In vitro method for detecting and quantifying HIV-2 DNA
The present invention relates to a method for detecting or quantifying deoxyribonucleic acid (DNA) of human immunodeficiency virus 2 (HIV-2) in a sample containing DNA comprising: a) performing a real-time polymerase chain reaction (PCR) on the sample, or a fraction thereof comprising DNA, with at least two sets of primers and probe each respectively comprising two primers and a labeled probe for the detection or quantification of HIV-2 DNA, at least one of the sets is selected from the group consisting of: a set comprising a primer comprising or consisting of a sequence SEQ ID NO: 1 or a sequence having al least 90% identity to SEQ ID NO: 1, a primer comprising or consisting of a sequence SEQ ID NO: 2 or a sequence having 90% identity to SEQ ID NO: 2 or the complement of these sequences, and a labeled probe comprising or consisting of a sequence SEQ ID NO: 3, or a sequence having at least 90% identify to SEQ ID NO: 3 or the complement of these sequences, and a set comprising a primer comprising or consisting of a sequence SEQ ID NO: 4 or a sequence having at least 90% identity to SEQ ID NO: 4, a primer comprising or consisting of a sequence SEQ ID NO: 5 or a sequence having 90% identity to SEQ ID NO: 5 or the complement of these sequences, and a labeled probe comprising or consisting of a sequence SEQ ID NO: 6 or a sequence having at least 90% identity to SEQ ID NO: 6 or the complement of these sequences, and b) determining therefrom the presence or absence and/or the quantity of HIV-2 DNA in the biological sample.
1. A method for detecting or quantifying deoxyribonucleic acid (DNA) of human immunodeficiency virus 2 (HIV-2) in a sample containing DNA comprising:
a) performing a real-time polymerase chain reaction (PCR) on the sample, or a fraction thereof comprising DNA, with at least two sets of primers and probe each respectively comprising two primers and a labeled probe for the detection or quantification of HIV-2 DNA, at least one of the sets being selected from the group consisting of:
a set comprising a primer comprising or consisting of a sequence SEQ ID NO: 1 or a sequence having at least 90% identity to SEQ ID NO: 1, a primer comprising or consisting of a sequence SEQ ID NO: 2 or a sequence having 90% identity to SEQ ID NO: 2 or the complement of these sequences, and a labeled probe comprising or consisting of a sequence SEQ ID NO: 3, or a sequence having at least 90% identity to SEQ ID NO: 3 or the complement of these sequences, and
a set comprising a primer comprising or consisting of a sequence SEQ ID NO: 4 or a sequence having at least 90% identity to SEQ ID NO: 4, a primer comprising or consisting of a sequence SEQ ID NO: 5 or a sequence having 90% identity to SEQ ID NO: 5 or the complement of these sequences, and a labeled probe comprising or consisting of a sequence SEQ ID NO: 6, or a sequence having at least 90% identity to SEQ ID NO: 6 or the complement of these sequences, and
b) determining therefrom the presence or absence and/or the quantity of HIV-2 DNA in the biological sample,
wherein the primers and probes are each present at a concentration between 300 and 500 nM.
2. The method according to claim 1 , wherein in step
a) of performing a real-time polymerase chain reaction (PCR) on the sample, or a fraction thereof comprising DNA, is performed with:
(i) at least 4 primers respectively comprising or consisting of:
a sequence SEQ ID NO: 1 or a sequence having at least 90% identity to SEQ ID NO: 1 or the complement of these sequences, and
a sequence SEQ ID NO: 2 or a sequence having 90% identity to SEQ ID NO: 2 or the complement of these sequences, and
a sequence SEQ ID NO: 4 or a sequence having 90% identity to SEQ ID NO: 4 or the complement of these sequences, and
a sequence SEQ ID NO: 5 or a sequence having 90% identity to SEQ ID NO: 5 or the complement of these sequences, and
(ii) at least 2 labelled probes respectively comprising or consisting of:
a sequence SEQ ID NO: 3, a sequence complement to SEQ ID NO: 3, or a sequence having at least 90% identity to SEQ ID NO: 3 or the complement thereof, and
a sequence SEQ ID NO: 6, a sequence complement to SEQ ID NO: 6, or a sequence having at least 90% identity to SEQ ID NO: 6 or the complement thereof.
