Molecular fingerprinting methods to detect and genotype DNA targets through polymerase chain reaction

A molecular fingerprinting method to detect and genotype at least two pathogen DNA targets in a sample through polymerase chain reaction (PCR), said method comprising:

• (a) providing a PCR mixture comprising an amplification buffer comprising an intercalating molecule or compound capable of incorporating into a double-stranded amplicon and emitting a detectable signal;
• wherein the PCR mixture comprises two or more pairs of amplification primers for amplifying in a multiplex approach two or more pathogen DNA targets, wherein said PCR mixture comprises a melting temperature calibrator to periodically check the effective melting temperature of the amplicons produced in a particular thermocycler machine, and compare it with an expected melting temperature,
• wherein said calibrator is a synthetic oligonucleotide having the same melting temperature as an amplicon generated by an amplification primer pair of the PCR mixture, wherein said amplification primers are designed in order to generate amplicons with a different melting temperature from each other in order to discriminate one amplicon from other amplicons by observing the specific melting temperature of each amplicon,
• (b) performing PCR amplification of the pathogen DNA targets using said PCR mixture in said sample to produce double-stranded amplicons;
• (c) performing a High Resolution Melting (HRM) analysis on the PCR mixture and the sample subjected to PCR amplification in step (b);
• (d) monitoring the change in the signal emission resulting from temperature-induced denaturation of the double-stranded amplicons into two single-stranded DNAs due to the release of the intercalating molecule or compound, and
• (e) discriminating between pathogen DNA targets in the sample, through a reader analysing the change in signal emission from step (d) and obtaining the result of the analysis through a graphic interface connected to said reader.