WO2011036328A2 - Method for early detection of resistance to antiretroviral therapy in hiv - Google Patents

Method for early detection of resistance to antiretroviral therapy in hiv Download PDF

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WO2011036328A2
WO2011036328A2 PCT/ES2010/070619 ES2010070619W WO2011036328A2 WO 2011036328 A2 WO2011036328 A2 WO 2011036328A2 ES 2010070619 W ES2010070619 W ES 2010070619W WO 2011036328 A2 WO2011036328 A2 WO 2011036328A2
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hiv
copies
rna
plasma
sample
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PCT/ES2010/070619
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French (fr)
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WO2011036328A3 (en
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Pilar Perez Romero
Julian Mier Mota
Jeronimo Pachon Diaz
Luis F. Lopez Cortes
Pompeyo Viciana Fernandez
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Fundación Pública Andaluza Para La Gestión De La Investigación En Salud De Sevilla
Universidad De Sevilla
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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 falls within the field of molecular biology and medicine and refers to a pair of primers capable of amplifying the pol gene of the human immunodeficiency virus (HIV) from plasma samples containing low levels of viral load. (between 20 and 1,000 copies of viral RNA / mL of plasma, and more preferably between 20 and 100 copies / mL).
  • these primers allow the detection of HIV in biological samples that have low viral load, with the minimum limit necessary for carrying out said detection 20 copies of viral RNA per ml_ of sample. HIV RNA thus detected can be genotyped to subsequently establish a profile of resistance mutations to antiretroviral therapy (ART) in HIV in patients with a low viral load.
  • ART antiretroviral therapy
  • the methods of the invention can, therefore, be used for the early detection of mutations that in HIV are responsible for ART failure even in cases where plasma viremia levels are low, which allows modifying the treatment early, avoid the appearance of new mutations and protect the efficacy of drugs from the current treatment regimen and others that may be administered in the future.
  • ART antiretroviral treatment
  • CV plasma viral load
  • Genotypic tests are based on the amplification by, first, an RT-PCR and secondly a PCR, of those regions of the viral genome involved in the development of resistance, usually the reverse transcriptase gene and the protease ( pol gene), for the subsequent analysis of its nucleotide sequence (sequencing) and its comparison with other HIV reference sequences.
  • These genotypic techniques are the most widespread due to their simplicity, low cost, speed and less technical complexity, so there are several commercial kits available (LIPA, TrueGene, Viroseq, VircoGen, etc.).
  • antiretroviral drugs are combined based on the history of previous ART and resistance genotypic tests that give information about possible drugs that still have antiviral activity.
  • genotypic methods for the detection of resistance in HIV focuses on the possibility of amplifying samples with ART failure and viral levels of less than 1,000 copies / mL. In this sense they have tried to develop several methods capable of overcoming this technical difficulty. Some of them are those carried out through modified commercial protocols, for example, by adding an optional centrifugation step to concentrate the virus, prior to the extraction of the RNA, so that a minimum threshold of 100 copies / mL has been established. for amplification (Howard B. Gale, et al., 2005, 45th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, Abstract H-1053).
  • genotypic resistance tests have been tested that allow the analysis of samples with viral loads below 75 copies / mL, for which the RNA is extracted using a lithic agent, a RT-PCR is carried out to obtain the cDNA and subsequently an amplification is performed by nested PCR of the protease and retrotranscriptase genes (Mitsuya Y., et al., 2006, Journal of Acquired Immune Deficiency Syndromes, 43 (1): 56-59). There are also genotypic HIV resistance tests for patients with viremia levels below 50 copies / mL.
  • HIV RNA sequences that can be genotyped from samples with viremia levels of 30-40 copies / mL, by using affinity columns to extract the RNA (Robert W. Shafer, et al., 1997 , Journal of Clinical Microbiology, 35 (2): 520-522).
  • the present invention provides a pair of primers capable of amplifying the pol gene of the human immunodeficiency virus (HIV) from plasma samples containing low levels of viral load (between 20 and 1,000 copies of viral RNA / mL of plasma, and more preferably between 20 and 100 copies / mL).
  • these primers allow the detection of HIV in biological samples that have low viral load, with the minimum limit necessary for carrying out said detection 20 copies of viral RNA per ml_ of sample. HIV RNA thus detected can be genotyped to subsequently establish a profile of resistance mutations to antiretroviral therapy (ART) in HIV in patients with a low viral load.
  • ART antiretroviral therapy
  • the methods of the invention can, therefore, be used for the early detection of mutations that in HIV are responsible for ART failure even in cases where plasma viremia levels are low, which allows modifying the treatment early, avoid the appearance of new mutations and protect the efficacy of drugs from the current treatment regimen and others that may be administered in the future.
  • the optimization of several steps integrated in the genotypic methods commonly used for the detection of ART resistance in HIV has been combined retrotranscriptases and polymerases used), in order to achieve greater sensitivity.
  • a pair of primers or primers capable of amplifying the pol gene has been designed even from plasma samples that have low viral copies (the minimum threshold required for said amplification of 20 RNA / mL copies).
  • the steps of isolation or extraction of the RNA from the virus have been optimized by concentrating the sample through the use of affinity columns for viral RNA, as well as the amplification, RT-PCR and PCR reactions, in the that a retrotranscriptase and a high affinity polymerase have been used.
  • the result is that the protocol thus designed allows the viral sequences of interest to be amplified from plasma samples of patients that are below the limit of minimum viral copies usually required with other genotypic methods for the detection of mutations in HIV.
  • a first aspect of the invention relates to a method of early detection of VI H, hereafter referred to as "first method of the invention", comprising: a. obtain an isolated biological sample,
  • step (b) extracting the HIV RNA present in the sample from step (a) using an RNA affinity column
  • step (b) contacting the RNA obtained in step (b) with a reaction mixture containing primers SEQ ID NO: 1 and SEQ ID NO:
  • step (d) Detect the RT-PCR products obtained in step (d).
  • the biological sample from step (a) presents between 10 and 1,000 copies of HIV RNA per measured sample. In a more preferred embodiment, the biological sample from step (a) has between 20 and 1,000 copies of HIV RNA per ml of sample. In an even more preferred embodiment, the biological sample from step (a) has between 20 and 100 copies of HIV RNA per ml of sample. In another preferred embodiment, the biological sample isolated from step (a) is blood plasma.
  • the polymerase employed in any of steps (c) and (d) is of high affinity.
  • the detection of the RT-PCR products of step (e) is performed by electrophoresis.
  • the "HIV or human immunodeficiency virus” is a retrovirus belonging to the subfamily Lentiviridae and is characterized by causing the slowly evolving disease called AIDS or Acquired Immune Deficiency Syndrome.
  • HIV virions are spherical particles between 80 and 100 nm in which three concentric layers can be distinguished: a lipidoprotein envelope, a central icosahedral nucleocapsid and inside it both the genetic material and the enzymes necessary to complete its life cycle (reverse transcriptase, integrase and protease).
  • Its genome is a single chain RNA consisting of two identical strands of positive polarity, of a length of 9,800 base pairs, presenting three structural genes (gag, pol and env) and at least six regulatory genes, which in the state of provirus they are limited by repeated sequences (LTR) that facilitate their integration into the genome of the host cell and that contain the regulatory elements of the onset of viral transcription.
  • LTR repeated sequences
  • an isolated biological sample includes, but is not limited to, cells, tissues and / or biological fluids of an organism, obtained by any method known to a person skilled in the art that serves this purpose.
  • the biological sample isolated from step (a) is preferably blood plasma, which can be obtained by blood extraction and subsequent separation of blood and plasma by centrifugation.
  • Blood plasma means the liquid and acellular fraction of the translucent yellowish blood, composed of 90% water and multiple substances dissolved in it, of which the most abundant are proteins. It also contains carbohydrates and lipids, as well as metabolic waste products. It is the major component of the blood, since it represents approximately 55% of the total blood volume. The remaining 45% corresponds to the formal elements (such magnitude is related to the hematocrit).
  • the virus RNA is extracted from it. Said extraction or isolation could be done through protocols of extraction of the viral genetic material, such as, but not limited to, the Qiagen protocol, by lysis agents, such as guanidine thiocyanate, by separation in the aqueous phase by saturation with phenol-chloroform, or by adsorption extraction in affinity columns by RNA, as for example, but not limited to, in silica columns ⁇ silica beads) or through the QlAamp Viral RNA Mini Kit system (QIAGEN, Valencia, CA, USA).
  • the viral genetic material such as, but not limited to, the Qiagen protocol
  • lysis agents such as guanidine thiocyanate
  • separation in the aqueous phase by saturation with phenol-chloroform
  • adsorption extraction in affinity columns by RNA as for example, but not limited to, in silica columns ⁇ silica beads
  • QIAamp Viral RNA Mini Kit system QIAGEN, Valencia, CA, USA.
  • RNA extraction is preferably performed by RNA affinity columns, and more preferably by the QlAamp Viral RNA Mini Kit system (QIAGEN, Valencia, CA, USA), since they provide greater effectiveness in the process of extraction and isolation of the genetic material, and also allow the concentration of the genetic material contained in the sample to be analyzed, which is especially desirable in those samples that have low levels of viremia (below 1,000 copies / mL and preferably below 100 copies / mL), so that RNA is obtained in a quality and quantity sufficient for subsequent analysis.
  • QlAamp Viral RNA Mini Kit system QIAGEN, Valencia, CA, USA
  • the pol gene of HIV can be retrotranscribed and amplified in an amplification reaction suitable for this purpose, such as, for example, RT-PCR or Polymerase Chain Reaction in Reverse Transcription, which consists of a laboratory technique commonly used in biology. molecular to generate a large number of cDNA copies.
  • RT-PCR an RNA strand is retrotranscribed in its complementary DNA (cDNA) by the action of an enzyme called reverse transcriptase or retrotranscriptase, and subsequently the cDNA resulting from this process can be amplified by a traditional PCR with an enzyme polymerase .
  • RNA or DNA RNA or DNA by RT-PCR or PCR, respectively.
  • two primers are necessary, one of them will be attached to one of the template chains (direct primer) and the other to the complementary chain (reverse primer), in a position that allows to obtain, by successive amplifications with a heat-resistant DNA retrotranscriptase enzyme and DNA polymerase, a fragment that can be detected and / or quantified.
  • RNA samples obtained from patients with plasma viremia levels between 1,000 and 20 copies. mL, and more preferably between 100 and 20 copies / mL, as the examples of the present invention demonstrate.
  • the primers used in the amplification reactions of the invention for the pol gene are those collected in SEQ ID NO: 1 (first 5 * pol gene) and in SEQ ID NO: 2 (first 3 * gene pol), since they allow the presence of HIV to be detected in patients with viremia levels below 1,000 copies / mL and more preferably below 100 copies / mL.
