WO2008136033A2

WO2008136033A2 - Method for the genotyping of hiv-1 and relevant diagnostic kits for the detection of drug resistances

Info

Publication number: WO2008136033A2
Application number: PCT/IT2008/000298
Authority: WO
Inventors: Fabio Tramuto; Nino Romano; Francesco Vitale
Original assignee: Universita' Degli Studi Di Palermo
Priority date: 2007-05-03
Filing date: 2008-05-02
Publication date: 2008-11-13
Also published as: ITRM20070254A1; WO2008136033A3

Abstract

The invention concerns a method for HIV-1 genotyping by gp41-env gene sequencing for the detection of mutations associated to resistances to fusion inhibitors antiretroviral drugs and related diagnostic kit.

Description

METHOD FOR THE GENOTYPING OF HIV-1 AND RELEVANT DIAGNOSTIC KITS FOR THE DETECTION OF DRUG RESISTANCES

The present invention concerns a method for genotyping human immunodeficiency virus type 1 (HIV-1) through gene sequencing of gp41- env and related diagnostic kits for the detection of resistance to antiretroviral drugs active to gp41-env (i.e. fusion inhibitors).

The clinical significance of resistance to antiretroviral drugs in the human immunodeficiency virus type 1 (HIV-1) infection is a much studied and well known phenomenon. HIV-1 seropositive patients, infected with HIV-1 strainsof which is documented the drug-resistance activity on the base of genotypic/phenotypic assays, if not properly treated develop an adequate virological response with difficulties (Deeks S. G et al., 1999; Zolopa A.R et al., 1999). On the contrary, the use of drugs for which the virus is sensitive, increases the probability to re-establish the virologic control in patients, where previous therapeutic schemes were not effective (DeGruttola V., et al, 2000).

The resistance to anti-retroviral drugs is a phenomenon associated with mutations, well characterised at the level of genetic segments of HIV- 1 encoding proteins that are the natural target of drugs currently used in clinical practice. On the base of pharmacological classes actually used, resistance to antiretroviral drugs is associated with mutations in the protease and retro-transcriptase domains of the HIV-1 pol gene (Eshleman S. H., et al., 2005). The use of genotyping assays for the detection and evaluation of these mutations resulting in resistance to antiretroviral drugs in HIV-1 virus genes (i.e pol) is much diffused in the clinical management of HIV-1 infection and particularly economic also (Zolopa A.R et al., 2006, Smith D et al., 2007). Two types of HIV-1 genotyping kits based on HIV-1 pol gene sequencing are commercially available, i.e. "ViroSeq Genotyping System" (Abbott/Celera Diagnostics) and "TRUGENE™ HIV-1 Genotyping System" (Bayer). These commercial diagnostic kits have a sensitivity limit of 10³ copies of HIV-RNA/mL, so it is difficult to detect mutations in samples with low viremia.

The international guidelines show that a proper use of the resistance genotypic tests may improve the clinical outcomes of HIV- infected patients; more recently, panel of experts in the field have reevaluated the recommendations on the use of such tests, suggesting a wider application in all clinical steps of the infection (Hirsch MS et al., 2003, Vandamme AM et al., 2004).

In the light of such evidences, it is considered that the use of the genotypic tests can be considered as a growing segment of diagnostics in the coming years, both in terms of spread on worldwide territory, and the number of tests requested.

Recently new anti-retroviral drugs (called fusion inhibitors) were developed directed towards a target different from the traditional genes gag/pol, that is the gene encoding for the gp41 protein within env gene; so it is expected that the use of such drugs over time leads to the development of drug resistances and, therefore, they need a fast and effective monitoring. To date, on the market there are no kits and/or methods available that allow genotyping the gp41-env gene of HIV-1.

It is evident the need to provide methods of genotyping HIV-1 that are applicable to all viral variants atually known and plasma samples with very low viremia (high sensitivity).

The authors of the present invention have now developed a method and related diagnostic kit for genotyping HIV-1 through amplification and sequencing of the gp41-env gene at high sensitivity that allows the analysis of low viremia samples at around 10¹-10² copies of HIV-RNA/mL (see Figure 5) and then lowering the limit of sensitivity of current commercial diagnostic systems (10³ copies of HIV-RNA/mL) aimed at genotyping of several genetic regions of HIV-1 (i.e pol).

This method also offers the advantage of being easily adaptable to both plasma and lymphocyte samples and is therefore able to value "in advance" the genetic mutations potentially involved in the resistance phenomena to anti-retroviral drugs, even in patients with optimal virological control (viral load < 47 copies of HIV-RNA/mL; an internationally accepted sensitivity limit), otherwise not able to undergo genotyping (presuntive resistances).

Moreover, taking advantage of one-step RT-PCR technologies, the method offers very short processing times as to operator and PCR protocol in the order of 5 hours.

