Detection method using hrpzyme

A method for detecting a target DNA polynucleotide in a sample, comprising:

• (a) obtaining a target DNA polynucleotide from a sample and a control DNA polynucleotide, wherein the target DNA polynucleotide comprises a target DNA sequence, and wherein the control DNA polynucleotide lacks the target DNA sequence;
• (b) providing a first primer and a second primer, wherein the first primer comprises a first protector, the HRPzyme sequence of GGGTAGGGCGGGTTGGGT (SEQ ID NO:
• 1), a first spacer and a forward primer sequence for the target DNA sequence in the order from the 5′ end to the 3′ end of the first primer, wherein the second primer comprises a second protector, the HRPzyme sequence, a second spacer and a reverse primer sequence for the target DNA sequence in the order from the 5′ end to the 3′ end of the second primer;
• (c) performing a polymerase chain reaction (PCR) in a test mixture, wherein the test mixture comprises the target DNA polynucleotide, the first primer, and the second primer, whereby a PRC product is produced in the test mixture;
• (d) exposing the test mixture from step (c) to hemin, a substrate and hydrogen peroxide;
• (e) making a test color measurement in the test mixture from step (d);
• (f) performing a PCR in a control mixture, wherein the control mixture comprises the control DNA polynucleotide, the first primer, and the second primer, whereby no PRC product is produced in the control mixture;
• (g) exposing the control mixture from step (f) to hemin, a substrate and hydrogen peroxide;
• (h) making a control color measurement in the control mixture from step (g);
• and
• (i) comparing the test color measurement with the control color measurement, wherein a color difference between the test color measurement and the control color measurement indicates the presence of the target DNA polynucleotide in the sample.