  • the term "specific" implies that the primers comprise a nucleotide sequence completely complementary to the sequence of the pol gene employed by the method of the present invention.
  • the " ⁇ HIV gene is one of the three main genes included in the HIV genome and codes for protease and reverse transcriptase.
  • the polymerase used in any of steps (c) and (d) is preferably of high affinity.
  • the mixture of enzymes for retrotranscription and PCR (RT-PCR) employed in any of steps (c) and (d) is from Invitrogen: SuperScriptI II One-Step RT-PCR System with Platinum Taq Polymerase (Invitrogen Corporation , Carlsbad, CA, USA).
  • RT-PCR One-Step RT-PCR System with Platinum Taq Polymerase
  • the products obtained in the reaction can be detected by, for example, but not limited to, quantitative PCR, fluorescent probes, or electrophoresis, among other methods.
  • the detection of the RT-PCR products obtained in step (d) is performed by separating them in an electrophoresis.
  • Electrophoresis is defined as a technique for the separation of molecules according to their mobility in an electric field.
  • the separation can be carried out on the hydrated surface of a solid support (for example, in paper or cellulose acetate electrophoresis), either through a porous matrix (gel electrophoresis), or in solution (free electrophoresis).
  • a solid support for example, in paper or cellulose acetate electrophoresis
  • gel electrophoresis gel electrophoresis
  • free electrophoresis free electrophoresis
  • the separation is due in different measure to the electrical charge of the molecules and their mass.
  • the most common variant for the analysis of mixtures of proteins or nucleic acids uses a gel, usually agarose or polyacrylamide, as support. Nucleic acids have a negative electrical charge that directs them to the positive pole.
  • another aspect of the invention relates to an HIV genotyping method, hereafter referred to as the "second method of the invention", which comprises all the steps of the first method of the invention and also: f. sequence the products detected in step (e).
  • the biological sample from step (a) presents between 10 and 1,000 copies of HIV RNA per mL. shows.
  • the biological sample from step (a) has between 20 and 1,000 copies of HIV RNA per ml of sample.
  • the biological sample from step (a) has between 20 and 100 copies of HIV RNA per ml of sample.
  • the biological sample isolated from step (a) is blood plasma.
  • the polymerase employed in any of steps (c) and (d) is of high affinity.
  • the detection of the RT-PCR products of step (e) is performed by electrophoresis.
  • the polymerase used in any of steps (c) and (d) is preferably of high affinity.
  • the mixture of enzymes for retrotranscription and PCR (RT-PCR) employed in any of steps (c) and (d) is from Invitrogen: SuperScriptIII One-Step RT-PCR System with Platinum Taq Polymerase (Invitrogen Corporation, Carlsbad, CA, USA).
  • Genotyping means the process of determining the genotype (genetic content) of an individual by means of a biological test. Genotyping is applied to a wide range of "individuals,” including microorganisms. For example, viruses or bacteria can be genotyped.
  • step (f) of the second method of the invention the bands obtained in the electrophoresis gel of step (e) are purified and sequenced.
  • the purification can be carried out by any known technique that serves this purpose, for example, but not limited to, by the QIAquick gel extraction gel purification kit (QIAGEN, Valencia, CA, USA); as well as sequencing, which could be done, for example, but not limited to, in an automatic sequencer.
  • QIAquick gel extraction gel purification kit QIAGEN, Valencia, CA, USA
  • sequencing which could be done, for example, but not limited to, in an automatic sequencer.
  • the term “sequencing” refers to the set of biochemical methods and techniques whose purpose is the determination of the order of nucleotides (A, C, G and T) in a DNA oligonucleotide.
  • the ordered information of the nucleotide sequence of the pol gene, present in the analyzed sample is available, so the comparison of this sequence obtained with a database that collects genetic information about this HIV sequence would allow to determine the nucleotide position of all mutations present in the sequence studied and thus a pattern of resistance mutations of the virus to antiretroviral treatment.
  • another aspect of the invention relates to a method of early detection of resistance mutations of VI H to antiretroviral treatment, hereafter referred to as "third method of the invention", which comprises all the steps of the first and second method of the invention, and also: g. compare the sequences obtained in step (f) with an HIV sequence database.
  • the biological sample from step (a) presents between 10 and 1,000 copies of HIV RNA per measured sample. In a more preferred embodiment, the sample has between 20 and 1,000 copies of HIV RNA per ml of sample. In an even more preferred embodiment, the biological sample from step (a) has between 20 and 100 copies of HIV RNA per ml of sample. In another preferred embodiment, the biological sample isolated from step (a) is blood plasma.
  • the polymerase employed in any of steps (c) and (d) is of high affinity.
  • the detection of the RT-PCR products of step (e) is performed by electrophoresis.
  • the polymerase used in any of steps (c) and (d) is preferably of high affinity.
  • the mixture of enzymes for retrotranscription and PCR (RT-PCR) employed in any of steps (c) and (d) is from Invitrogen: SuperScriptI II One-Step RT-PCR System with Platinum Taq Polymerase (Invitrogen Corporation , Carlsbad, CA, USA).
  • the term "early” refers to the detection of HIV, genotyping of VI H and / or detection of resistance mutations to antiretroviral treatment in HIV in biological samples isolated from patients presenting between 10 and 1,000 copies of viral RNA / mL of sample, more preferably between 20 and 1,000 copies of viral RNA / mL of sample, and even more preferably, between 20 and 100 copies of viral RNA / mL of sample.
  • the first, second and third method of the invention thanks to the primers SEQ ID NO: 1 and SEQ ID NO: 2, the retrotranscriptase and polymerase used and the method of extracting or isolating viral RNA based on the use of affinity columns for RNA, allows amplifying, in the first method of the invention, for subsequent genotyping in the second method of the invention, HIV RNA obtained from samples with low viral loads, being understood as those between 10 and 1,000 copies of viral RNA / mL of sample, more preferably between 20 and 1,000 copies of viral RNA / mL of sample, and even more preferably, between 20 and 100 copies of viral RNA / mL of sample, which is an improvement in sensitivity with respect to other HIV detection and genotyping methods, therefore, once the nucleotide sequence of the pol gene of the strain of the virus that infects the sample to be analyzed is available, it is possible to obtain its pattern of mutations if compared to a suitable database in the third method of the invention.
  • HIV sequences useful for carrying out the third method of the invention are collected, including, but not limited to, HIV-1 Resistance Mutation Datábase, HIV Sequence Datábase or HIV Drug Resistance Datábase.
  • HIV sequence database used to compare the sequences obtained in step (f) of the second method of the invention is preferably HIV Drug Resistance Datábase (Standford University), a public database designed for the storage and analysis of divergent sequences that produce resistance to HIV available at http://hivdb.stanford.edu/.
  • resistance mutations encompasses all possible alterations of the nucleotide sequence of HIV RNA or the nucleotide sequence of its cDNA that affect the sensitivity of the virus to drugs used in antiretroviral treatment.
  • Resistors means the set of mechanisms that a pathogenic microorganism (virus, bacteria or parasite) can develop in order to avoid the pressure exerted by the drugs supplied to the infected patient, which results in the total or partial loss of activity of the medication
  • Antiretroviral therapy or ART is one that involves the administration of medications or combinations of medications to combat the effects caused as a result of HIV retrovirus infection, which causes AIDS.
  • the biological sample isolated from the first, second and third method of the invention comes, preferably, from patients who are receiving ART, and more preferably, from patients who are receiving ART and who also present a failure of said treatment, defining as "ART failure" those levels of HIV RNA greater than 20 copies per ml_ of sample, in samples from patients receiving ART.
  • the biological sample isolated from the first, second and third method of the invention preferably has between 20 and 1,000 copies of HIV RNA per ml of sample and more preferably between 20 and 100 copies of HIV RNA per ml_ of sample, however, as demonstrated in the examples of the present invention, it is possible to obtain amplification product from plasma samples with 14 copies / mL, so one skilled in the art might think that it may also be possible get it for a lower minimum copy threshold, such as 10 copies / mL.
  • the biological sample isolated from the first, and consequently, from the second and third method of the invention preferably has between 10 and 1,000 copies of HIV RNA per ml of sample, more preferably between 20 and 1,000. copies of HIV RNA per ml_ of sample and even more preferably, between 20 and 100 copies of HIV RNA per mL of sample.
  • primers SEQ ID NO: 1 and SEQ ID NO: 2 used in appropriate reactions, are capable of amplifying the pol gene of HIV, so they are useful for carrying out any of the three methods of the invention. Therefore, another aspect of the invention relates to primers SEQ ID NO: 1 and SEQ ID NO: 2, hereinafter "primers of the invention”.
  • a “primer or primer” is a sequence of a specific oligonucleotide complementary to a target nucleotide sequence with which it is capable of hybridizing and serving as a starting point for a nucleotide polymerization catalyzed by RNA polymerase, DNA polymerase or reverse transcriptase.
  • Another aspect of the invention relates to the use of the primers of the invention to amplify the HIV pol gene in a plasma sample that has between 10 and 1,000 copies of HIV RNA per mL of plasma.
  • the plasma sample has between 20 and 1,000 copies of HIV RNA per mL of plasma.
  • the plasma sample has between 20 and 100 copies of HIV RNA per mL of plasma.
  • kit of the invention Another aspect of the invention relates to an HIV detection and / or genotyping kit, hereinafter "kit of the invention", which comprises the primers of the invention.
  • kit of the invention further comprises a high affinity polymerase.
  • high affinity polymerases are understood, for example but not limited to, Platinum Taq polymerase (Invitrogen Corporation, Carlsbad, CA, USA), EuroTaq polymerase (Genycell Biotech) or BlueTaq polymerase (Genycell Biotech).
  • the kit of the invention preferably comprises Platinum Taq polymerase included in the SuperScriptl ll One-Step RT-PCR System (Invitrogen) mix.
  • Said kit may comprise, without limiting us, the means necessary to carry out the extraction of the isolated biological sample, as well as affinity columns for viral RNA, such as, but not limited to, the QlAamp Viral RNA Mini Kit (QIAGEN, Valencia, CA, USA), also washing and elution solutions, primers, probes, dNTPs, media and columns necessary for purification, such as but not limited to, the QIAquick gel extraction gel band purification kit (QIAGEN, Valencia , CA, USA) and all those reagents necessary to carry out any of the methods of the invention.
