Detection of apolipoprotein E genotypes by a real-time fluorescent PCR assay based on rapid solidified primers technolo-gy
Objective To establish a new rapid and low-cost technology for solidifying primers and to detect human apolipoprotein E genotypes conveniently by real time fluorescent PCR based on the solidified primers.Methods The properties of different adhesive colorants were studied to realize rapid drying and solidifying of primers.A real-time fluorescent PCR assay with solidified primer was established for the detection of six apolipoprotein E genotypes.In addition,the storage performance at room temperature,accuracy,specificity,sensitivity and the consistency with the sequencing method were evaluated.Results The solidified primers method only need high temperature,and the solidified primers can be stored and transported at room tempera-ture,with high stability and without influence on PCR amplification.The real-time fluorescent PCR assay based on solidified primers had high accuracy,good specificity.And the sensitivity for the assay was 0.63 ng/μL which was consistent with the sensi-tivity of traditional liquid PCR reagent.The verification results for clinical samples showed that the coincidence rate between the assay and the gene sequencing method was 100％.Conclusion The real-time fluorescent PCR assay with solidified primer could improve the convenience of multi-mutation gene detection,and flexibility of detection throughput selection.
Solidified primersMultiple mutation sitesReal-time fluorescent PCRApolipoprotein E genotypes
万方数据
维普