Finally, the use of one-step method for cDNA reverse-transcription and successive amplification in dsDNA, limits the operator intervention and reduces the risk of sample contamination. It is, therefore, an object of the present invention a method for genotyping HIV-1 comprising the following steps: a) HIV-1 viral RNA reverse-transcription from a biological sample, using specific anti-sense primer consisting of nucleotide sequence 5'-TGC TTW TAT GCA GCA TCT GAG GG-3¹ (SEQ ID NO: 1) (or alternatively 5'- GAA AGT CCC CAG CGG AAA GTC C-3¹ (SEQ ID NO: 2)) and subsequent amplification of dsDNA fragment through PCR reaction, using a pair of external target-specific primers consisting in the nucleotide sequences selected from the group consisting of: a1) Fw 5'-AGT TTY AAT TGT RGA GGR GAA TTT-3' (SEQ ID NO:

3)/ Rv 5'-TGC TTW TAT GCA GCA TCT GAG GG -3" (SEQ ID NO: 4); or alternatively: a2) Fw 5'-AGT TTT AAT TGT RGA GGR GAA TTT-3¹ (SEQ ID NO: 5)/Rv 5'-GAA AGT CCC CAG CGG AAA GTC C-3' (SEQ ID NO: 6); wherein the reverse-transcription and amplification occur through in a single step; b) sequencing of the amplicon obtained in step a) in independent reactions using each of the following primers consisting in the following nucleotide sequences: b1) Fw 5'- TTR AAC CAY TAG GAR TAG CAC CCA-3' (SEQ ID

NO: 7) or alternatively, Fw 5'-TTG AAC CAY TAG GAG TAG CAC CCA-3¹ (SEQ ID NO: 8); b2) Fw 5'-GGC AAA GAG AAG AGT GGT-3¹ (SEQ ID NO: 9) or alternatively, Fw 5'-GGC AAR RAG AAG AGT GGT-3¹ (SEQ ID NO: 10); b3) Fw 5'-TTG GGG YTG CTC TGG AAA AC-3¹ (SEQ ID NO: 11) or alternatively, Fw 5'-TTG GGG YTG CTC TGG AAA-3¹ (SEQ ID NO: 12); b4) Fw 5'-ATA ATG ATA GTA GGA GG-3¹ (SEQ ID NO: 13); b5) Fw 5'-GGA RCC TGT GCC TCT TCA-3¹ (SEQ ID NO: 14); b6) FW 5'-TAC CTA GAA GAA TMA GAC A-3¹ (SEQ ID NO: 15); b7) Rv 5'-TCT YAT TCT TTC CCT WA-3¹ (SEQ ID NO: 16) or alternatively, Rv 5'-TCT YAT TCT TTC CCT TA-3' (SEQ ID NO: 17); bδ) Rv 5'-GTC CCA GAA GTT CCA CA-3' (SEQ ID NO: 18); b9) Rv 5'-TYT TAT ATA CCA YAG CCA-3' (SEQ ID NO: 19); b10) Rv 5'-GTA TCT TTC YAC AGC CAG-3¹ (SEQ ID NO: 20) or alternatively, Rv 5'-GTA TCT TTC YAY AGC CAG-3¹ (SEQ ID NO: 21). c) assembling of the internal segments obtained in step b) in a consensus sequence. According to an embodiment of the method according to the invention, when there is no RT-PCR amplification in step a), it is possible in order to improve the sensitivity that the step a) further comprises an amplification step through a nested-PCR, using a pair of internal target- specific primers selected from the following nucleotide sequences: c1) Fw 5'-TTR AAC CAY TAG GAR TAG CAC CCA-3¹ (SEQ ID NO: 22)/Rv 5'-TAC TAG CTT GWA GCA CCA RCC AA-3¹ (SEQ ID NO: 23); or alternatively, c2) Fw 5'-TTG AAC CAY TAG GAG TAG CAC CCA-3¹ (SEQ ID NO: 24)/Rv 5'-GTC AGC AGT CCT TGT AGT ACT CCG GAT-3¹ (SEQ ID NO: 25).

Based on the IUPAC-IUB nomenclature the following letters identify the contemporary presence of different nucleotides: R (A or G)-Y (C or T)- M (A or C)-K (G or T)-S (G or C)-W (A or T)-H (A or C or T)-B (G or T or C)-V (G or C or A)-D (G or T or A)-N (G or A or T or C).

This genotyping method may find a useful practical application as a method for identifying mutations in the gp41-env gene that may confer resistance to anti-retroviral drugs. In this case the step c) of the genotyping method comprising previously described a)-c) steps, consists in the production of a consensus sequence obtained from the different nucleotide generated segments and in its comparison with reference sequences, for the interpretation of mutations present in the gp41-env gene which are eventually associated with resistance to anti-retroviral drugs (according to the credited scientific knownledge). At the web site http://www.iasusa.org, heading publications

August/September 2006 (Volume 14, Issue 3), it is possible to download the last survey of mutations for which, up to now, is known the association to pharmacological resistance (Johnson VA et al. 2006).

The method according to the invention is very versatile and can be used on different biological samples, including plasma and lymphocyte pellet.