  • affinity columns for viral RNA such as, but not limited to, the QlAamp Viral RNA Mini Kit (QIAGEN, Valencia, CA, USA), also washing and elution solutions, primers, probes, dNTPs, media and columns necessary for purification, such as but not limited to, the QIAquick gel extraction gel band purification kit (QIAGEN, Valencia , CA, USA) and all those reagent
  • the kit can also include, without any limitation, the use of buffers, including lysis buffers, polymerases and retrotranscriptases, preferably of high affinity and low number of errors, and more preferably the enzymatic mixture SuperScriptl ll One-Step RT-PCR System with Platinum Taq polymerase (Invitrogen Corporation, Carlsbad, CA, USA), cofactors for optimal activity of these, agents to prevent contamination, etc.
  • the kit can include all the supports and containers necessary for commissioning and optimization.
  • the kit further comprises instructions for carrying out the methods of the invention.
  • the kit will contain all the elements necessary for the early detection of HIV, the genotyping of HIV and the early detection of HIV resistance to antiretroviral treatment from isolated biological samples, preferably blood plasma, by the techniques described above.
  • Fig. 1 Represents the design of the protocol for retrotranscription and amplification of the pol gene from plasma samples with viral load less than 1,000 copies.
  • the RNA extracted from the plasma was used as a template for the back-transcription reaction followed by PCR (RT + PCR) using the combination of primers 5 -191 1 and 3 ' -3602 (previously tested as the best combination and specified in the described examples) with homology at the 3 ' and 5 ' ends of the pol region, in different dilutions containing different concentrations of viral RNA (D1, D2, D3, D4, D5 ).
  • Fig. 2. Shows the 1% electrophoresis gel with the samples resulting from the back transcription reaction followed by PCR amplification.
  • PM molecular weight marker in base pairs.
  • Fig. 3. Represents the amplification of the pol gene from a sample with a viral load of 85 copies / ml_.
  • the extracted RNA was used for the retrotranscription-PCR reaction (RT-PCR).
  • the reaction products were loaded on a 1% electrophoresis gel and the amplification band is shown in the figure along with the molecular weight marker (PM).
  • RNA-HIV-1 / mL RNA-HIV-1 / mL .
  • ART failure is considered when the viral load or copies of RNA-HIV-1 / mL in plasma is greater than 20 copies / mL. All candidates are requested to participate in the study through informed consent.
  • Figure 1 shows the design protocol. Starting from a plasma sample with known viral load and genotype, viral RNA extraction was performed using the QlAamp Viral RNA Mini Kit viral RNA extraction protocol (QIAGEN, Valencia, CA, USA). Starting from the 1,743 copies of isolated viral RNA, serial dilutions were made in base two (mixing 15 ⁇ _ of the RNA and 15 ⁇ _ of H 2 0 free of RNase) until reaching an approximate dilution of 5 copies / mL.
  • 10 ⁇ _ were used as a template together with the specific primers in a retrotranscription reaction followed by PCR amplification using high fidelity enzymes (retrotranscriptase and polymerase) (Invitrogen).
  • the retrotranscription reaction was incubated at 55 ° C for 30 minutes, followed by PCR amplification with the following amplification conditions: 1 cycle of 2 minutes at 94 ° C, 40 cycles of 15 seconds at 94 ° C; 30 seconds at 55 ° C; 90 seconds at 68 ° C and a last 5 minute cycle at 68 ° C, after which the samples were kept at 4 ° C until use.
  • the amplification products were separated by electrophoresis on a 1% agarose gel.
  • dilution 7 was the smallest dilution at which amplification product was obtained, which corresponds to 14 total copies of viral RNA.
  • the sample was concentrated by passing the volume of plasma necessary to obtain the minimum viral load through an RNA affinity column of the QlAamp Viral RNA Mini Kit system (QIAGEN, Valencia, CA, USA).
  • the retrotranscription reaction was incubated at 55 ° C for 30 minutes, followed by PCR amplification with the following amplification conditions: 1 cycle of 2 minutes at 94 ° C, 40 cycles of 15 seconds at 94 ° C; 30 seconds at 55 ° C; 90 seconds at 68 ° C and a last cycle of 5 minutes at 68 ° C, after which the samples were kept at 4 ° C until use.
  • the amplification products were separated by electrophoresis on a 1% agarose gel.
  • the amplification protocol was successfully carried out in a total of 50 samples with viral load below 1 000 copies / mL.
  • An example using plasma from a patient with viral load of 85 copies / mL is shown in Figure 3.
  • Amplification product was obtained in all cases shown in Table 2, which represents the use of this protocol in a total of 14 samples of patients with viral load less than 200 copies / mL.
  • the designed protocol allows, therefore, to obtain an amplification detection limit of 20 copies of total RNA per mL of plasma.
  • Example 2 Genotyping of HIV from plasma samples that have 20 to 1,000 copies of RNA / mL. To verify the quality of the pol gene sequences, obtained after amplification, the products obtained were sequenced. For this, the bands obtained and separated in the electrophoresis gel as explained in the previous section were cut and purified using the QIAquick gel extraction gel band purification kit (QIAGEN, Valencia, CA, USA). In all cases the concentration of the purified bands ranged between 35 and 40 ng ⁇ L. For the sequencing (Secugen, Spain) of each amplification product a minimum volume of 15 ⁇ _ was necessary.
  • Example 3 Early determination of resistance mutations from plasma samples that have 20 to 1,000 copies of RNA / mL.
  • sequences obtained corresponding to the pol gene were analyzed using the free database and available through Stanford University ⁇ HIV Drug Resistance Datábase: http://hivdb.stanford.edu/) to obtain the resistance pattern.
  • the quality of the sequences obtained was compared with those previously obtained for the same patient using the NTI 10.3.0 vector program (Invitrogen Corporation).
  • the alignment of the sequences obtained with those previously available showed a 100% identity, demonstrating that the sequences obtained in the amplification products do not differ from the genotype obtained using conventional methods, thus obtaining high fidelity in the final genotyping and, by so much so that sequences can be obtained from samples with a minimum number of 20 copies of the viral genome, demonstrating the optimization of the method.
  • Example 4 Optimization of antiretroviral treatment based on the early detection of resistance mutations.
  • plasma samples were collected from patients who presented virological failure with viral loads of less than 1,000 copies / ml. From the plasma samples, it was carried out by RT-PCR, the retrotranscription of the isolated HIV RNA and the subsequent amplification of the retrotranscriptase and protease genes for sequencing and obtaining the viral genotype. Based on the previous history of treatment and the results of the genotypic resistance test, ART was modified so that the new regimen had> 2 fully active drugs. Finally, the efficacy of the antiretroviral treatment optimization program in patients with virological failure with low viral load was evaluated, assessing the percentage of patients included who achieve undetectable CV after the change of treatment. All candidates were asked to participate in the study through informed consent.
  • Plasma samples were collected from which viral RNA was used which was used as a template for amplify the retrotranscriptase and protease genes by RT-PCR. The amplified products were sequenced and the viral genotype was obtained.

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Abstract

The invention relates to a pair of primers capable of amplifying the pol gene of the human immuno deficiency virus (HIV) from plasma samples that contain low levels of viral load (between 10 and 1000 copies of viral RNA/mL of plasma, more preferably between 20 and 1000 copies/mL and even more preferably between 20 and 100 copies/mL). Thus, said primers allow the detection of HIV in biological samples that exhibit a low viral load. The HIV RNA thus detected may be genotyped in order subsequently to establish a mutation profile showing resistance to antiretroviral therapy (ART) in HIV, in patients presenting a low viral load. The methods of the invention may therefore be used for the early detection of mutations which, in HIV, are responsible for the failure of ART, even in cases where plasma viremia levels are low, which makes it possible to modify the therapy early on to prevent the appearance of new mutations and to safeguard the efficacy of drugs in the current therapy regimen and of others that might be administered in the future.

Description

MÉTODO DE DETECCIÓN PRECOZ DE RESISTENCIAS AL TRATAMIENTO METHOD OF EARLY DETECTION OF TREATMENT RESISTORS
ANTIRRETROVIRAL EN VIH ANTIRRETROVIRAL IN HIV
La presente invención se encuadra en el campo de la biología molecular y de la medicina y se refiere a una pareja de cebadores capaces de amplificar el gen pol del virus de inmunodeficiencia humana (VIH) a partir de muestras plasmáticas que contienen bajos niveles de carga viral (entre 20 y 1 .000 copias de RNA viral/mL de plasma, y más preferiblemente entre 20 y 100 copias/mL). Así, estos cebadores permiten la detección de VIH en muestras biológicas que presentan baja carga viral, siendo el límite mínimo necesario para llevar a cabo dicha detección 20 copias de ARN viral por ml_ de muestra. El ARN del VIH así detectado se puede genotipar para, posteriormente, establecer un perfil de mutaciones de resistencia al tratamiento antirretroviral (TAR) en el VIH en pacientes que presentan una carga viral baja. Los métodos de la invención pueden, por tanto, ser empleados para la detección temprana de las mutaciones que en el VIH son responsables del fracaso del TAR incluso en los casos en los que los niveles de viremia en plasma son bajos, lo que permite modificar el tratamiento precozmente, evitar la aparición de nuevas mutaciones y proteger la eficacia de fármacos del régimen de tratamiento actual y de otros que puedan ser administrados en un futuro. The present invention falls within the field of molecular biology and medicine and refers to a pair of primers capable of amplifying the pol gene of the human immunodeficiency virus (HIV) from plasma samples containing low levels of viral load. (between 20 and 1,000 copies of viral RNA / mL of plasma, and more preferably between 20 and 100 copies / mL). Thus, these primers allow the detection of HIV in biological samples that have low viral load, with the minimum limit necessary for carrying out said detection 20 copies of viral RNA per ml_ of sample. HIV RNA thus detected can be genotyped to subsequently establish a profile of resistance mutations to antiretroviral therapy (ART) in HIV in patients with a low viral load. The methods of the invention can, therefore, be used for the early detection of mutations that in HIV are responsible for ART failure even in cases where plasma viremia levels are low, which allows modifying the treatment early, avoid the appearance of new mutations and protect the efficacy of drugs from the current treatment regimen and others that may be administered in the future.
