Molecular assay methods

A method of conducting an amplification assay for detection of a target nucleic acid, the method comprising:

• (a) contacting a biological sample with a control amplification reagent and a target amplification reagent to form a reaction mixture, wherein the control amplification reagent comprises two or more pairs of amplification oligonucleotides capable of amplifying a medium chain acyl-coenzyme A dehydrogenase (MCAD) nucleic acid molecule or a complement thereof, and wherein the target amplification reagent comprises a pair of amplification oligonucleotides capable of amplifying a non-MCAD target nucleic acid of interest or a complement thereof, wherein said non-MCAD target nucleic acid is a chromosomal DNA;
• (b) subjecting the reaction mixture to amplification conditions to produce an amplification mixture, whereby
• (i) two or more regions of the MCAD nucleic acid molecule, if present in the biological sample, are amplified to produce two or more detectably distinguishable MCAD amplicons, each having a different length, wherein the two or more different length MCAD amplicons form a size control ladder comprised of amplicons of increasing length, and
• (ii) a region of the target nucleic acid, if present in the biological sample, is amplified to produce at least one detectable target amplicon;
• (c) evaluating the amplification mixture to detect the two or more MCAD amplicons and the target amplicon;
• and
• (d) comparing the detected MCAD amplicons with the detected target amplicon to evaluate the amount and quality of the target nucleic acid and/or the MCAD nucleic acid molecule present in the biological sample.