| 主权项 |
1. 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; andb) 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; andb) 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; andb) 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; andb) detecting a change in fluorescence, the wavelengths and magnitude thereof determining the quantity and/or presence of HPV type 31, 33, 35. |