ESTADO DE LA TÉCNICA ANTERIOR STATE OF THE PREVIOUS TECHNIQUE
El objetivo actual del tratamiento antirretroviral (TAR) para el virus de inmunodeficiencia humana o VIH, es conseguir alcanzar una carga viral plasmática (CV) indetectable. En pacientes sin TAR previo y sin mutaciones de resistencia a los fármacos utilizados esto suele conseguirse en alrededor del 60-85% de los pacientes al año de iniciar el TAR. Sin embargo, en pacientes pretratados este objetivo no siempre es fácil de conseguir y estos porcentajes descienden considerablemente, incluso con la utilización de los nuevos fármacos. En los casos en los que ocurre una replicación viral persistente a pesar del TAR, progresivamente van apareciendo y acumulándose mutaciones de resistencia a los distintos fármacos, habitualmente cruzadas dentro de una misma familia, que limitan las opciones terapéuticas, hasta el punto de que en un número significativo de pacientes éstas acaban agotándose con claras repercusiones en la morbilidad y mortalidad de la infección por el VIH. Tanto es así, que ante un fracaso del TAR con un régimen de varios fármacos con frecuencia se puede constatar que se ha perdido la eficacia de al menos 1 o más de ellos, por lo que la siguiente combinación terapéutica se ha de diseñar en base a estos datos. Por ello, la detección de resistencias en el virus puede permitir correlacionar dicho fracaso con los distintos fármacos administrados y optimizar la terapia. En este sentido, existen dos tipos de técnicas para determinar resistencias en el VIH al tratamiento antirretroviral: las genotípicas, que detectan mutaciones en el genoma del virus, y las fenotípicas, que analizan la sensibilidad del virus a los distintos fármacos. Los tests genotípicos se basan en la amplificación mediante, en primer lugar, una RT-PCR y en segundo lugar una PCR, de aquellas regiones del genoma viral implicadas en el desarrollo de resistencias, habitualmente el gen de la transcriptasa inversa y de la proteasa (gen pol), para el posterior análisis de su secuencia nucleotídica (secuenciación) y su comparación con otras secuencias de referencia del VIH. Estas técnicas genotípicas son las más extendidas debido a su simplicidad, su bajo coste, su rapidez y su menor complejidad técnica, por lo que existen varios kits comerciales disponibles (LIPA, TrueGene, Viroseq, VircoGen, etc.). En la práctica clínica, los fármacos antirretrovirales se combinan basándose en la historia de TAR previos y en los tests genotípicos de resistencias que dan información sobre los posibles fármacos que aún tienen actividad antiviral. No obstante, la principal limitación de los tests de resistencias actuales es que con viremias inferiores a 1 .000 copias de ARN/mL de plasma la muestra es muy difícil de amplificar y, por tanto, o bien no se solicitan o con frecuencia no ofrecen resultados, tanto más cuanto menor sea el nivel de viremia. En esta situación, se suele mantener el mismo TAR hasta que la CV supera las 1 .000 copias de ARN/mL y se puede realizar dicho estudio y orientar el siguiente régimen de fármacos aunque el paciente esté en fracaso virológico mayor. Es decir, existe un vacío metodológico entre el nivel de viremia indicativo de fracaso del TAR (más de 20 copias de ARN/mL de plasma) y el nivel de viremia que la mayoría de los métodos genotípicos de detección de resistencias son capaces de detectar (superior o igual a 1 .000 copias/mL), por lo que es complejo detectar resistencias en aquellos pacientes que sufren fracaso del TAR y que presentan entre 1 .000 y 20 copias de ARN por mL de plasma. En los casos en los que existen resistencias, esta viremia mantenida durante el TAR, aunque sea de bajo grado, no impide la acumulación de más mutaciones de resistencia que hacen que pueda perder eficacia algún otro fármaco de los que se están administrando. Por tanto, el objetivo actual de los métodos genotípicos de detección de resistencias en VIH se centra en la posibilidad amplificar muestras con fracaso del TAR y niveles víricos inferiores a 1 .000 copias/mL. En este sentido se han tratado de desarrollar varios métodos capaces de superar esta dificultad técnica. Algunos de ellos son los llevados a cabo mediante protocolos comerciales modificados, por ejemplo, añadiendo un paso opcional de centrifugación para concentrar el virus, previamente a la extracción del ARN, con lo que se ha podido establecer un umbral mínimo necesario de 100 copias/mL para la amplificación (Howard B. Gale, et al. , 2005, 45th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, Abstract H-1053). Incluso se han ensayado tests genotípicos de resistencias que permiten el análisis de muestras con cargas virales inferiores a 75 copias/mL, para cuya realización, se extrae el ARN utilizando un agente lítico, se lleva a cabo una RT-PCR para obtener el cDNA y posteriormente se realiza una amplificación mediante PCR anidada de los genes de la proteasa y de la retrotranscriptasa (Mitsuya Y., et al., 2006, Journal of Acquired Immune Deficiency Syndromes, 43(1 ): 56-59). También existen los tests genotípicos de resistencias del VIH para pacientes con niveles de viremia inferiores a 50 copias/mL. El éxito de este procedimiento se debe a la introducción de un paso de ultracentrifugación para concentrar las partículas del virus y a una PCR anidada que emplea una polimerasa correctora de errores (Hermankova M., et al., 2001 , JAMA, 286 (2):196-207). The current goal of antiretroviral treatment (ART) for human immunodeficiency virus or HIV is to achieve an undetectable plasma viral load (CV). In patients without prior ART and without resistance mutations to the drugs used, this is usually achieved in about 60-85% of patients a year after starting ART. However, in pretreated patients this goal is not always easy to achieve and these percentages decrease considerably, even with the use of new drugs. In cases where persistent viral replication occurs despite ART, mutations progressively appear and accumulate. of resistance to the different drugs, usually crossed within the same family, that limit the therapeutic options, to the point that in a significant number of patients they end up depleting with clear repercussions on the morbidity and mortality of HIV infection. So much so, that in the event of a failure of ART with a regimen of several drugs it can often be seen that the efficacy of at least 1 or more of them has been lost, so the following therapeutic combination has to be designed based on these dates. Therefore, the detection of resistance in the virus can correlate this failure with the different drugs administered and optimize the therapy. In this sense, there are two types of techniques to determine resistance in HIV to antiretroviral treatment: genotypic, which detect mutations in the genome of the virus, and phenotypic, which analyze the sensitivity of the virus to different drugs. Genotypic tests are based on the amplification by, first, an RT-PCR and secondly a PCR, of those regions of the viral genome involved in the development of resistance, usually the reverse transcriptase gene and the protease ( pol gene), for the subsequent analysis of its nucleotide sequence (sequencing) and its comparison with other HIV reference sequences. These genotypic techniques are the most widespread due to their simplicity, low cost, speed and less technical complexity, so there are several commercial kits available (LIPA, TrueGene, Viroseq, VircoGen, etc.). In clinical practice, antiretroviral drugs are combined based on the history of previous ART and resistance genotypic tests that give information about possible drugs that still have antiviral activity. However, the main limitation of current resistance tests is that with viremias less than 1 000 copies of RNA / mL of plasma the sample is very difficult to amplify and, therefore, either is not requested or often does not offer results, all the more the lower the viremia level. In this situation, the same TAR is usually maintained until the CV exceeds 1,000. RNA / mL copies and you can perform this study and guide the next drug regimen even if the patient is in major virological failure. That is, there is a methodological gap between the level of viremia indicative of ART failure (more than 20 copies of RNA / mL of plasma) and the level of viremia that most genotypic resistance detection methods are able to detect ( greater than or equal to 1 000 copies / mL), so it is complex to detect resistance in those patients who suffer from ART failure and who present between 1 and 20 and 20 copies of RNA per mL of plasma. In cases where there are resistances, this viremia maintained during ART, although it is low grade, does not prevent the accumulation of more resistance mutations that make it possible to lose efficacy some other drug than those being administered. Therefore, the current objective of genotypic methods for the detection of resistance in HIV focuses on the possibility of amplifying samples with ART failure and viral levels of less than 1,000 copies / mL. In this sense they have tried to develop several methods capable of overcoming this technical difficulty. Some of them are those carried out through modified commercial protocols, for example, by adding an optional centrifugation step to concentrate the virus, prior to the extraction of the RNA, so that a minimum threshold of 100 copies / mL has been established. for amplification (Howard B. Gale, et al., 2005, 45th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, Abstract H-1053). Even genotypic resistance tests have been tested that allow the analysis of samples with viral loads below 75 copies / mL, for which the RNA is extracted using a lithic agent, a RT-PCR is carried out to obtain the cDNA and subsequently an amplification is performed by nested PCR of the protease and retrotranscriptase genes (Mitsuya Y., et al., 2006, Journal of Acquired Immune Deficiency Syndromes, 43 (1): 56-59). There are also genotypic HIV resistance tests for patients with viremia levels below 50 copies / mL. The success of this procedure is due to the introduction of an ultracentrifugation step to concentrate the virus particles and to a nested PCR using an error-corrective polymerase (Hermankova M., et al., 2001, JAMA, 286 (2): 196-207).
Asimismo, es posible obtener secuencias de ARN del VIH susceptibles de ser genotipadas desde muestras con niveles de viremia de 30-40 copias/mL, mediante el empleo de columnas de afinidad para extraer el ARN (Robert W. Shafer, et al., 1997, Journal of Clinical Microbiology, 35(2):520-522). Likewise, it is possible to obtain HIV RNA sequences that can be genotyped from samples with viremia levels of 30-40 copies / mL, by using affinity columns to extract the RNA (Robert W. Shafer, et al., 1997 , Journal of Clinical Microbiology, 35 (2): 520-522).
En otra aproximación la detección de resistencias en el VIH se ha demostrado viable a partir de muestras con 5-10 copias de ARN/mL (WO20051 121379 A2). Sin embargo, este método requiere la utilización de un conjunto de cebadores y sondas específicas de cada mutación. In another approach the detection of resistance in HIV has been proven viable from samples with 5-10 copies of RNA / mL (WO20051 121379 A2). However, this method requires the use of a set of primers and probes specific to each mutation.
Por todo ello, sería deseable poder disponer de un test genotípico de resistencias en VIH más sensible que permita determinar mutaciones lo antes posible tras detectar un fracaso en el tratamiento antirretroviral aunque la viremia sea de bajo grado, que además sea estándar para poder ser aplicado en la práctica clínica. Con ello, se podría modificar el TAR más precozmente, evitar la aparición de nuevas mutaciones y "proteger" fármacos del régimen actual y otros adicionales cuya actividad se vería comprometida con la aparición o acumulación de mutaciones. Therefore, it would be desirable to have a more sensitive genotypic test of resistance in HIV that allows to determine mutations as soon as possible after detecting a failure in antiretroviral treatment even if the viremia is low grade, which is also standard to be applied in clinical practice With this, the ART could be modified more early, avoid the appearance of new mutations and "protect" drugs from the current regime and additional ones whose activity would be compromised with the appearance or accumulation of mutations.
