Evaluation Of Virtual Standard Curve Functionality Of The HID Real-Time Pcr Analysis Software By Comparison To Assay Specific Standard Curves And An External Standard Curve Generated In- House

Abstract

Quantitative PCR (qPCR) plays a critical role in the field of forensic biology to determine the amount of “amplifiable” human specific DNA. If too much or too little DNA is present, it can result in profiles difficult to interpret. Therefore, qPCR is beneficial in determining the quality and quantity of DNA to generate an interpretable profile from a forensic sample. To determine the DNA quantity, a set of standard with known DNA concentrations are used to make a standard curve using ordinary least squares which is compared to samples with unknown quantities of DNA to determine the concentration. The goal of this research project was to examine two alternative methods in determining the quantity of DNA that does not require a standard curve for each run to minimize run-to-run variation and reduce costs and analyst time. These two methods are the use of an external standard curve and a virtual standard curve using different lot numbers, curve preparations by different analysts, and between instrument calibrations. Samples were quantified in duplicate, and a linear regression was determined utilizing the of the average of all runs to calculate the slope and y-intercept per variable and target to generate a virtual standard curve in the new HID Real-Time PCR Analysis Software v1.3. It was determined that the external standard curve method and virtual standard curve method were identical. Results showed there was no significance between instrument calibration and no difference between kit lots when comparing the assay specific curve to the virtual/external curve methods. For the virtual standard curve, there was no significant difference to the assay specific method. There was significant differences between pipetting from different analysts when looking at the different standards prepared. A recommendation from this research regarding the use of these techniques is to have as many analysts as possible pipetting. If more than one variable is introduced throughout the process, a new virtual standard curve needs to be generated. This study demonstrates the feasibility of the implementation of the virtual standard curve function into a case working laboratory workflow and that a laboratory can benefit using these methods.