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

1 . 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.

2 . The method according to claim 1 , wherein said amplification primers are designed to amplify pathogen DNA targets to produce amplicons with a length between 38 to 1500 bp.

3 . The method according to claim 1 , wherein each of the amplification primers is designed to generate an amplicon, wherein the melting peak of the amplicon is between 68 and 95° C.

4 . The method according to claim 1 , wherein each amplification primer is present in the PCR mixture at a final concentration of 50 to 1000 nanomolar (nM).

5 . The method according to claim 1 , wherein the amplification buffer of the PCR mixture further comprises a DNA polymerase, deoxynucleoside triphosphates (dNTPs) or analogues, water, a source of monovalent or bivalent cations, and bovine serum albumin (BSA).

6 . The method according to claim 5 , wherein the amplification buffer of the PCR mixture further comprises one or more additives selected from the group consisting of: DMSO, TMAC (Tetramethylammonium Chloride), acetamide, a detergent, formamide, betaine, E. coli ssDNA binding protein, glycerol, L-Carnitine and gelatine.

7 . The method according to claim 1 , wherein performing PCR amplification using said PCR mixture includes amplifying the pathogen DNA targets using said PCR mixture to generate at least two amplicons, wherein amplifying the pathogen DNA targets includes thermocycling by repeatedly performing a ramp of temperature, wherein, during thermocycling in the PCR amplification, monitoring the change in the signal emission resulting from the temperature-induced denaturation of the double-stranded amplicons into two single-stranded DNAs, due to the release of the intercalating molecule or compound, is performed, wherein said ramp of temperature comprises performing the following temperature steps:

denaturation at 95-98° C. from 1 to 30 seconds;

annealing in a range between 50° C. and 70° C. from 1 to 60 seconds; and

extension in a range between 60° C. and 75° C. from 0 seconds to 5 minutes.

8 . The method according to claim 1 , wherein performing HRM analysis includes performing a ramp of temperature for HRM on the PCR mixture subjected to PCR amplification in step (b) and, during the ramp of temperature, performing said monitoring the change in the signal emission resulting from the temperature-induced denaturation of the double-stranded amplicons into two single-stranded DNA, due to the release of the intercalating molecule or compound, wherein said ramp of temperature comprises includes performing the following temperature steps:

incubation at 95° C. from 1 second to 60 seconds;

incubation at 60° C. from 30 seconds to 2 minutes; and

ramping up to 95° C. increasing the temperature 0.1° C./second or less.

9 . The method according to claim 1 , wherein the PCR amplification occurs in a real-time PCR machine or in a thermocycling machine able to acquire said signal emission each 0.1° C./second or less.

10 . The method according to claim 1 , wherein said PCR amplification is performed in a thermocycler or in a real-time PCR thermocycler, wherein, when the PCR amplification is performed in a thermocycler, HRM analysis is performed in a separate instrument, while when the PCR amplification is performed in a real-time PCR thermocycler, HRM analysis is performed in the same real-time PCR thermocycler.

11 . The method according to claim 1 , wherein two or more further sets of primers are used, wherein a first set of primers is present in a PCR mixture in one well, at least a second set of primers is present in a PCR mixture in another well, and wherein each set of primers comprises primers each recognizing one specific pathogen DNA target, wherein the pathogen DNA targets recognized by the first set of primers are the same as the pathogen DNA targets recognized by the second set of primers, wherein the melting temperature of an amplicon generated by a primer of one set of primers recognizing a specific pathogen DNA target is different from the melting temperature of an amplicon generated by a primer of the other set of primers recognizing said specific pathogen DNA target, and wherein each pathogen DNA target is defined by a single melting peak in each well.

12 . The method according to claim 1 , wherein each of the pathogens is responsible for a sexually transmitted disease or infection.

13 . The method according to claim 12 , wherein the pathogens are different types of human papilloma virus (HPV).

14 . The method according to claim 13 , wherein the amplification primers are selected from SEQ ID NO: 20 to SEQ ID NO: 616 and from SEQ ID NO: 655 to SEQ ID NO: 706.

15 . The method according to claim 12 , wherein the pathogens are selected from pathogens responsible for chlamydia, pathogens responsible for syphilis, or pathogens responsible for gonorrhoea.

16 . The method according to claim 15 , wherein said amplification primers include one or more primers selected from the following primers: SEQ ID NOs: 1-19 and SEQ ID NOs: 636-654.

17 . The method according to claim 1 , wherein a set of normalizing primers is further used for amplification of human genomic DNA serving as an internal PCR validation control and/or as a control for normalization of the amplified pathogen DNA obtained.

18 . The method according to claim 17 , wherein said amplified pathogen DNA targets include HPV DNA and a pair of said normalizing primers targets a fragment from the human beta-globin gene and is selected from the primers set forth in SEQ ID NO: 617 to SEQ ID NO: 629.