微滴式数字PCR技术用于生物样品种属鉴定和绝对定量

Species Identification and Absolute Quantification of Biological Samples by Droplet Digital PCR

目的运用微滴式数字PCR技术进行生物样品的种属鉴定和绝对定量。方法选择人mtDNA两个编码基因ND4和16S rRNA,设计特异性引物与探针,用人来源及常见动物样本验证种属特异性,再用重组质粒和2组共16份人来源生物检材,倍比稀释。使用微滴式数字PCR技术进行种属检验和绝对定量,验证其灵敏度和稳定性。结果人重组质粒FAM(ND4)可进行人来源样品的检测,其检测结果与各级稀释梯度基本吻合,微滴式数字PCR技术可以检测出反应体系中低至单拷贝的DNA。结论微滴式数字PCR技术可以进行生物样品的种属鉴定和绝对定量,并具有很高的灵敏度和特异性,可应用于日常法医物证检验。

Objective To test droplet digital PCR for species identification and absolute quantification of biological sample. Methods Specific primers and probes for human mtDNA encoding gene ND4 and 16S rRNA were designed, and the species-specificity was assessed on DNA samples derived from human and common animals. To determine the sensitivity and stability of droplet digital PCR for species identifica- tion and absolute quantification, gradient dilution series of recombinant plasmid and 16 human DNA samples were analyzed. Results Human recombinant plasmid FAM （ND4） could be used in detecting the samples of human. And the results of detecting were consistent with all levels of diluted concentra- tions. Droplet digital PCR was able to detect low and single copy of target DNA. Conclusion Droplet digital PCR, with high sensitivity and specificity, is fully amenable for species identification and absolute quantification of biological samples, also it can be applied on routine forensic examination.