Establishment and Verification of miniSTR Loci Double Multiplex Amplification System

Degradation of samples is one of the difficulties in forensic evidence examination. Affected by environmental factors such as high temperature, humidity, exposure, and microorganisms, the structure of DNA molecules is destroyed and fragmented. Large fragments of DNA are prone to failure during amplification, and complete amplification products cannot be obtained. The miniSTR technology can provide an effective solution to such problems. In this paper, a double multiplex amplification system for 25 miniSTR loci was established. Its performance indicators were tested, and its application value in degraded samples was evaluated. The technical performance indicators of the system, including balance, sensitivity, species specificity, and consistency, were compared with the VeriFiler^TM Plus PCR Amplification Kit to test its detection ability for both simulated degraded samples and case work samples. The results showed that the average intra-color and inter-color balance of the miniSTR double multiplex amplification system were both more than 0.7, and the detection sensitivity for the standard 9948 DNA reached 0.05 ng. No specific amplification was observed in species detection. All 100 extracted blood samples achieved accurate typing, and all simulated degraded samples were successfully detected. The detection rate of case work samples was higher than that of the VeriFiler^TM Plus PCR Amplification Kit. The miniSTR double multiplex amplification system exhibits good balance, high sensitivity, strong species specificity, and accurate typing. Its detection ability for simulated degraded samples and trace degraded samples is superior to that of the VeriFiler^TM Plus PCR Amplification Kit. Particularly, the use of this amplification system can significantly increase the detection of loci in large, severely degraded fragments that are otherwise difficult to detect. At present, there are few commercial miniSTR products available, making this research even more valuable for advancing forensic DNA analysis.