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.