Method for detection of human papillomavirus (HPV) type

A method for simultaneously detecting and/or typing and/or quantifying HPV 18, 52, 59, 39, 51, 56, 66, 16, 45, 58, 31, 33 and 35 in a biological sample, the method comprising the following four parallel reactions:

• Reaction 1
• a) amplification of nucleic acid fragments of HPV 18, 52 and 59 in the presence of
• (i) a nucleic acid polymerase
• (ii) a forward PCR primer with the oligonucleotide sequence of SEQ ID NO:
• 1,
• (iii) a reverse PCR primer with the oligonucleotide sequence of SEQ ID NO:
• 2, and
• (iv) probes comprising SEQ ID NO:
• 7, 8 and 9, each labelled with a different fluorophore and a quencher molecule;
• and
• b) detecting a change in fluorescence, the wavelengths and magnitude thereof determining the quantity and/or presence of HPV type 18, 52 and 59;
• Reaction 2
• a) amplification of nucleic acid fragments of HPV 39, 51, 56 and 66 in the presence of
• (i) a nucleic acid polymerase
• (ii) a forward PCR primer with the oligonucleotide sequence of SEQ ID NO:
• 1,
• (iii) a reverse PCR primer with the oligonucleotide sequence of SEQ ID NO:
• 3, and
• (iv) probes comprising SEQ ID NO:
• 10, 11, 12 and 13, each labelled with a different fluorophore and a quencher molecule;
• and
• b) detecting a change in fluorescence, the wavelengths and magnitude thereof determining the quantity and/or presence of HPV type 39, 51, 56 and 66;
• Reaction 3
• a) amplification of nucleic acid fragments of HPV 16, 45 and 58 in the presence of
• (i) a nucleic acid polymerase
• (ii) a forward PCR primer with the oligonucleotide sequence of SEQ ID NO:
• 1,
• (iii) a reverse PCR primer with the oligonucleotide sequence of SEQ ID NO:
• 4, and
• (iv) probes comprising SEQ ID NO:
• 14, 15 and 16, each labelled with a different fluorophore and a quencher molecule;
• and
• b) detecting a change in fluorescence, the wavelengths and magnitude thereof determining the quantity and/or presence of HPV type 16, 45 and 58;
• and
• Reaction 4
• a) amplification of nucleic acid fragments of HPV 31, 33 and 35 and an internal control in the presence of
• (i) a nucleic acid polymerase
• (ii) a forward PCR primer with the oligonucleotide sequence of SEQ ID NO:
• 1,
• (iii) a reverse PCR primer with the oligonucleotide sequence of SEQ ID NO:
• 5,
• (iv) probes comprising SEQ ID NO:
• 17, 18, 19 and 20, each labelled with a different fluorophore and a quencher molecule;
• and
• (v) an internal control;
• and
• b) detecting a change in fluorescence, the wavelengths and magnitude thereof determining the quantity and/or presence of HPV type 31, 33, 35.