Half-volume validation of the NGM Detect™ PCR Amplification Kit and its application on degraded casework samples

Area of Science:

• Forensic Science
• Molecular Biology
• Genetics

Background:

• Degraded DNA samples from casework present significant challenges in forensic analysis.
• Existing DNA amplification kits may struggle to generate complete profiles from low-quality DNA.
• The NGM Detect™ PCR Amplification Kit was developed to address these limitations.

Purpose of the Study:

• To validate the performance of the NGM Detect™ Kit using a half-volume polymerase chain reaction (PCR) system.
• To assess the kit's long-term application and effectiveness in genotyping degraded casework samples.
• To compare manual and robotic PCR preparation methods for DNA amplification.

Main Methods:

• Validation was conducted following guidelines from the Scientific Working Group on DNA analysis and the European Network of Forensic Science Institutes.
• Sensitivity, reproducibility, and repeatability were assessed using varying DNA input amounts (0.0375 ng to 0.5 ng).
• PCRs were performed with 29 cycles, increased to 31 for sub-optimal DNA input (≤0.5 ng).
• Eighty-four degraded casework samples were analyzed using manual PCR preparation.

Main Results:

• Optimal allelic peak heights and no allelic dropout were observed with 0.5 ng DNA input, irrespective of preparation method.
• Allelic dropout began to appear at 0.0375 ng DNA input, with manual PCRs showing fewer dropouts.
• Increased cycle numbers (31) resulted in 1.9% (manual) and 4.4% (robotic) dropout.
• Informative profiles were obtained for 78.57% (lowly), 70.37% (moderately), and 69.77% (highly) degraded samples.
• Allelic dropout rates were 26.05%, 44.88%, and 51.23% for lowly, moderately, and highly degraded samples, respectively.

Conclusions:

• The NGM Detect™ Kit is recommended for use in a half-volume PCR system for genotyping degraded DNA.
• The kit demonstrates successful long-term application and provides informative profiles from challenging forensic samples.
• Its usage is encouraged in cases where other kits fail to yield complete DNA profiles, particularly for degraded samples.