DESCRIPCIÓN DE LA INVENCIÓN La presente invención proporciona una pareja de cebadores capaces de amplificar el gen pol del virus de inmunodeficiencia humana (VIH) a partir de muestras plasmáticas que contienen bajos niveles de carga viral (entre 20 y 1.000 copias de RNA viral/mL de plasma, y más preferiblemente entre 20 y 100 copias/mL). Así, estos cebadores permiten la detección de VIH en muestras biológicas que presentan baja carga viral, siendo el límite mínimo necesario para llevar a cabo dicha detección 20 copias de ARN viral por ml_ de muestra. El ARN del VIH así detectado se puede genotipar para, posteriormente, establecer un perfil de mutaciones de resistencia al tratamiento antirretroviral (TAR) en el VIH en pacientes que presentan una carga viral baja. Los métodos de la invención pueden, por tanto, ser empleados para la detección temprana de las mutaciones que en el VIH son responsables del fracaso del TAR incluso en los casos en los que los niveles de viremia en plasma son bajos, lo que permite modificar el tratamiento precozmente, evitar la aparición de nuevas mutaciones y proteger la eficacia de fármacos del régimen de tratamiento actual y de otros que puedan ser administrados en un futuro. En la presente invención, se ha combinado la optimización de varios pasos integrados en los métodos genotípicos comúnmente empleados para la detección de resistencias al TAR en el VIH (los relacionados con la concentración del RNA vírico, la combinación de primers y las características de las enzimas retrotranscriptasas y polimerasas utilizadas), para así alcanzar una mayor sensibilidad de los mismos. DESCRIPTION OF THE INVENTION The present invention provides a pair of primers capable of amplifying the pol gene of the human immunodeficiency virus (HIV) from plasma samples containing low levels of viral load (between 20 and 1,000 copies of viral RNA / mL of plasma, and more preferably between 20 and 100 copies / mL). Thus, these primers allow the detection of HIV in biological samples that have low viral load, with the minimum limit necessary for carrying out said detection 20 copies of viral RNA per ml_ of sample. HIV RNA thus detected can be genotyped to subsequently establish a profile of resistance mutations to antiretroviral therapy (ART) in HIV in patients with a low viral load. The methods of the invention can, therefore, be used for the early detection of mutations that in HIV are responsible for ART failure even in cases where plasma viremia levels are low, which allows modifying the treatment early, avoid the appearance of new mutations and protect the efficacy of drugs from the current treatment regimen and others that may be administered in the future. In the present invention, the optimization of several steps integrated in the genotypic methods commonly used for the detection of ART resistance in HIV (those related to the concentration of viral RNA, the combination of primers and the characteristics of enzymes) has been combined retrotranscriptases and polymerases used), in order to achieve greater sensitivity.
Para ello, se ha diseñado una pareja de cebadores o primers capaces de amplificar el gen pol incluso desde muestras plasmáticas que presentan bajas copias virales (siendo el umbral mínimo necesario para dicha amplificación de 20 copias de ARN/mL). For this, a pair of primers or primers capable of amplifying the pol gene has been designed even from plasma samples that have low viral copies (the minimum threshold required for said amplification of 20 RNA / mL copies).
Por otro lado, se han optimizado los pasos de aislamiento o extracción del ARN del virus mediante la concentración de la muestra a través del empleo de columnas de afinidad por ARN viral, así como las reacciones de amplificación, RT-PCR y PCR, en las que se han empleado una retrotranscriptasa y una polimerasa de alta afinidad. El resultado es que el protocolo así diseñado permite amplificar las secuencias víricas de interés a partir de muestras de plasma de pacientes que se encuentran por debajo del límite de copias virales mínimas requeridas habitualmente con otros métodos genotípicos para la detección de mutaciones en VIH. On the other hand, the steps of isolation or extraction of the RNA from the virus have been optimized by concentrating the sample through the use of affinity columns for viral RNA, as well as the amplification, RT-PCR and PCR reactions, in the that a retrotranscriptase and a high affinity polymerase have been used. The result is that the protocol thus designed allows the viral sequences of interest to be amplified from plasma samples of patients that are below the limit of minimum viral copies usually required with other genotypic methods for the detection of mutations in HIV.
Por tanto, un primer aspecto de la invención se refiere a un método de detección precoz de VI H, de ahora en adelante "primer método de la invención", que comprende: a. obtener una muestra biológica aislada, Therefore, a first aspect of the invention relates to a method of early detection of VI H, hereafter referred to as "first method of the invention", comprising: a. obtain an isolated biological sample,
b. extraer el ARN del VIH presente en la muestra del paso (a) mediante una columna de afinidad por ARN,  b. extracting the HIV RNA present in the sample from step (a) using an RNA affinity column,
c. poner en contacto el ARN obtenido en el paso (b) con una mezcla de reacción que contiene los cebadores SEQ ID NO: 1 y SEQ ID C. contacting the RNA obtained in step (b) with a reaction mixture containing primers SEQ ID NO: 1 and SEQ ID
NO: 2, NO: 2,
d. retrotranscribir el gen pol del VIH y amplificar el ADNc obtenido mediante RT-PCR, y  d. retrotranscribe the pol gene of HIV and amplify the cDNA obtained by RT-PCR, and
e. detectar los productos de la RT-PCR obtenidos en el paso (d).  and. Detect the RT-PCR products obtained in step (d).
En una realización preferida de este aspecto de la invención, la muestra biológica del paso (a) presenta entre 10 y 1 .000 copias de ARN de VIH por mide muestra. En una realización más preferida la muestra biológica del paso (a) presenta entre 20 y 1 .000 copias de ARN de VIH por ml_ de muestra. En una realización aún más preferida, la muestra biológica del paso (a) presenta entre 20 y 100 copias de ARN de VIH por ml_ de muestra. En otra realización preferida, la muestra biológica aislada del paso (a) es plasma sanguíneo. In a preferred embodiment of this aspect of the invention, the biological sample from step (a) presents between 10 and 1,000 copies of HIV RNA per measured sample. In a more preferred embodiment, the biological sample from step (a) has between 20 and 1,000 copies of HIV RNA per ml of sample. In an even more preferred embodiment, the biological sample from step (a) has between 20 and 100 copies of HIV RNA per ml of sample. In another preferred embodiment, the biological sample isolated from step (a) is blood plasma.
En otra realización preferida, la polimerasa empleada en cualquiera de los pasos (c) y (d) es de alta afinidad. En otra realización preferida, la detección de los productos de la RT-PCR del paso (e) se realiza mediante electroforesis. El "VIH o virus de inmunodeficiencia humana" es un retrovirus perteneciente a la subfamilia Lentiviridae y se caracteriza por provocar la enfermedad de evolución lenta denominada SIDA o Síndrome de Inmunodeficiencia Adquirida. Los viriones del VIH son partículas esféricas de entre 80 y 100 nm en las que se pueden distinguir tres capas concéntricas: una envoltura lipidoproteica, una nucleocápside icosaédrica central y en el interior de ésta tanto el material genético como las enzimas necesarias para completar su ciclo vital (transcriptasa inversa, integrasa y proteasa). Su genoma es un RNA de cadena única constituido por dos hebras idénticas de polaridad positiva, de una longitud de 9.800 pares de bases, presentando tres genes estructurales (gag, pol y env) y al menos seis genes reguladores, que en el estado de provirus están limitados por unas secuencias repetidas (LTR) que facilitan su integración en el genoma de la célula huésped y que contienen los elementos reguladores del inicio de la transcripción viral. In another preferred embodiment, the polymerase employed in any of steps (c) and (d) is of high affinity. In another preferred embodiment, the detection of the RT-PCR products of step (e) is performed by electrophoresis. The "HIV or human immunodeficiency virus" is a retrovirus belonging to the subfamily Lentiviridae and is characterized by causing the slowly evolving disease called AIDS or Acquired Immune Deficiency Syndrome. HIV virions are spherical particles between 80 and 100 nm in which three concentric layers can be distinguished: a lipidoprotein envelope, a central icosahedral nucleocapsid and inside it both the genetic material and the enzymes necessary to complete its life cycle (reverse transcriptase, integrase and protease). Its genome is a single chain RNA consisting of two identical strands of positive polarity, of a length of 9,800 base pairs, presenting three structural genes (gag, pol and env) and at least six regulatory genes, which in the state of provirus they are limited by repeated sequences (LTR) that facilitate their integration into the genome of the host cell and that contain the regulatory elements of the onset of viral transcription.
Una muestra biológica aislada incluye, pero sin limitarnos a, células, tejidos y/o fluidos biológicos de un organismo, obtenidos mediante cualquier método conocido por un experto en la materia que sirva para tal fin. En el primer método de la invención, la muestra biológica aislada del paso (a) es, preferiblemente, plasma sanguíneo, cuya obtención se puede llevar a cabo mediante extracción sanguínea y posterior separación de la sangre y el plasma por centrifugación. Se entiende por "plasma sanguíneo" la fracción líquida y acelular de la sangre de color amarillento translúcido, compuesto en un 90% por agua y múltiples sustancias disueltas en ella, de las cuales las más abundantes son las proteínas. También contiene glúcidos y lípidos, así como los productos de desecho del metabolismo. Es el componente mayoritario de la sangre, puesto que representa aproximadamente el 55% del volumen sanguíneo total. El 45% restante corresponde a los elementos formes (tal magnitud está relacionada con el hematocrito). An isolated biological sample includes, but is not limited to, cells, tissues and / or biological fluids of an organism, obtained by any method known to a person skilled in the art that serves this purpose. In the first method of the invention, the biological sample isolated from step (a) is preferably blood plasma, which can be obtained by blood extraction and subsequent separation of blood and plasma by centrifugation. "Blood plasma" means the liquid and acellular fraction of the translucent yellowish blood, composed of 90% water and multiple substances dissolved in it, of which the most abundant are proteins. It also contains carbohydrates and lipids, as well as metabolic waste products. It is the major component of the blood, since it represents approximately 55% of the total blood volume. The remaining 45% corresponds to the formal elements (such magnitude is related to the hematocrit).
