del_96 region). The average signal concentration of VIC dye detected in Korean beef samples was 243 (copies/μL). Although VIC dye signals with an average concentration of 0.12 (copies/μL) were also detected in Holstein samples, these signals were negligible compared to Korean cattle.
▲Comparison of absolute copy numbers of regions between Korean Del_96 and Holstein samples detected by the naica® Droplet Chip Digital PCR System. The concentration graph indicates the sample number on the X-axis and the logarithmic scale bar (copy/μL) on the Y-axis. (A) FAM fluorescence was detected in all samples. The absolute copy number of Korean cattle samples was approximately twice that of Holstein samples. (B) VIC fluorescence was strongly detected only in Korean cattle samples.
Finally, the article Results and Discussion shows that digital PCR is suitable as a platform for verifying species-specific markers.
In summary, for naica️® droplet chip digital PCR technology, accurate quantification of absolute copy number is a key feature. Compared with qPCR, naica️® droplet chip digital PCR has higher accuracy. It provides favorable support for the detection in this article and verifies this feature. In the near future, by applying species identification tools to the naica️® droplet chip digital PCR system, it has great potential as a large sample size species identification platform. Therefore, the naica️® droplet chip digital PCR system is suitable as a platform for verifying species-specific markers.
Original link：https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7120350/
Journal Introduction:
Genomics & Informatics is a journal published by the Korean Genome Organization covering the fields of agriculture and biological sciences, biochemistry, genetics, molecular biology, health informatics, etc.
