Back to results
US 8,012,719 B2Granted
Method for the quantitative detection of nucleic acids
OwnerPaolo Lusso, Mauro Malnati +1inventors
InventorsPaolo Lusso, Mauro Malnati, Giulia Cassina
Life of this patent
Estimating the term…
Abstract
Provided herein is a method for the quantitative detection of HHV-6 subtypes A and/or B based on the use of a calibrator, suitable primers and probes, and a nucleic acid polymerase with 5′-3′ nuclease activity.
Claims6
A method for the quantitative detection of a target nucleic acid from HHV-6 subtype variants A and/or B in a sample, which comprises the following steps:
- a) extraction of the nucleic acid from the sample with a calibrator previously added to the sample itself, wherein said calibrator is a nucleic acid containing two regions annealing to the primers SEQ ID NOS:
- 1 and 2, a third region annealing to the probe SEQ ID NO:
- 7 that is located in between said two regions and two spacers containing from 1 to 30 nucleotides located between the primer- and probe-annealing regions, respectively, wherein said spacers are mismatched with both HHV-6 A and B nucleic acid sequences;
- b) mixing the extracted target nucleic acid and calibrator with:
- i) the following primers pairs:
- a) SEQ ID NOs:
- 1 and 2 for the calibrator, and
- b) SEQ ID NOs:
- 3 and 4 for HHV-6 type A, and
- c) SEQ ID NOs:
- 5 and 6 for HHV-6 type B;
- ii) the following probes:
- SEQ ID NO:7 for the calibrator, SEQ ID NO:8 for HHV-6A and SEQ ID NO:9 for HHV-6B, wherein said probes carry a fluorescent reporter label able to generate a fluorescence signal and a quencher label at their 5′ and 3′ ends, respectively;
- iii) a nucleic acid polymerase with 5′-3′ nuclease activity;
- c) performing a polymerization reaction whereby a signal associated with the reporters label is detected;
- and
- d) quantification of the copy number of the extracted target nucleic acid and of the calibrator copy number recovered upon extraction.
14 more elements
Kit for the quantification of a HHV-6 subtype variants A and/or B nucleic acid from a sample, containing:
- i) a calibrator that is a nucleic acid containing two regions annealing to the primers SEQ ID NOS:
- 1 and 2, a third region annealing to the probe SEQ ID NO:
- 7 that is located in between said two regions and two spacers containing from 1 to 30 nucleotides located between the primers- and probe-annealing regions, respectively, wherein said spacers are mismatched with both HHV-6 A and B nucleic acid sequences;
- ii) the following primers pairs:
- a) SEQ ID NOs:
- 1 and 2, and
- b) SEQ ID NOs:
- 3 and 4, and
- c) SEQ ID NOs:
- 5 and 6;
- iii) the following probes:
- SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 (HHV-6B), wherein said probes carry a fluorescent reporter label and a quencher label at their 5′ and 3′ ends, respectively;
- iv) a nucleic acid polymerase with 5′-3′ nuclease activity.
10 more elements
Dependencies are inferred from the claim text.