Una vez obtenida la muestra de plasma, se extrae de ella el ARN del virus. Dicha extracción o aislamiento se podría realizar mediante protocolos de extracción del material genético viral, como por ejemplo, pero sin limitarnos, el protocolo Qiagen, mediante agentes de lisis, como el tiocianato de guanidina, mediante separación en fase acuosa mediante saturación con fenol-cloroformo, o mediante extracción por adsorción en columnas de afinidad por ARN, como por ejemplo, pero sin limitarnos, en columnas de sílica {silica beads) o mediante el sistema de QlAamp Viral RNA Mini Kit (QIAGEN, Valencia, CA, USA). En la presente invención, la extracción del ARN se realiza, preferiblemente, mediante columnas de afinidad por ARN, y más preferiblemente mediante el sistema de QlAamp Viral RNA Mini Kit (QIAGEN, Valencia, CA, USA), ya que proporcionan una mayor efectividad en el proceso de extracción y aislamiento del material genético, y además permiten la concentración del material genético contenido en la muestra a analizar, lo que es deseable especialmente en aquellas muestras que presentan bajos niveles de viremia (por debajo de 1 .000 copias/mL y preferiblemente por debajo de 100 copias/mL), por lo que se obtiene ARN en una calidad y cantidad suficiente para su posterior análisis. Once the plasma sample is obtained, the virus RNA is extracted from it. Said extraction or isolation could be done through protocols of extraction of the viral genetic material, such as, but not limited to, the Qiagen protocol, by lysis agents, such as guanidine thiocyanate, by separation in the aqueous phase by saturation with phenol-chloroform, or by adsorption extraction in affinity columns by RNA, as for example, but not limited to, in silica columns {silica beads) or through the QlAamp Viral RNA Mini Kit system (QIAGEN, Valencia, CA, USA). In the present invention, RNA extraction is preferably performed by RNA affinity columns, and more preferably by the QlAamp Viral RNA Mini Kit system (QIAGEN, Valencia, CA, USA), since they provide greater effectiveness in the process of extraction and isolation of the genetic material, and also allow the concentration of the genetic material contained in the sample to be analyzed, which is especially desirable in those samples that have low levels of viremia (below 1,000 copies / mL and preferably below 100 copies / mL), so that RNA is obtained in a quality and quantity sufficient for subsequent analysis.
El gen pol del VIH puede ser retrotranscrito y amplificado en una reacción de amplificación adecuada para tal fin, como por ejemplo, la RT-PCR o Reacción en Cadena de la Polimerasa en Trascripción Reversa, que consiste en una técnica de laboratorio comúnmente usada en biología molecular para generar una gran cantidad de copias de ADNc. En la RT-PCR, una hebra de ARN es retrotranscrita en su ADN complementario (ADNc) mediante la acción de una enzima llamada transcriptasa reversa o retrotranscriptasa, y posteriormente el ADNc resultado de este proceso se puede amplificar mediante una PCR tradicional con una enzima polimerasa. The pol gene of HIV can be retrotranscribed and amplified in an amplification reaction suitable for this purpose, such as, for example, RT-PCR or Polymerase Chain Reaction in Reverse Transcription, which consists of a laboratory technique commonly used in biology. molecular to generate a large number of cDNA copies. In RT-PCR, an RNA strand is retrotranscribed in its complementary DNA (cDNA) by the action of an enzyme called reverse transcriptase or retrotranscriptase, and subsequently the cDNA resulting from this process can be amplified by a traditional PCR with an enzyme polymerase .
Para amplificar un fragmento nucleotídico de ARN o ADN por RT-PCR o PCR, respectivamente, son necesarios dos cebadores, uno de ellos se unirá a una de las cadenas moldes (cebador directo) y el otro a la cadena complementaria (cebador reverso), en una posición que permita obtener, mediante sucesivas amplificaciones con una enzima retrotranscriptasa y ADN polimerasa termorresistente, un fragmento que pueda ser detectado y/o cuantificado. To amplify a nucleotide fragment of RNA or DNA by RT-PCR or PCR, respectively, two primers are necessary, one of them will be attached to one of the template chains (direct primer) and the other to the complementary chain (reverse primer), in a position that allows to obtain, by successive amplifications with a heat-resistant DNA retrotranscriptase enzyme and DNA polymerase, a fragment that can be detected and / or quantified.
Para la amplificación del gen pol del VIH se podrían diseñar numerosos cebadores. Sin embargo, cuando se diseñan cebadores es necesario hacer una serie de predicciones, como la temperatura de fusión (Tm), la presencia de pares indeseables (a) o la posibilidad de formación de horquillas (b) que puedan reducir, por competencia, la efectividad de emparejamiento con la secuencia de ARN diana. Los cebadores específicos del gen pol podrían diseñarse mediante el alineamiento de secuencias de distintas estirpes del VIH, no obstante, no todos los cebadores diseñados permiten amplificar muestras de ARN obtenidas de pacientes con niveles de viremia en plasma de entre 1 .000 y 20 copias/mL, y más preferiblemente de entre 100 y 20 copias/mL, como demuestran los ejemplos de la presente invención. Así pues, los cebadores empleados en las reacciones de amplificación de la invención para el gen pol son los que se recogen en la SEQ ID NO: 1 (primer 5* gen pol) y en la SEQ ID NO: 2 (primer 3* gen pol), ya que permiten detectar la presencia de VIH en pacientes con niveles de viremia inferiores a 1 .000 copias/mL y más preferiblemente inferiores a 100 copias/mL. Numerous primers could be designed for the amplification of the HIV gene. However, when designing primers it is necessary to make a series of predictions, such as melting temperature (Tm), the presence of undesirable pairs (a) or the possibility of formation of forks (b) that can reduce, by competition, the pairing effectiveness with the target RNA sequence. Pol gene specific primers could be designed by aligning sequences of different strains of HIV, however, not all primers designed allow amplification of RNA samples obtained from patients with plasma viremia levels between 1,000 and 20 copies. mL, and more preferably between 100 and 20 copies / mL, as the examples of the present invention demonstrate. Thus, the primers used in the amplification reactions of the invention for the pol gene are those collected in SEQ ID NO: 1 (first 5 * pol gene) and in SEQ ID NO: 2 (first 3 * gene pol), since they allow the presence of HIV to be detected in patients with viremia levels below 1,000 copies / mL and more preferably below 100 copies / mL.
En la presente descripción, el termino "específicos" implica que los cebadores comprenden una secuencia nucleotídica totalmente complementaria a la secuencia del gen pol empleado por el método de la presente invención. In the present description, the term "specific" implies that the primers comprise a nucleotide sequence completely complementary to the sequence of the pol gene employed by the method of the present invention.
El "gen ροΓ del VIH es uno de los tres genes principales comprendidos en el genoma del VIH y codifica para la proteasa y para la transcriptasa inversa. The "ροΓ HIV gene is one of the three main genes included in the HIV genome and codes for protease and reverse transcriptase.
La polimerasa empleada en cualquiera de los pasos (c) y (d) es, preferiblemente, de alta afinidad. Preferiblemente, la mezcla de enzimas para la retrotranscripción y PCR (RT-PCR) empleada en cualquiera de los pasos (c) y (d) es de Invitrogen: SuperScriptI I I One-Step RT-PCR System con la polimerasa Platinum Taq (Invitrogen Corporation, Carlsbad, CA, USA). Una vez amplificado el ADNc mediante la RT-PCR del paso (d) los productos obtenidos en la reacción se pueden detectar mediante, por ejemplo, pero sin limitarnos, PCR cuantitativa, mareaje con sondas fluorescentes, o electroforesis, entre otros métodos. En una realización preferida, la detección de los productos de la RT-PCR obtenidos en el paso (d) se realiza mediante su separación en una electroforesis. Se define "electroforesis" como una técnica para la separación de moléculas según la movilidad de éstas en un campo eléctrico. La separación puede realizarse sobre la superficie hidratada de un soporte sólido (por ejemplo, electroforesis en papel o en acetato de celulosa), o bien a través de una matriz porosa (electroforesis en gel), o bien en disolución (electroforesis libre). Dependiendo de la técnica que se use, la separación obedece en distinta medida a la carga eléctrica de las moléculas y a su masa. La variante de uso más común para el análisis de mezclas de proteínas o de ácidos nucleicos utiliza como soporte un gel, habitualmente de agarosa o de poliacrilamida. Los ácidos nucleicos disponen de una carga eléctrica negativa que los dirige al polo positivo. Al poner la mezcla de moléculas y aplicar un campo eléctrico, éstas se mueven y deben ir pasando por la malla del gel (una red tridimensional de fibras cruzadas), por lo que las pequeñas se mueven mejor, más rápidamente. Así, las más pequeñas avanzan más y las más grandes quedan cerca del lugar de partida. Tras la electroforesis, se obtiene un patrón de bandas en el soporte empleado, preferiblemente, un gel de agarosa, correspondiente a los fragmentos de ADNc amplificados. Estas bandas podrían ser purificadas y secuenciadas para obtener así el genotipo del virus. The polymerase used in any of steps (c) and (d) is preferably of high affinity. Preferably, the mixture of enzymes for retrotranscription and PCR (RT-PCR) employed in any of steps (c) and (d) is from Invitrogen: SuperScriptI II One-Step RT-PCR System with Platinum Taq Polymerase (Invitrogen Corporation , Carlsbad, CA, USA). Once the cDNA is amplified by the RT-PCR of step (d) the products obtained in the reaction can be detected by, for example, but not limited to, quantitative PCR, fluorescent probes, or electrophoresis, among other methods. In a preferred embodiment, the detection of the RT-PCR products obtained in step (d) is performed by separating them in an electrophoresis. "Electrophoresis" is defined as a technique for the separation of molecules according to their mobility in an electric field. The separation can be carried out on the hydrated surface of a solid support (for example, in paper or cellulose acetate electrophoresis), either through a porous matrix (gel electrophoresis), or in solution (free electrophoresis). Depending on the technique used, the separation is due in different measure to the electrical charge of the molecules and their mass. The most common variant for the analysis of mixtures of proteins or nucleic acids uses a gel, usually agarose or polyacrylamide, as support. Nucleic acids have a negative electrical charge that directs them to the positive pole. When putting the mixture of molecules and applying an electric field, they move and must go through the mesh of the gel (a three-dimensional network of crossed fibers), so that the small ones move better, more quickly. Thus, the smallest advance more and the largest are close to the place of departure. After electrophoresis, a band pattern is obtained on the support, preferably an agarose gel, corresponding to the amplified cDNA fragments. These bands could be purified and sequenced to obtain the genotype of the virus.
Por ello, otro aspecto de la invención se refiere a un método de genotipado del VIH, de ahora en adelante "segundo método de la invención", que comprende todos los pasos del primer método de la invención y además: f. secuenciar los productos detectados en el paso (e). Therefore, another aspect of the invention relates to an HIV genotyping method, hereafter referred to as the "second method of the invention", which comprises all the steps of the first method of the invention and also: f. sequence the products detected in step (e).
En una realización preferida de este aspecto de la invención, la muestra biológica del paso (a) presenta entre 10 y 1 .000 copias de ARN de VIH por mL de muestra. En una realización más preferida la muestra biológica del paso (a) presenta entre 20 y 1 .000 copias de ARN de VIH por ml_ de muestra. En una realización aún más preferida, la muestra biológica del paso (a) presenta entre 20 y 100 copias de ARN de VIH por ml_ de muestra. En otra realización preferida, la muestra biológica aislada del paso (a) es plasma sanguíneo. In a preferred embodiment of this aspect of the invention, the biological sample from step (a) presents between 10 and 1,000 copies of HIV RNA per mL. shows. In a more preferred embodiment, the biological sample from step (a) has between 20 and 1,000 copies of HIV RNA per ml of sample. In an even more preferred embodiment, the biological sample from step (a) has between 20 and 100 copies of HIV RNA per ml of sample. In another preferred embodiment, the biological sample isolated from step (a) is blood plasma.