3. The method according to claim 1 , wherein the sample containing DNA is a biological sample or a sample comprising cells infected in vitro by HIV-2.
4. The method according to claim 1 , wherein the primers respectively consist of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5, and the labeled probes respectively consist of sequences SEQ ID NO: 3 and SEQ ID NO: 6.
5. The method according to claim 1 , wherein the labelled probes are labelled with 6-carboxyfluorescein (FAM) at their 5′end and with Black Hole Quencher-1 (BHQ1) at their 3′ end.
6. The method according to claim 1 , wherein at least one DNA virus or at least one DNA molecule having essentially no sequence similarity with the genomic sequence of HIV-2 is added to the sample or the fraction thereof, as well as primers and probe necessary for its amplification and detection by real-time PCR, as an internal control of extraction and/or inhibition.
7. The method according to claim 1 , wherein the PCR comprises the following thermocycling conditions:
2 min at 50° C., followed by
10 min at 95° C., followed by
50 cycles of 95° C. for 15 s and 60° C. for 1 min.
8. The method according to claim 1 , further comprising determining or quantifying HIV-1 nucleic acids in the sample or a fraction thereof.
9. A kit or a mix for detecting or quantifying HIV-2 DNA, comprising:
a) at least two sets of primers and probe each respectively comprising two primers and one probe for the detection or quantification of HIV-2 DNA, the primers and probe being each present at a concentration between 300 and 500 nM, and at least one of the sets being selected from the group consisting of:
a set comprising a primer comprising or consisting of a sequence SEQ ID NO: 1 or a sequence having at least 90% identity to SEQ ID NO: 1, a primer comprising or consisting of a sequence SEQ ID NO: 2 or the complement of these sequences, and a labelled probe comprising or consisting of a sequence SEQ ID NO: 3, or a sequence having at least 90% identity to SEQ ID NO: 3 or the complement of these sequences, and
a set comprising a primer comprising or consisting of a sequence SEQ ID NO: 4 or a sequence having at least 90% identity with SEQ ID NO: 4, a primer comprising or consisting of a sequence SEQ ID NO: 5 or a sequence having 90% identity to SEQ ID NO: 5 or the complement of these primers, and a labelled probe comprising or consisting of a sequence SEQ ID NO: 6, or a sequence having at least 90% identity to SEQ ID NO: 6 or the complement of these sequences, and
b) at least one DNA virus or at least one DNA molecule having essentially no sequence similarity with the genomic sequence of HIV-2, as well as primers and probe necessary for its amplification and detection by real-time PCR, as an internal control of extraction and/or inhibition, and
c) optionally additional reagents for performing a PCR.
10. The kit or the mix according to claim 9 , comprising:
a1) at least 4 primers, at a concentration between 300 and 500 nm, respectively comprising or consisting of:
a sequence SEQ ID NO: 1 or a sequence having at least 90% identity to SEQ ID NO: 1 or the complement of these sequences, and
a sequence SEQ ID NO: 2 or a sequence having 90% identity to SEQ ID NO: 2 or the complement of these sequences, and
a sequence SEQ ID NO: 4 or a sequence having 90% identity to SEQ ID NO: 4 or the complement of these sequences, and
a sequence SEQ ID NO: 5 or a sequence having 90% identity to SEQ ID NO: 5 or the complement of these sequences, and
a2) at least 2 labelled probes at a concentration between 300 and 500 nM, respectively comprising or consisting of:
a sequence SEQ ID NO: 3, a sequence complement to SEQ ID NO: 3, or a sequence having at least 90% identity to SEQ ID NO: 3 or the complement thereof, and
a sequence SEQ ID NO: 6, a sequence complement to SEQ ID NO: 6, or a sequence having at least 90% identity to SEQ ID NO: 6 or the complement thereof.
11. The kit or the mix according to claim 9 , further comprising primers and at least one labelled probe for detecting or quantifying HIV-1 nucleic acids.
12. An in vitro method for determining HIV-2 viral load, in an individual, comprising the steps of:
(c) performing the method for detecting or quantifying HIV-2 DNA in a biological sample taken from the individual as defined in claim 1 ; and
(d) determining therefrom the HIV-2 viral load of the individual.