Accurate nucleic acid quantification in a single sample tube without the need for calibration.

Convenient and accurate nucleic acid quantification (NAQ) is crucial to clinical diagnosis, forensic medicine, veterinary medicine and food analysis. However, traditional NAQ relies on the preparation of a laborious, time-consuming and expensive calibration curve, which would also propagate pipette errors through serially dilutions. Besides, traditional NAQ is run in different tubes, which introduces bias from random tube-to-tube variations and is unable to detect inhibitors from biological samples. To solve these problems, a single-tube quantitative PCR (stqPCR) technique is proposed which enables accurate quantification without the need for a calibration curve. In this method, an internal quantitative standard DNA (IQS-DNA) for quantification was screened out by co-amplification with the target DNA. Then the difference between the quantification cycle value (ΔCq) of the IQS-DNA and the target DNA was used for NAQ. The method permitted high accuracy quantification with reliable data for concentrations in plasmid, serum standard, and clinical samples being confirmed (R

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Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.