En otra realización preferida, la polimerasa empleada en cualquiera de los pasos (c) y (d) es de alta afinidad. En otra realización preferida, la detección de los productos de la RT-PCR del paso (e) se realiza mediante electroforesis. In another preferred embodiment, the polymerase employed in any of steps (c) and (d) is of high affinity. In another preferred embodiment, the detection of the RT-PCR products of step (e) is performed by electrophoresis.
La polimerasa empleada en cualquiera de los pasos (c) y (d) es, preferiblemente, de alta afinidad. Preferiblemente, la mezcla de enzimas para la retrotranscripción y PCR (RT-PCR) empleada en cualquiera de los pasos (c) y (d) es de Invitrogen: SuperScriptIII One-Step RT-PCR System con la polimerasa Platinum Taq (Invitrogen Corporation, Carlsbad, CA, USA). The polymerase used in any of steps (c) and (d) is preferably of high affinity. Preferably, the mixture of enzymes for retrotranscription and PCR (RT-PCR) employed in any of steps (c) and (d) is from Invitrogen: SuperScriptIII One-Step RT-PCR System with Platinum Taq Polymerase (Invitrogen Corporation, Carlsbad, CA, USA).
Se entiende por "genotipado" el proceso de determinación del genotipo (contenido genético) de un individuo mediante una prueba biológica. El genotipado se aplica a un amplio rango de "individuos", incluyendo los microorganismos. Por ejemplo, se pueden genotipar virus o bacterias. "Genotyping" means the process of determining the genotype (genetic content) of an individual by means of a biological test. Genotyping is applied to a wide range of "individuals," including microorganisms. For example, viruses or bacteria can be genotyped.
En el paso (f) del segundo método de la invención, se purifican las bandas obtenidas en el gel de electroforesis del paso (e) y se secuencian. La purificación puede llevarse a cabo mediante cualquier técnica conocida que sirva para tal fin, como por ejemplo, aunque sin limitarnos, mediante el kit de purificación de bandas en gel QIAquick gel extraction (QIAGEN, Valencia, CA, USA); así como la secuenciación, la cual podría realizarse, por ejemplo, pero sin limitarnos, en un secuenciador automático. El término "secuenciación" hace referencia al conjunto de métodos y técnicas bioquímicas cuya finalidad es la determinación del orden de los nucleótidos (A, C, G y T) en un oligonucleótido de ADN. Tras la secuenciación se dispone de la información ordenada de la secuencia de nucleótidos del gen pol, presente en la muestra analizada, por lo que la comparación de esta secuencia obtenida con una base de datos que recoja información genética sobre esta secuencia del VIH, permitiría determinar la posición nucleotídica de todas las mutaciones presentes en la secuencia estudiada y así un patrón de mutaciones de resistencia del virus al tratamiento antirretroviral. In step (f) of the second method of the invention, the bands obtained in the electrophoresis gel of step (e) are purified and sequenced. The purification can be carried out by any known technique that serves this purpose, for example, but not limited to, by the QIAquick gel extraction gel purification kit (QIAGEN, Valencia, CA, USA); as well as sequencing, which could be done, for example, but not limited to, in an automatic sequencer. The term "sequencing" refers to the set of biochemical methods and techniques whose purpose is the determination of the order of nucleotides (A, C, G and T) in a DNA oligonucleotide. After sequencing, the ordered information of the nucleotide sequence of the pol gene, present in the analyzed sample, is available, so the comparison of this sequence obtained with a database that collects genetic information about this HIV sequence would allow to determine the nucleotide position of all mutations present in the sequence studied and thus a pattern of resistance mutations of the virus to antiretroviral treatment.
Por tanto, otro aspecto de la invención se refiere a un método de detección precoz de mutaciones de resistencia del VI H al tratamiento antirretroviral, de ahora en adelante "tercer método de la invención", que comprende todos los pasos del primer y segundo método de la invención, y además: g. comparar las secuencias obtenidas en el paso (f) con una base de datos de secuencias del VIH. Therefore, another aspect of the invention relates to a method of early detection of resistance mutations of VI H to antiretroviral treatment, hereafter referred to as "third method of the invention", which comprises all the steps of the first and second method of the invention, and also: g. compare the sequences obtained in step (f) with an HIV sequence database.
En una realización preferida de este aspecto de la invención, la muestra biológica del paso (a) presenta entre 10 y 1 .000 copias de ARN de VIH por mide muestra. En una realización más preferida la muestra presenta entre 20 y 1 .000 copias de ARN de VIH por ml_ de muestra. En una realización aún más preferida, la muestra biológica del paso (a) presenta entre 20 y 100 copias de ARN de VIH por ml_ de muestra. En otra realización preferida, la muestra biológica aislada del paso (a) es plasma sanguíneo.  In a preferred embodiment of this aspect of the invention, the biological sample from step (a) presents between 10 and 1,000 copies of HIV RNA per measured sample. In a more preferred embodiment, the sample has between 20 and 1,000 copies of HIV RNA per ml of sample. In an even more preferred embodiment, the biological sample from step (a) has between 20 and 100 copies of HIV RNA per ml of sample. In another preferred embodiment, the biological sample isolated from step (a) is blood plasma.
En otra realización preferida la polimerasa empleada en cualquiera de los pasos (c) y (d) es de alta afinidad. En otra realización preferida, la detección de los productos de la RT-PCR del paso (e) se realiza mediante electroforesis. In another preferred embodiment the polymerase employed in any of steps (c) and (d) is of high affinity. In another preferred embodiment, the detection of the RT-PCR products of step (e) is performed by electrophoresis.
La polimerasa empleada en cualquiera de los pasos (c) y (d) es, preferiblemente, de alta afinidad. Preferiblemente, la mezcla de enzimas para la retrotranscripción y PCR (RT-PCR) empleada en cualquiera de los pasos (c) y (d) es de Invitrogen: SuperScriptI I I One-Step RT-PCR System con la polimerasa Platinum Taq (Invitrogen Corporation, Carlsbad, CA, USA). Tal y como aparece en la presente descripción, el término "precoz" se refiere a la detección de VIH, genotipado de VI H y/o detección de mutaciones de resistencia al tratamiento antirretroviral en el VIH en muestras biológicas aisladas de pacientes que presentan entre 10 y 1 .000 copias de ARN viral/mL de muestra, más preferiblemente ente 20 y 1 .000 copias de ARN viral/mL de muestra, y aún más preferiblemente, entre 20 y 100 copias de ARN viral/mL de muestra. Es decir, el primer, segundo y tercer método de la invención, gracias a los cebadores SEQ ID NO: 1 y SEQ ID NO: 2, a la retrotranscriptasa y a la polimerasa empleadas y al método de extracción o aislamiento de ARN viral basado en la utilización de columnas de afinidad por ARN, permite amplificar, en el primer método de la invención, para su posterior genotipado en el segundo método de la invención, ARN del VIH obtenido a partir de muestras con bajas cargas virales, entendiéndose como tales las comprendidas entre 10 y 1 .000 copias de ARN viral/mL de muestra, más preferiblemente entre 20 y 1 .000 copias de ARN viral/mL de muestra, y aún más preferiblemente, entre 20 y 100 copias de ARN viral/mL de muestra, lo cual supone una mejora en la sensibilidad con respecto a otros métodos de detección y genotipado de VIH, por ello, una vez que se dispone de la secuencia nucleotídica del gen pol de la estirpe del virus que infecta la muestra a analizar es posible obtener su patrón de mutaciones si se compara con una base de datos adecuada en el tercer método de la invención. En este sentido, existen numerosas bases de datos donde se encuentran recogidas las secuencias del VIH útiles para llevar a cabo el tercer método de la invención, entre ellas, pero sin limitarnos, HIV-1 Resistance Mutation Datábase, HIV Sequence Datábase o HIV Drug Resistance Datábase. En el paso (g) del tercer método de la invención, la base de datos de secuencias del VIH empleada para comparar las secuencias obtenidas en el paso (f) del segundo método de la invención es, preferiblemente, la HIV Drug Resistance Datábase (Standford University), base de datos de carácter público diseñada para el almacenamiento y análisis de secuencias divergentes que producen resistencias al VIH disponible en http://hivdb.stanford.edu/. El término "mutaciones de resistencia" engloba toda las posibles alteraciones de la secuencia nucleotídica de ARN del VIH o de la secuencia nucleotídica de su ADNc que afectan a la sensibilidad del virus a los fármacos empleados en el tratamiento antirretroviral. Se entiende por "resistencias" el conjunto de mecanismos que un microorganismo patógeno (virus, bacteria o parásito) puede desarrollar con el fin de evadir la presión ejercida por los fármacos suministrados al paciente infectado, cuyo resultado es la pérdida total o parcial de la actividad del medicamento. El "tratamiento antirretroviral o TAR" es aquel que supone la administración de medicamentos o combinaciones de medicamentos para combatir los efectos causados como consecuencia de la infección por el retrovirus del VIH, causante del SIDA. The polymerase used in any of steps (c) and (d) is preferably of high affinity. Preferably, the mixture of enzymes for retrotranscription and PCR (RT-PCR) employed in any of steps (c) and (d) is from Invitrogen: SuperScriptI II One-Step RT-PCR System with Platinum Taq Polymerase (Invitrogen Corporation , Carlsbad, CA, USA). As it appears in the present description, the term "early" refers to the detection of HIV, genotyping of VI H and / or detection of resistance mutations to antiretroviral treatment in HIV in biological samples isolated from patients presenting between 10 and 1,000 copies of viral RNA / mL of sample, more preferably between 20 and 1,000 copies of viral RNA / mL of sample, and even more preferably, between 20 and 100 copies of viral RNA / mL of sample. That is, the first, second and third method of the invention, thanks to the primers SEQ ID NO: 1 and SEQ ID NO: 2, the retrotranscriptase and polymerase used and the method of extracting or isolating viral RNA based on the use of affinity columns for RNA, allows amplifying, in the first method of the invention, for subsequent genotyping in the second method of the invention, HIV RNA obtained from samples with low viral loads, being understood as those between 10 and 1,000 copies of viral RNA / mL of sample, more preferably between 20 and 1,000 copies of viral RNA / mL of sample, and even more preferably, between 20 and 100 copies of viral RNA / mL of sample, which is an improvement in sensitivity with respect to other HIV detection and genotyping methods, therefore, once the nucleotide sequence of the pol gene of the strain of the virus that infects the sample to be analyzed is available, it is possible to obtain its pattern of mutations if compared to a suitable database in the third method of the invention. In this sense, there are numerous databases where the HIV sequences useful for carrying out the third method of the invention are collected, including, but not limited to, HIV-1 Resistance Mutation Datábase, HIV Sequence Datábase or HIV Drug Resistance Datábase. In step (g) of the third method of the invention, the HIV sequence database used to compare the sequences obtained in step (f) of the second method of the invention is preferably HIV Drug Resistance Datábase (Standford University), a public database designed for the storage and analysis of divergent sequences that produce resistance to HIV available at http://hivdb.stanford.edu/. The term "resistance mutations" encompasses all possible alterations of the nucleotide sequence of HIV RNA or the nucleotide sequence of its cDNA that affect the sensitivity of the virus to drugs used in antiretroviral treatment. "Resistors" means the set of mechanisms that a pathogenic microorganism (virus, bacteria or parasite) can develop in order to avoid the pressure exerted by the drugs supplied to the infected patient, which results in the total or partial loss of activity of the medication "Antiretroviral therapy or ART" is one that involves the administration of medications or combinations of medications to combat the effects caused as a result of HIV retrovirus infection, which causes AIDS.