It is an object of the present invention a diagnostic kit comprising the following components: a) at least one primer pair for amplification of the dsDNA fragment by RT-PCR, consisting in the nucleotide sequences selected from: a1) Fw 5'-AGT TTY AAT TGT RGA GGR GAA TTT-3¹ (SEQ ID NO: 3)/Rv 5'-TGC TTW TAT GCA GCA TCT GAG GG -3' (SEQ ID NO: 4); or alternatively: a2) Fw 5'-AGT TTT AAT TGT RGA GGR GAA TTT-3¹ (SEQ ID NO: 5)/Rv 5'-GAA AGT CCC CAG CGG AAA GTC C-3¹ (SEQ ID NO: 6); b) a set of primers for the sequencing of the amplicon obtained by amplification with primers a1) or a2) consisting in the following nucleotide sequences: b1) Fw 5'-TTR AAC CAY TAG GAR TAG CAC CCA-3' (SEQ ID NO: 7) or alternatively, Fw 5'-TTG AAC CAY TAG GAG TAG CAC CCA-3¹ (SEQ ID NO: 8); b2) Fw 5'-GGC AAA GAG AAG AGT GGT-3¹ (SEQ ID NO: 9) or alternatively, Fw 5'-GGC AAR RAG AAG AGT GGT-3' (SEQ ID NO: 10); b3) Fw 5'-TTG GGG YTG CTC TGG AAA AC-3¹ (SEQ ID NO: 11) or alternatively, Fw 5'-TTG GGG YTG CTC TGG AAA-3¹ (SEQ ID NO: 12); b4) Fw 5'-ATA ATG ATA GTA GGA GG-3¹ (SEQ ID NO: 13); b5) Fw 5'-GGA RCC TGT GCC TCT TCA-3 (SEQ ID NO: 14); b6) Fw 5'-TAC CTA GAA GAA TMA GAC A-3¹ (SEQ ID NO: 15); b7) Rv 5'-TCT YAT TCT TTC CCT WA-3' (SEQ ID NO: 16) or alternatively, Rv 5'-TCT YAT TCT TTC CCT TA-3¹ (SEQ ID NO: 17); b8) Rv 5'-GTC CCA GAA GTT CCA CA-3¹ (SEQ ID NO: 18); b9) Rv 5'-TYT TAT ATA CCA YAG CCA-3¹ (SEQ ID NO: 19); b10) Rv 5'-GTA TCT TTC YAC AGC CAG-3¹ (SEQ ID NO: 20) or alternatively, Rv 5'-GTA TCT TTC YAY AGC CAG-3¹ (SEQ ID NO: 21).

According to a preferred embodiment of the kit, the invention comprises a further pair of primers for the amplification of more internal segment through nested-PCR, selected from the following nucleotide sequences: c1) Fw 5'-TTR AAC CAY TAG GAR TAG CACC CA-3' (SEQ ID NO: 22)/Rv 5'-TAC TAG CTT GWA GCA CCA RCC AA-3¹ (SEQ ID NO: 23); or alternatively, c2) Fw 5'-TTG AAC CAY TAG GAG TAG CAC CCA-3¹ (SEQ ID

NO: 24)/Rv 5'-GTC AGC AGT CCT TGT AGT ACT CCGGAT-3¹ (SEQ ID NO: 25).

The present invention will be now described by way of illustration, but not limitation, according to preferred embodiments thereof, with particular reference to the enclosed drawings, wherein:

Figure 1 shows the pattern of reaction one-step RT-PCR and nested-PCR; Figure 2 shows the map of amplified genetic segment of gp41-env gene ;

Figure 3 shows the pattern of contribution of "primer sequence" in achieving the final consensus sequence; Figure 4 shows a summary scheme of laboratory procedures applied in the genotyping resistance test of gp41-env gene;

Figure 5, shows electrophoresis agarose gel of a panel of samples obtained by 1 :10 serial dilutions of a reference sample with known viral concentration; legend: 1 kb) Reference standard of molecular weight r;

-) Negative sample (H1V-1 negative human plasma); 1 :1) Whole sample. Viraemia: 110,000 copies of HIV-1 RNA/mL;

1 :10) diluted sample. Viremia estimated: 11 ,000 copies of HIV-1 RNA/mL;

1 :100) diluted sample. Viremia estimated: 1 ,100 copies HIV-1 RNA/mL;

1 :1000) diluted sample. Viremia estimated: 110 copies of HIV-1 RNA/mL; 1 :10.000) diluted sample. Viremia estimated: 11 copies of HIV-1 RNA/mL;

1:100.000) diluted sample. Viremia estimated: 1.1 copies of HIV-1 RNA/mL; p.HIVI) positive sample (HIV-1 near-full genome plasmid).

EXAMPLE 1 : Development of the method of HIV-1 genotyping The method, after nucleic acid extraction, is based on original reverse-transcription of HIV-1 viral RNA to a complementary DNA (cDNA) and on the subsequent amplification of a double-stranded DNA fragment (dsDNA).

For this purpose, in one step (One-step Reverse Transcription- Polimerase Chain Reaction), a single reaction tube is used both for the reaction of the reverse-transcription, using a specific "anti-sense primer" called FT1gp41_Rev, and for the subsequent PCR amplification of a dsDNA "target", using a pair of target-specific primers (external primer, called FT1gp41_For and FT1gp41_Rev) (Figure 1a and 3). The test uses a facultative second step of amplification (nested-PCR), to improve its sensitivity, using a different pair of target-specific primers (internal primers, called FT2gp41_For and FT2gp41_Rev) (Figure 1b and 3). The details of the primer sequences are depicted in Table 1.

