高通量测序技术在动物源性物种鉴定中的研究

Application of High-Throughput Sequencing Technology in Identification of Animal-Derived Species

目的:利用高通量测序技术建立动物源性物种鉴定方法。方法:对9种纯动物组织样本分别按比例进行肉糜和DNA混合,对2组混合提取的DNA样本进行条形码标记和文库制备。随后进行高通量测序和生物信息学分析,最后对DNA含量为1%的组合进行实时荧光PCR方法检测。结果:根据Summay结果分析,测序结果良好,6组肉糜混合的9种动物源性均被检出。8组DNA混合样本中,DM 7-2中的马未被检出,其他组合物种均被检出。各物种reads数比例与DNA最初混样比例基本一致。在实时荧光PCR实验中,DNA比例为1%的物种均能检测到,CT值在32~35之间。结论:初步建立动物源性鉴定的高通量测序方法,具有极高的准确性和稳定性,检测限达到1%(质量分数)。

Objective:High-throughput sequencing technology was used to establish an identification method of animal-derived species.Methods:Nine types of pure animal tissue samples were mixed with meat mince and DNA in proportion.The DNA samples extracted from the two mixed groups were labeled with barcodes and library preparation,followed by high-throughput sequencing and bioinformatics analysis.Finally,the combination with 1%DNA content was detected by real-time fluorescent PCR.Results:According to the Summary results,the sequencing results were good,9 kinds of animal origin mixed with 6 groups of meat paste were detected.Horses in DM 7-2 was not detected in 8 groups of DNA mixed samples,while other combined species were detected.And the proportion of reads in each species were basically as same as the proportion of initial DNA mixing.In the real-time fluorescence PCR experiment,the species with DNA ratio of 1%could be detected,and the CT value was between 32-35.Conclusion:A high-throughput sequencing method for identification of animal is preliminary established,with high accuracy and stability,and detection limit of 1%(mass fraction).