Development of a duplex fluorescence RT-PCR assay for identifying SRBⅠgene knockout mice / 天津医药

ABSTRACT

Objective To develop a duplex fluorescence RT-PCR assay for detection of scavenger receptor class B, typeⅠ(SRBⅠ) knockout mice. Methods Primers and probes were designed according to knockout region of SRBⅠgene and related substituted sequence. DNA samples were extracted from tails of mice and performed amplification using real-time PCR. SRBⅠgenotypes of mice were analyzed according to amplification curves of FAM and CY5 channels. Finally, the sensitivity of the method was detected and the accuracy was verified by the direct sequencing. Results The homozygous SRBⅠwild genotype showed an amplification curve only in FAM channel. When the homozygous SRBⅠknockout genotype was present, the typical S amplification curve appeared only in the CY5 channel. Heterozygous genotype showed two typical S amplification curves in both FAM and CY5 channels, respectively. The results showed that the sensitivity reached 4×101 copies/μL, and there was complete concordance between this method and direct DNA sequencing. Conclusion The new method is simple, rapid and accurate, which is suitable for genotyping SRBⅠknockout mice.