As an alternative to FT1gp41_For /FT1gp41_Rev and FT2gp41_For/FT2gp41_Rev, FT1gp41.b_For/FT1gp41.-b_Rev and FT2gp41.b_For/FT2gp41.b_Rev primer pairs can be used (Table 1).

The obtained PCR product consists in a fragment of about 1720 nucleotide base pairs (bp), corresponding to a region located between

HIV-1 env gene and 3'-LTR, which includes the whole gp41-env domain (Figure 2). In particular, the proposed method amplifies a genetic fragment included between the nucleotide positions 7300 and 9500 (referring to the

HIV-1 subtype B sequence, HXB2 - GenBank Accession number K03455), which totally includes the gp41 env domain (see Figure 2). Although clinically mutations associated with resistance to fusion inhibitors must be sought in HR1 domain of gp41-env gene, were recently described mutations in the HR2 domain potentially involved in the susceptibility to these drugs (Xu L et al. 2004; Poveda E et al. 2002); therefore it has been chosen to assess the gp-41-env gene in its entirety.

The method can be applied indiscriminately: - to plasma samples, for the detection of viral RNA circulating variants (expression of viral replication);

- to cellular samples, for the detection of viral cDNA variants integrated at lymphocyte level, even in subjects with effective virological control (viral load <47 copies HIV-RNA/mL) (search for "presumptive resistances").

The PCR fragment obtained is then sequenced, using "Big Dye terminator Cycle Sequencing" technology, to show in detail the nucleotide composition (genotyping); at this purpose, a set of 11 different " sequence primers" called FT2gp41_For (or FT2gp41.b_For), FT2gp41_Rev (or FT2gp41.b_Rev), gp41Seq1_For (or gp41Seq1.b_For), gp41Seq2_For (or gp41Seq2.b_For), gp41Seq3_For, gp41Seq4_For, gp41Seq5_For, gp41Seq6_Rev (or gp41Seq6.b_Rev), gp41Seq7_Rev, gp41Seq8_Rev and gp41Seq9_Rev (or gp41Seq9.b_Rev) are used (Table 1).

Table 1

Therefore, independent reactions conducted each of different primer, allow to obtain nucleotide sequences that, compared to a reference sequence, are partially overlapped and oriented in both directions (forward and reverse) (Figure 3).

The obtained segments are "assembled" in a "consensus" sequence that can be compared to the HIV-1 gp41-env gene mutations of which is known their association (or is being to validate) with resistance to fusion inhibitors. Also, the system is able to evaluate the corresponding viral genotype.

An overview of the laboratory procedure is depicted in Figure 4. The following describes in detail the laboratory procedure applied for genotyping the HIV-1 gp41 env gene. It should be considered as a simple example of the invention and not as a limitation.

EXPERIMENTAL PROCEDURE The following protocol is used for the amplification of a region of about 1.7 kb of the env-gp41 gene of HIV-1. Step 1 - RNA preparation:

Plasma samples, either fresh or thawed at room temperature, were mixed for agitation through vortex, at low speed for 3-5 seconds, and then briefly centrifuged to collect the sample at the bottom of the tube.

For the viral RNA extraction was preferred to adopt commercial kit based on column with silica-gel membrane.

Extraction from plasma/serum or other acellular fluids. The extraction kit is designed for the rapid, efficient and at highly sensitive recovery of DNA/RNA, from plasma or serum and is able to heavily concentrate nucleic acids, even when present at extremely low level. Contaminants and enzyme inhibitors are efficiently removed with the purification procedure based on membranes of silica-gel without the use of phenol, chloroform or other organic solvents.

The kit allows to obtain viral DNA/RNA at high concentration, from 1 mL of plasma or serum sample equilibrated to room temperature (15- 25°C) into a 2 mL microcentrifuge tubes. Samples smaller than 1 mL (> 200 μL) can be adjusted to 1 mL using PBS solution (phoshate-buffered saline). In the sample, are added 800 μL of buffer containing a reagent able to constitute complexes with nucleic acids which can sediment by centrifugation at low force of gravity to form a pellet. To facilitate the recovery of nucleic acids at very low title, 5.6 μL of RNA carrier solution (1 ng/μL) is added to the surface of the sample. The tube with the mixture sample/reagents is mixed thoroughly by fisrt inverting the microcentrifuge tube (3 times) and then by vortexing (10 s); therefore, put to incubate at room temperature (15-25⁰C) for 10 min.

After centrifugation at 1200xg for 3 min, the supernatant is completely removed and both 300 μL of a lysis buffer (pre-warmed to 6O⁰C) containing guanidine hydrochloride and 20 μL of a proteinase K solution are added to the solution. The sample, mixed on a vortex until the pellets at the bottom is completely resuspended, is incubated in agitation at 40⁰C for 10 min.

Then, a brief centrifugation is applied to remove the drops at the top of the tube and 300 μl_ of a solution containing ethanol and a non-ionic surfactant type Nonidet P40 are added to the sample, mixed on vortex and briefly centrifuged.

The mixture of lysed sample is applied on a column containing a silica-gel membrane and centrifuged for 1 min at 3000-5000xg.

