Abstract
Quantification of nucleic acid targets at low copy number is problematic with the limit of detection at 95 percent confidence predicted to be 3 molecules or higher for quantitative PCR. Conversely the accuracy of digital PCR is diminished at higher concentrations of template approaching 100 percent positive partitions, with the Poisson distribution showing that an average of only 3 molecules per partition represents an amplification frequency of greater than 95 percent. Therefore a full range of template concentrations cannot be quantified accurately with these methods alone without dilution. Here we report the development of quantification metrics for use with loop-mediated amplification (LAMP) as a bridge between concentrated and dilute template concentrations. The basis for this is that real-time monitoring of LAMP reactions either by bioluminescent reporting (BART) or by fluorescent dye binding shows increasing variation in timings between replicates at low copy number due to the LAMP amplification mechanism. This effect increases with decreasing copy number, closely associated with the amplification frequency. The use of an artificial template showed that the increasing variation is not linked to the use of displacement primers during the initiation of amplification and is therefore a fundamental feature of the LAMP initiation event. Quantification between 1 and 10 copies of a template was successfully achieved with a number of methods with a low number of replicates with the strongest correlation to timing variance. These ultra-quantification methods for LAMP amplification either singularly or in combination have potential in a full dynamic range quantification strategy based on LAMP, in a closed tube, undiluted sample molecular diagnostic.
Highlights
The benchmark quantification method for nucleic acid targets in molecular diagnostics is the quantitative real-time polymerase chain reaction
The association of the variance metric with amplification frequency suggests a methodology to bridge between digital loop-mediated amplification (LAMP) and higher copy number quantification creating the possibility of full dynamic range quantification within a closed tube format without requirement for sample dilution
The timing of LAMP amplification measured by fluorescence (Fig. 2C i,C ii) or bioluminescence (Fig. 2A i,A ii) detection is proportional to target copy number over a broad range, but we have previously reported that variance increased at low copy number
Summary
The benchmark quantification method for nucleic acid targets in molecular diagnostics is the quantitative real-time polymerase chain reaction (qPCR). Over the last decade a number of new DNA amplification methods have been developed that do not require thermocycling These isothermal amplification methods[345] have attracted particular interest in molecular biology due to reduced equipment costs associated with maintaining a single temperature, as well as high specificity and sensitivity. One such method is Loop-mediated amplification (LAMP)[6] which uses loop forming and displacement oligonucleotide primers and a strand displacing polymerase to initiate and cycle amplification (Fig. 1 panel A to C). The association of the variance metric with amplification frequency suggests a methodology to bridge between digital LAMP and higher copy number quantification creating the possibility of full dynamic range quantification within a closed tube format without requirement for sample dilution
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