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WO2008136033A2 - Procédé pour le génotypage du vih-1 et trousses de diagnostic pertinentes pour la détection de résistances à des médicaments - Google Patents

Procédé pour le génotypage du vih-1 et trousses de diagnostic pertinentes pour la détection de résistances à des médicaments Download PDF

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Publication number
WO2008136033A2
WO2008136033A2 PCT/IT2008/000298 IT2008000298W WO2008136033A2 WO 2008136033 A2 WO2008136033 A2 WO 2008136033A2 IT 2008000298 W IT2008000298 W IT 2008000298W WO 2008136033 A2 WO2008136033 A2 WO 2008136033A2
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seq
tag
alternatively
cca
tct
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PCT/IT2008/000298
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WO2008136033A3 (fr
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Fabio Tramuto
Nino Romano
Francesco Vitale
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Universita' Degli Studi Di Palermo
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Publication of WO2008136033A2 publication Critical patent/WO2008136033A2/fr
Publication of WO2008136033A3 publication Critical patent/WO2008136033A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS

Definitions

  • 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).
  • HIV-1 human immunodeficiency virus type 1
  • diagnostic kits for the detection of resistance to antiretroviral drugs active to gp41-env (i.e. fusion inhibitors).
  • 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).
  • 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).
  • 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 "TRUGENETM HIV-1 Genotyping System” (Bayer). These commercial diagnostic kits have a sensitivity limit of 10 3 copies of HIV-RNA/mL, so it is difficult to detect mutations in samples with low viremia.
  • 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.
  • fusion inhibitors anti-retroviral drugs
  • 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 10 1 -10 2 copies of HIV-RNA/mL (see Figure 5) and then lowering the limit of sensitivity of current commercial diagnostic systems (10 3 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).
  • the method offers very short processing times as to operator and PCR protocol in the order of 5 hours.
  • 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-3 1 (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-3 1 (SEQ ID NO: 5)/Rv 5'-GAA AGT CCC CAG CGG AAA GTC C-3 1 (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
  • 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 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;
  • Viremia estimated: 11 ,000 copies of HIV-1 RNA/mL;
  • Viremia estimated: 1 ,100 copies HIV-1 RNA/mL;
  • Viremia 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;
  • 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).
  • cDNA complementary DNA
  • dsDNA double-stranded DNA fragment
  • 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).
  • 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).
  • the proposed method amplifies a genetic fragment included between the nucleotide positions 7300 and 9500 (referring to the
  • 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.
  • RNA extraction was preferred to adopt commercial kit based on column with silica-gel membrane.
  • the mixture of lysed sample is applied on a column containing a silica-gel membrane and centrifuged for 1 min at 3000-5000xg.
  • the filtrate is eliminated and the column is placed in a clean collection tube. After adding 500 ⁇ l_ 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 ⁇ l_ of a solution containing ethanol, the column is centrifuged for 3 min at 20000xg.
  • the purified DNA/RNA can be used immediately or kept at -80 0 C.
  • 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.
  • PBMCs lymphocyte pellets
  • Step 2 One step RT-PCR
  • 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.
  • RNA molecules 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 MgSO 4 (final concentration 1.7 mM); b) forward primer (FT1gp41_For) and anti-sense (FT1gp41_Rev) (each a final concentration of 0.4 ⁇ M), c) RNA template (> 1 pg) d) RNAse free H 2 O to a total of 50 ⁇ L Thermal protocol:
  • the obtained dsDNA can be used for the next steps or stored at -2O 0 C.
  • a second step of 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.
  • the nested-PCR reaction is conducted using the DNA polymerase
  • 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-SO 4 - pH 8.9, 180 mM ammonium sulfate; b) dATP, dCTP, dGTP and dTTP (each a final concentration of 0.2 mM); c) MgSO 4 (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 ⁇ M); e) Platinum® Taq DNA Polymerase High Fidelity (2 U); f) dsDNA template (RT-PCR) 5
  • the obtained dsDNA can be used for the next steps or stored at -20 0 C.
  • Step 4 purification and analysis of PCR products
  • 300 ⁇ l_ of sterile H 2 O and 50 ⁇ l_ 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 ⁇ l_ of sterile H 2 O on the membrane. After centrifugation for 5 minutes at 550xg, the dsDNA purified sample is transferred to a clean tube.
  • 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).
  • the amplified sample is diluted with sterile H 2 O 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)
  • Sequences are purified using the Centri-SepTM columns (Princeton Separation, consisting of resin SephadexTM 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 ⁇ l_ of SDS to each of 20 ⁇ l_ 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.
  • the mixture is purified using a Centri-SepTM column or by alternative procedures, as recommended by the manufacturer
  • 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.
  • 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:
  • Viraemia (dispensed): 110,000 copies HIV-1 RNA/mL; 1 :10) Diluted sample. Viraemia (estimated): 11 ,000 copies HIV-1 RNA/mL;
  • Viraemia (estimated): 1 ,100 copies HIV-1 RNA/mL;
  • 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 can be eventually improved by a subsequent amplification- nested PCR step able to genotyping plasma samples with very low viraemia (10 1 -10 2 HIV-RNA/mL copies), lowering the sensitivity of current commercial diagnostic systems directed to other genetic regions (10 3 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.

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Abstract

L'invention concerne un procédé pour le génotypage du VIH-1 par séquençage du gène gp41-env afin de détecter des mutations associées à des résistances à des médicaments antirétroviraux inhibiteurs de fusion, et une trousse de diagnostic associée.
PCT/IT2008/000298 2007-05-03 2008-05-02 Procédé pour le génotypage du vih-1 et trousses de diagnostic pertinentes pour la détection de résistances à des médicaments WO2008136033A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ITRM20070254 ITRM20070254A1 (it) 2007-05-03 2007-05-03 Metodo per la genotipizzazione di hiv-1 e relativi kit diagnostici per la rilevazione di farmaco-resistenze.
ITRM2007A000254 2007-05-03

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WO2008136033A2 true WO2008136033A2 (fr) 2008-11-13
WO2008136033A3 WO2008136033A3 (fr) 2009-02-26

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WO1994023069A1 (fr) * 1993-03-26 1994-10-13 Gen-Probe Incorporated Detection du virus de type 1 de l'immunodeficience humaine

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