To wash and purify the extracted DNA/RNA, the filtrate is eliminated and the column is placed in a clean collection tube. After adding 500 μl_ of a solution containing ethanol and guanidine hydrochloride, the column is centrifuged for 1 minute at 6000xg.

The eluate is eliminated and the column is placed in a clean collection tube. After adding 500 μl_ of a solution containing ethanol, the column is centrifuged for 3 min at 20000xg.

To ensure the drying of the membrane, after elimination of the filtrate, centrifugation is repeated for 3 min at 20000xg.

After removal of the tube containing the filtrate, the column is placed in a new sterile tube, 30 μL of elution buffer are added, centrifuged for 1 min at 6000xg and, the purified DNA/RNA recovered.

The purified DNA/RNA can be used immediately or kept at -80⁰C.

Alternatively, the viral DNA integrated into the human genomic DNA can be assessed after extraction from lymphocyte pellets (PBMCs), with a similar extraction procedure in the presence of proteinase K and subsequent purification/washing with ethanol.

Step 2 - One step RT-PCR

Reverse transcription and polymerization reactions (first step of

PCR) are conducted in a single step and in the same tube (0.2 ml_) on a thermal cycler type Applied Biosystems GenAmp ® PCR System 9700, in order to simplify the laboratory procedures, to increase the sensitivity of the method and to minimise the risk of contamination.

For RT-PCR reaction a commercial kit was chosen specifically designed for the analysis and endpoint detection of RNA molecules by RT- PCR, with high sensitivity and high fidelity.

The system uses a mixture of Superscript III ® Reverse Transciptase ® and Platinum Taq DNA Polymerase High Fidelity (Invitrogen) for high yields and fidelity of RT-PCR, even from long templates.

The reaction conditions are as follows: a) buffer reaction containing dATP, dCTP, dGTP and dTTP (each a final concentration of 0.2 mM) and MgSO₄ (final concentration 1.7 mM); b) forward primer (FT1gp41_For) and anti-sense (FT1gp41_Rev) (each a final concentration of 0.4 μM), c) RNA template (> 1 pg) d) RNAse free H₂O to a total of 50 μL Thermal protocol:

52°C 60 min

94°C 2 min

10 cycles:

94°C 15 sec 52°C 30 sec

68°C 3 min

30 cycles:

94°C 15 sec

52°C 30 sec 68°C 3 min + 5 sec/cycle

68°C 10 min

4°C °°

After checking whether the amplification occurred (see Step 4), the obtained dsDNA can be used for the next steps or stored at -2O⁰C. When the single step RT-PCR is not sufficient to provide an adequate amplification, a second step of PCR (nested-PCR) using the "inner primers" (forward, FT2gp41_For or, alternatively, FT2gp41.b_For, and anti-sense, FT2gp41_Rev or, alternatively, FT2gp41.b_Rev), according to the protocol which follows.

Step 3 - nested-PCR

The nested-PCR reaction is conducted using the DNA polymerase

® Platinum Taq DNA Polymerase High Fidelity (5 U/μL) (Invitrogen), which consists of a mixture of Taq Polymerase recombinant DNA, Pyrococcus species GB-D polymerase (with proofreading exonuclease activity) and

Platinum Taq ® Antibody. This enzyme, allow to use a "hot-start" method that prevents polymerization at room temperature, with high yields of DNA, high sensitivity and specificity, even from long templates. The reaction conditions are as follows: a) buffer reaction containing: 600 mM TrJs-SO₄ - pH 8.9, 180 mM ammonium sulfate; b) dATP, dCTP, dGTP and dTTP (each a final concentration of 0.2 mM); c) MgSO₄ (final concentration 1.7 mM); d) sense primer (FT2gp41_For or, alternatively, FT2gp41.b_For) and antisense (FT2gp41_Rev or, alternatively, FT2gp41.b_Rev) (each a final concentration of 0.4 μM); e) Platinum® Taq DNA Polymerase High Fidelity (2 U); f) dsDNA template (RT-PCR) 5 μl_; g) RNAsi free H₂O to a total of 50 μl_. Thermal protocol:

94°C 2 min

10 cycles:

94°C 15 sec

58°C 30 sec 68°C 3 min

20 cycles:

94°C 15 sec

58°C 30 sec

68°C 3 min + 5 sec/cycle 68°C 10 min

4°C °°

After checking whether the amplification occurred (see Step 4), the obtained dsDNA can be used for the next steps or stored at -20⁰C.

Step 4 - purification and analysis of PCR products

The PCR products obtained are then purified and displayed by agarose gel electrophoresis, before the subsequent genotype analysis. Procedure:

300 μl_ of sterile H₂O and 50 μl_ of the RT-PCR/nested-PCR product are applied to a Microcon 100 (Millipore) column and, centrifuged for 15 minutes at 550xg. The filtrate is discarded and the Microcon 100 column is placed in inverted position on a clean 1.5 mL Eppendorf tube with 35 μl_ of sterile H₂O on the membrane. After centrifugation for 5 minutes at 550xg, the dsDNA purified sample is transferred to a clean tube.

