Reproducibility of Quantifiler® Trio Assay-Specific Standard Curves Over a Range of Variables to Generate Guidelines for Crime Laboratory Workflows

Abstract

Quantitative PCR (qPCR) is the preferred method of quantitation in forensic DNA analysis, used to determine the amount of amplifiable human DNA present in evidence or reference samples. A standard curve is created via quantitation of a serial dilution containing known DNA concentrations. Quantifiler® Trio manufacturer guidelines were followed for the creation of five concentrations varying from 50 ng/µL to 0.005 ng/µL. The primary goal of this research was to generate laboratory guidelines and recommendations for quantitation standards for forensic laboratories hoping to streamline their workflows, and to determine how long standards are valid to decrease the amount of time and money spent on assay-specific standard curves. Most often, a standard curve is generated every time an assay is performed. The research data was generated over a two-month period. Through multiples runs, including two analysts involved in plating of samples, standard curves were analyzed for variation in curve parameters, e.g., has the slope or quantitation range changed over time. Mock case samples were prepared and analyzed to check the efficacy of the assay-specific standards versus a virtual curve to validate the suitability in DNA crime laboratories. In order to illustrate the relevance of laboratory generated internal standards (assay standard curves). First, if the standard curve is slightly different every time a new curve is generated, how could it affect the laboratory DNA results? Secondly, how variable are these standard curves overtime when performed by the same or multiple individuals; this will be evaluated by a comparison of linear regression values calculated by the software for each standard curve, between analysts. Results indicated little to no difference in the values for T.Y, small, and large autosomal targets. Linearity remained consistent beyond recommended discard of 14-days. Whether a laboratory prefers ASC or VSC the data provided shows that there is little to no difference between the two curve methods prior to amplification. The use of a Quantifiler® Trio kit to generate laboratory standard curves remains effective for a maximum of 21-days, experimentation will prove that the length of laboratory generated standard curves will last well beyond the manufactured data.