Rapid genotyping of 32 insertion/deletion panel for human identification using fluorogenic probes-based multiplex real-time PCR

Insertion and deletion polymorphisms (InDels) have considerable potential in the field of forensic genetics because of their low mutation rate and small amplicons. At present, InDel polymorphisms detection based on the technique of capillary electrophoresis is the main technique used in forensic DNA laboratory. However, this method is complicated and time-consuming, and is not suitable for rapid on-site paternity and personal identification. Next-generation sequencing analysis of InDels polymorphisms requires expensive instruments, large upfront reagent and supply costs, computational requirements and complex bioinformatics, increased the time to obtain results. Thus, there is an urgent need to establish a method to provide reliable, rapid, sensitive and economical genotyping for InDels. A rapid InDels (32 InDels) panel was established using fluorogenic probes-based multiplex real-time PCR with microfluidic test cartridge and portable real-time PCR instrument. Then, we performed several validation studies including concordance, accuracy, sensitivity, stability, species specificity. It showed that the complete genotypes could be obtained from ≥100 pg of input DNA and from a series of challenging samples with high accuracy and specificity within 90 min. This method provides a rapid and cost-effective solution for InDels genotyping and personal identification in portable format.