A sample aliquot (5-10 μl_), mixed with a loading gel electrophoresis buffer (containing 40% sucrose w/v, bromophenol blue 0.25% w/v, xylene- cyanol FF 0.25% w/v), is analyzed on 1% agarose gel containing 0.5 μg/mL of ethidium bromide. Amplified products are compared to a standard reference - High mass DNA ladder (Invitrogen, DNA markers fragments of 10, 6, 4, 3, 2 and 1 kb. Electrophoresis of 4 μL of High Mass DNA Ladder produces bands each containing 200, 120, 80, 60, 40 and 20 ng - 520 ng of total DNA). PCR products of the target sequence undergoes to electrophoresis at 10 V/cm for 45-60 min. After electrophoresis the gel is illuminated with an UV light and, the amplified target are displayed corresponding at the 3 kb band (between 2 kb and 3 kb).

Before continuing with the genotyping, the amplified sample is diluted with sterile H₂O in proportion to the intensity of the band displayed on the gel. If the intensity of the band is less than those corresponding to 3 kb and 4 kb, the amplified sample may not be sufficient for subsequent sequencing procedure.

Step 5 - sequencing of amplified target sequence (genotyping)

Genetic sequencing of the whole fragment, can be obtained using up to 11 different primers (Figure 3 and Table 1) identified with labels FT2gp41_For (or FT2gp41.b_For), FT2gp41_Rev (or FT2gp41.b_Rev), gp41Seq1_For (or gp41Seq1.b_For), gp41Seq2_For (or gp41Seq2.b_For), gp41Seq3_For, gp41Seq4_For, gp41Seq5_For, gp41Seq6_Rev (or gp41Seq6.b_Rev), gp41Seq7_Rev, gp41Seq8_Rev and gp41Seq9_Rev (or gp41Seq9.b_Rev) (Table 1). The primers tagged with "b" should be considered an alternative to the original one.

To the purpose, independent reactions (separated 0.2 ml_ tubes) are conducted on a thermal cycler type Applied Biosystems GenAmp ® PCR System 9700, adding each primer sequence in a mixture of Big Dye Terminator ® Ready Mix Reaction (Big Dye ® Terminator v3.1 Cycle Sequencing Kit, Applied Biosystems) to the final concentration of 3.2 pmol (according to the manufacturer's specifications). The thermal protocol is as follows: 96°C 1 min 25 cycles: 96°C 10 sec 50⁰C 5 sec 6O⁰C 4 min

4°C ∞

Step 6 - purification and analysis of products sequence

Sequences are purified using the Centri-Sep™ columns (Princeton Separation, consisting of resin Sephadex™ G-50 Fine - Amersham), to remove excess of terminators Big Dye ®, salts and impurities at low molecular weight.

Sequences are pre-emptively treated with SDS (2.2% sodium dodecyl sulphate solution) by adding 2 μl_ of SDS to each of 20 μl_ sequencing product (final concentration 0.2%) and submitting the mixture to a thermal cycle at 98°C for 5 min followed by 10 min at 25°C.

Then, the mixture is purified using a Centri-Sep™ column or by alternative procedures, as recommended by the manufacturer

(Ethanol/EDTA, Ethanol/EDTA/Sodium acetate). By using the Hi-Di™ Formamide, the purified sequencing products are denatured by heating and analyzed through a capillary electrophoresis automatic sequencer.

The consensus sequences are compared with HIV-1 "wild-type" pol sequences for the evaluation of mutations associated with resistance to antiretroviral drugs and any possible polymorphisms.

Sensitivity of the method Electrophoresis Different agarose gel electrophoresis runs were conducted on a panel of experimental samples obtained by serial dilutions (10-fold) of a sample with a fixed viral concentration. The panel includes the following samples:

1kb) Reference standard of molecular weight;

-) Negative sample (HIV-1 negative human plasma);

1 :1) Whole sample. Viraemia (dispensed): 110,000 copies HIV-1 RNA/mL; 1 :10) Diluted sample. Viraemia (estimated): 11 ,000 copies HIV-1 RNA/mL;

1 :100) Diluted sample. Viraemia (estimated): 1 ,100 copies HIV-1 RNA/mL;

1 :1.000) Diluted sample. Viraemia (estimated): 110 copies HIV-1 RNA/mL; 1 :10.000) Diluted sample. Viraemia (estimated): 11 copies HIV-1 RNA/mL; 1 :100.000) Diluted sample. Viraemia (estimated): 1.1 copies HIV-1 RNA/mL; p.HIVI) Positive sample (HIV-1 near-full genome plasmid). The estimated viraemia of each diluted sample was also determined using a diagnostic commercial system US FDA-approved based on "real-time" technology and with a sensitivity of 47 HIV-1 RNA/mL copies.

The high sensitivity of tests, checked in this experimental phase (see Figure 5), can be eventually improved by a subsequent amplification- nested PCR step able to genotyping plasma samples with very low viraemia (10¹-10² HIV-RNA/mL copies), lowering the sensitivity of current commercial diagnostic systems directed to other genetic regions (10³ HIV- RNA/mL copies). The present invention has been described by an illustrative, but not limitative way, according to preferred embodiments thereof, but it is to be understood that variations/improvements (i.e reduction of working times, PCR thermal profile, calibration of chemical concentrations of used reagents, etc.) can be carried out by those skilled in the art without departing from the scope thereof, as defined in the enclosed claims.

BIBLIOGRAPHY

- Deeks S. G., Hllmann N.S., Grant R.M., Parkin NT., Petropoulos CJ. , Becker M., Symonds W., Chesney M., Volberding P.A. J. Infect. Dis. 1999; 179:1375-1381.

- Zolopa A.R., Shafer R.W., Warford A., Montoya J.G., Hsu P., Katzenstein D., Merigan T.C., Efron B. Ann. Intern. Med. 1999; 131 :813- 821.

- DeGruttola V., Dix L., D'Aquila R., Holder D., Philips A.N., AitKhaled M., Baxter J. D., Clevenbergh P., Hammer S., Harrigan R.P.,

Katzenstein D., Lanier R., Miller M. D., Para M., Yerly S., Zolopa A.R., Murray J. S., Patick A., Miller V., Castillo S., Pedneault L₁ Mellors J. Antivir. Ther. 2000; 5:41-48.

- Eshleman S. H., Crutcher G., Petrauskene O., Kunstman K., Cunningham S. P., Trevino C₁ Davis C₁ Kennedy J., Fairman J., Foley B.,

Kop J. Journal of Clinical Microbiology, 2005; 43:813-817. - Xu L, Pozniak A, Wildfire A, et a/. Antimicrob Agents Chemother 2005; 49:1113-1119.

- Kliger Y, Shai Y. J MoI Biol 2000; 295:163-168.

- Rimsky LT₁ Shugars DC, Matthews TJ. J Virol 1998; 72:986-993. - Derdeyn CA, Decker JM, Sfakianos JN. et al. J Virol 2000,

74:8358-8367.

- Greenberg ML, Cammack N, Salgo M, et al. Rev Med Virol 2004; 14:1-17.

- Kemp SD, Ruiz L, Lucas AM, et al. Antivir Ther 2002; 7:S11. - Sista P, Melby T, Davison D, et al. AIDS 2004; 18:1-8.

- Greenberg ML, Cammack N. J Antimicrob Chemother 2004; 54:333-340.

- Xu L, Pozniak A, Wildfire A, et al. In: Proceedings of the 11th Conference on Retroviruses and Opportunistic Infections, 2004 [Abstract 659].

- Poveda E, Rodes B, Toro C, et al. AIDS 2002; 16:1959-1961.

- Hirsch MS, Brun-Vezinet F, Clotet B, et al. Clin Infect Dis 2003; 37:113-128.

- Vandamme AM, Sonnerborg A, Ait-Khaled M, et al. Antivir Ther 2004; 9:829-848.

- Zolopa AR. J Infect Dis 2006; 194 Suppl 1 :S59-64. Review.

- Johnson VA, Brun-Vezinet F₁ Clotet B, et al. Top HIV Med 2006; 14:125-130.

- Smith D, Moini N, Pesano R, et al. Clin Infect Dis 2007; 44:456- 458.

Claims

CLAIMS 1. Method for HIV-1 genotyping comprising the following steps: a) reverse-transcription of HIV-1 viral RNA from a biological sample, using a specific anti-sense primer consisting in the nucleotide sequence 5'-TGC TTW TAT GCA GCA TCT GAG GG-3¹ (SEQ ID NO: 1) or 5'-GAA AGT CCC CAG CGG AAA GTC C-3¹ (SEQ ID NO: 2)) and subsequent PCR amplification of dsDNA fragment by using a pair of target-specific external primers consisting in the nucleotide sequences selected from the group consisting of: a1) Fw 5'-AGT TTY AAT TGT RGA GGR GAA TTT-3¹ (SEQ ID NO:

3)/Rv 5'-TGC TTW TAT GCA GCA TCT GAG GG-3¹ (SEQ ID NO: 4); or alternatively: a2) Fw 5'-AGT TTT AAT TGT RGA GGR GAA TTT-3' (SEQ ID NO: 5)/Rv 5'-GAA AGT CCC CAG CGG AAA GTC C-3' (SEQ ID NO: 6); wherein the reverse-transcription and amplification occur in a single step; b) sequencing the amplicon obtained in step a) in independent reactions each of the following primers comprising or consisting in anyone of the following nucleotide sequences: b1) Fw 5'- TTR AAC CAY TAG GAR TAG CAC CCA-3¹ (SEQ ID NO: 7) or alternatively, Fw 5'-TTG AAC CAY TAG GAG TAG CAC CCA-3¹ (SEQ ID NO: 8); b2) Fw 5'-GGC AAA GAG AAG AGT GGT-3' (SEQ ID NO: 9) or alternatively, Fw 5'-GGC AAR RAG AAG AGT GGT-3' (SEQ ID NO: 10); b3) Fw 5'-TTG GGG YTG CTC TGG AAA AC-3' (SEQ ID NO: 11) or alternatively, Fw 5'-TTG GGG YTG CTC TGG AAA-3' (SEQ ID NO: 12); b4) Fw 5'-ATA ATG ATA GTA GGA GG-3¹ (SEQ ID NO: 13); b5) Fw 5'-GGA RCC TGT GCC TCT TCA-3¹ (SEQ ID NO: 14); b6) Fw 5'-TAC CTA GAA GAA TMA GAC A-3' (SEQ ID NO: 15); b7) Rv 5'-TCT YAT TCT TTC CCT WA-3¹ (SEQ ID NO: 16) or alternatively, Rv 5'-TCT YAT TCT TTC CCT TA-3' (SEQ ID NO: 17); b8) Rv 5'-GTC CCA GAA GTT CCA CA-3¹ (SEQ ID NO: 18); b9) Rv 5'-TYT TAT ATA CCA YAG CCA-3' (SEQ ID NO: 19); b10) Rv 5'-GTA TCT TTC YAC AGC CAG-3' (SEQ ID NO: 20) or alternatively, Rv 5'-GTA TCT TTC YAY AGC CAG-3' (SEQ ID NO: 21). c) assembling of internal segments obtained in step b) in a consensus sequence.

2. Method according to claim 1 , wherein when amplification by RT-PCR is not obtained, the step a) comprises a further nested-PCR amplification step, using a pair of internal target-specific primers selected between the following nucleotide sequences: c1) Fw 5'-TTR AAC CAY TAG GAR TAG CAC CCA-3¹ (SEQ ID NO:

22)/Rv 5'-TAC TAG CTT GWA GCA CCA RCC AA-3¹ (SEQ ID NO: 23); or alternatively, c2) Fw 5'-TTG AAC CAY TAG GAG TAG CAC CCA-3¹ (SEQ ID NO: 24)/Rv 5'-GTC AGC AGT CCT TGT AGT ACT CCG GAT-3¹ (SEQ ID NO: 25).

3. Method for the detection of the mutations that confer resistance to anti- retroviral drugs comprising a)-c) steps as defined according to anyone of claims 1-2, wherein step c) consists in the production of a consensus sequence obtained from the different generated nucleotide segments and in its comparison with reference sequences, for the interpretation of mutations occurring in gp41-env gene eventually associated with resistance to anti-retroviral drugs.

4. Method according to anyone of claims 1-3, wherein said biological sample is a plasma or a cell lymphocyte sample.

5. Diagnostic kit comprising the following components: a) at least one pair of primers r for RT-PCR amplification of the dsDNA fragment, comprising or consisting in the nucleotide sequences selected from: a1) Fw 5'-AGT TTY AAT TGT RGA GGR GAA TTT-3¹ (SEQ ID NO: 3)/Rv 5'-TGC TTW TAT GCA GCA TCT GAG GG -3' (SEQ ID NO: 4); or alternatively, a2) Fw 5'-AGT TTT AAT TGT RGA GGR GAA TTT-3¹ (SEQ ID NO: 5)/Rv 5'-GAA AGT CCC CAG CGG AAA GTC C-3¹ (SEQ ID NO: 6); b) a set of primer for sequencing the amplicon obtained by amplification with a1) or a2) primers consisting in the following nucleotide sequences: b1) Fw 5'-TTR AAC CAY TAG GAR TAG CAC CCA-3¹ (SEQ ID NO: 7) or alternatively,, Fw 5'-TTG AAC CAY TAG GAG TAG CAC CCA-3¹ (SEQ ID NO: 8); b2) Fw 5'-GGC AAA GAG AAG AGT GGT-3¹ (SEQ ID NO: 9) or alternatively, Fw 5'-GGC AAR RAG AAG AGT GGT-3¹ (SEQ ID NO: 10); b3) Fw 5'-TTG GGG YTG CTC TGG AAA AC-3¹ (SEQ ID NO: 11) or alternatively, Fw 5'-TTG GGG YTG CTC TGG AAA-3¹ (SEQ ID NO: 12); b4) Fw 5'-ATA ATG ATA GTA GGA GG-3' (SEQ ID NO: 13); b5) Fw 5'-GGA RCC TGT GCC TCT TCA-3' (SEQ ID NO: 14); b6) Fw 5'-TAC CTA GAA GAA TMA GAC A-3¹ (SEQ ID NO: 15); b7) Rv 5'-TCT YAT TCT TTC CCT WA-3¹ (SEQ ID NO: 16) or alternatively, Rv 5'-TCT YAT TCT TTC CCT TA-3¹ (SEQ ID NO: 17); b8) Rv 5'-GTC CCA GAA GTT CCA CA-3¹ (SEQ ID NO: 18); b9) RV 5'-TYT TAT ATA CCA YAG CCA-3¹ (SEQ ID NO: 19); b10) Rv 5'-GTA TCT TTC YAC AGC CAG-3¹ (SEQ ID NO: 20) or alternatively, Rv 5'-GTA TCT TTC YAY AGC CAG-3' (SEQ ID NO: 21).

6. Diagnostic kit according to claim 5, comprising a further pair of primers for the possible amplification by nested-PCR, selected from the following nucleotide sequences: c1) Fw 5"-TTR AAC CAY TAG GAR TAG CACC CA-3¹ (SEQ ID NO:

22)/Rv 5'-TAC TAG CTT GWA GCA CCA RCC AA-3¹ (SEQ ID NO: 23); or alternatively, c2) Fw 5'-TTG AAC CAY TAG GAG TAG CAC CCA-3¹ (SEQ ID NO: 24)/Rv 5'-GTC AGC AGT CCT TGT AGT ACT CCGGAT-3¹ (SEQ ID NO: 25).