La muestra biológica aislada del primer, segundo y tercer método de la invención proviene, preferiblemente, de pacientes que se encuentran recibiendo un TAR, y más preferiblemente, de pacientes que se encuentran recibiendo un TAR y que además presentan un fracaso de dicho tratamiento, definiéndose como "fracaso del TAR" aquellos niveles de ARN de VIH superiores a 20 copias por ml_ de muestra, en muestras procedentes de pacientes recibiendo TAR. The biological sample isolated from the first, second and third method of the invention comes, preferably, from patients who are receiving ART, and more preferably, from patients who are receiving ART and who also present a failure of said treatment, defining as "ART failure" those levels of HIV RNA greater than 20 copies per ml_ of sample, in samples from patients receiving ART.
Por otro lado, la muestra biológica aislada del primer, segundo y tercer método de la invención presenta, preferiblemente, entre 20 y 1 .000 copias de ARN de VIH por ml_ de muestra y más preferiblemente entre 20 y 100 copias de ARN de VIH por ml_ de muestra, no obstante, como se demuestra en los ejemplos de la presente invención, es posible obtener producto de amplificación a partir de muestras plasmáticas con 14 copias/mL, por lo que un experto en la materia podría pensar que también puede ser posible obtenerlo para un umbral mínimo de copias inferior, como 10 copias/mL. Por tanto, la muestra biológica aislada del primer, y por consiguiente, del segundo y tercer método de la invención presenta, preferiblemente, entre 10 y 1 .000 copias de ARN de VIH por ml_ de muestra, más preferiblemente entre 20 y 1 .000 copias de ARN de VIH por ml_ de muestra y aún más preferiblemente, entre 20 y 100 copias de ARN de VIH por mL de muestra. On the other hand, the biological sample isolated from the first, second and third method of the invention preferably has between 20 and 1,000 copies of HIV RNA per ml of sample and more preferably between 20 and 100 copies of HIV RNA per ml_ of sample, however, as demonstrated in the examples of the present invention, it is possible to obtain amplification product from plasma samples with 14 copies / mL, so one skilled in the art might think that it may also be possible get it for a lower minimum copy threshold, such as 10 copies / mL. Thus, the biological sample isolated from the first, and consequently, from the second and third method of the invention preferably has between 10 and 1,000 copies of HIV RNA per ml of sample, more preferably between 20 and 1,000. copies of HIV RNA per ml_ of sample and even more preferably, between 20 and 100 copies of HIV RNA per mL of sample.
Como se ha explicado anteriormente, los cebadores SEQ ID NO: 1 y SEQ ID NO: 2, utilizados en reacciones adecuadas, son capaces de amplificar el gen pol del VIH, por lo que son de utilidad para llevar a cabo cualquiera de los tres métodos de la invención. Por ello, otro aspecto de la invención se refiere a los cebadores SEQ ID NO: 1 y SEQ ID NO: 2, de ahora en adelante "cebadores de la invención". As explained above, primers SEQ ID NO: 1 and SEQ ID NO: 2, used in appropriate reactions, are capable of amplifying the pol gene of HIV, so they are useful for carrying out any of the three methods of the invention. Therefore, another aspect of the invention relates to primers SEQ ID NO: 1 and SEQ ID NO: 2, hereinafter "primers of the invention".
Un "cebador o primer" es una secuencia de un oligonucleótido específico complementario a una secuencia nucleotídica diana con la que es capaz de hibridar y servir como punto de iniciación para una polimerización nucleotídica catalizada por ARN polimerasa, ADN polimerasa o transcriptasa reversa. A "primer or primer" is a sequence of a specific oligonucleotide complementary to a target nucleotide sequence with which it is capable of hybridizing and serving as a starting point for a nucleotide polymerization catalyzed by RNA polymerase, DNA polymerase or reverse transcriptase.
Otro aspecto de la invención se refiere al uso de los cebadores de la invención para amplificar el gen pol del VIH en una muestra de plasma que presenta entre 10 y 1 .000 copias de ARN de VIH por mL de plasma. En una realización preferida la muestra de plasma presenta entre 20 y 1 .000 copias de ARN de VIH por mL de plasma. En una realización más preferida, la muestra de plasma presenta entre 20 y 100 copias de ARN de VIH por mL de plasma. Another aspect of the invention relates to the use of the primers of the invention to amplify the HIV pol gene in a plasma sample that has between 10 and 1,000 copies of HIV RNA per mL of plasma. In a preferred embodiment, the plasma sample has between 20 and 1,000 copies of HIV RNA per mL of plasma. In a more preferred embodiment, the plasma sample has between 20 and 100 copies of HIV RNA per mL of plasma.
Otro aspecto de la invención se refiere a un kit de detección y/o genotipado del VIH, de ahora en adelante "kit de la invención", que comprende los cebadores de la invención. En una realización preferida, el kit de la invención además comprende una polimerasa de alta afinidad. Another aspect of the invention relates to an HIV detection and / or genotyping kit, hereinafter "kit of the invention", which comprises the primers of the invention. In a preferred embodiment, the kit of the invention further comprises a high affinity polymerase.
En la presente invención se entiende por polimerasas de alta afinidad, por ejemplo pero sin limitarnos, la polimerasa Platinum Taq (Invitrogen Corporation, Carlsbad, CA, USA), la polimerasa EuroTaq (Genycell Biotech) o la polimerasa BlueTaq (Genycell Biotech). El kit de la invención comprende, preferiblemente, la polimerasa Platinum Taq incluida en la mezcla SuperScriptl l l One-Step RT- PCR System (Invitrogen). In the present invention, high affinity polymerases are understood, for example but not limited to, Platinum Taq polymerase (Invitrogen Corporation, Carlsbad, CA, USA), EuroTaq polymerase (Genycell Biotech) or BlueTaq polymerase (Genycell Biotech). The kit of the invention preferably comprises Platinum Taq polymerase included in the SuperScriptl ll One-Step RT-PCR System (Invitrogen) mix.
Dicho kit puede comprender, sin limitarnos, los medios necesarios para llevar a cabo la extracción de la muestra biológica aislada, así como columnas de afinidad por ARN vírico, como por ejemplo pero sin limitarnos, el sistema de QlAamp Viral RNA Mini Kit (QIAGEN, Valencia, CA, USA), también soluciones de lavado y elución, cebadores, sondas, dNTPs, medios y columnas necesarios para la purificación, como por ejemplo pero sin limitarnos, el kit de purificación de bandas en gel QIAquick gel extraction (QIAGEN, Valencia, CA, USA) y todos aquellos reactivos necesarios para llevar a cabo cualquiera de los métodos de la invención. El kit además puede incluir, sin ningún tipo de limitación, el uso de tampones, incluyendo tampones de lisis, polimerasas y retrotranscriptasas, preferiblemente de alta afinidad y bajo número de errores, y más preferiblemente la mezcla enzimática SuperScriptl l l One-Step RT-PCR System con la polimerasa Platinum Taq (Invitrogen Corporation, Carlsbad, CA, USA), cofactores para obtener una actividad óptima de éstas, agentes para prevenir la contaminación, etc. Por otro lado el kit puede incluir todos los soportes y recipientes necesarios para su puesta en marcha y optimización. Preferiblemente, el kit comprende además las instrucciones para llevar a cabo los métodos de la invención. A título ilustrativo y sin que limite el alcance de la invención, el kit contendrá todos los elementos necesarios para la detección precoz de VIH, el genotipado del VIH y la detección precoz de resistencias del VIH al tratamiento antirretroviral a partir de muestras biológicas aisladas, preferiblemente plasma sanguíneo, por las técnicas que se han descrito anteriormente. Said kit may comprise, without limiting us, the means necessary to carry out the extraction of the isolated biological sample, as well as affinity columns for viral RNA, such as, but not limited to, the QlAamp Viral RNA Mini Kit (QIAGEN, Valencia, CA, USA), also washing and elution solutions, primers, probes, dNTPs, media and columns necessary for purification, such as but not limited to, the QIAquick gel extraction gel band purification kit (QIAGEN, Valencia , CA, USA) and all those reagents necessary to carry out any of the methods of the invention. The kit can also include, without any limitation, the use of buffers, including lysis buffers, polymerases and retrotranscriptases, preferably of high affinity and low number of errors, and more preferably the enzymatic mixture SuperScriptl ll One-Step RT-PCR System with Platinum Taq polymerase (Invitrogen Corporation, Carlsbad, CA, USA), cofactors for optimal activity of these, agents to prevent contamination, etc. On the other hand, the kit can include all the supports and containers necessary for commissioning and optimization. Preferably, the kit further comprises instructions for carrying out the methods of the invention. By way of illustration and without limiting the scope of the invention, the kit will contain all the elements necessary for the early detection of HIV, the genotyping of HIV and the early detection of HIV resistance to antiretroviral treatment from isolated biological samples, preferably blood plasma, by the techniques described above.
A lo largo de la descripción y las reivindicaciones la palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en la materia, otros objetos, ventajas y características de la invención se desprenderán en parte de la descripción y en parte de la práctica de la invención. Los siguientes ejemplos y dibujos se proporcionan a modo de ilustración, y no se pretende que sean limitativos de la presente invención. Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are they are provided by way of illustration, and are not intended to be limiting of the present invention.
DESCRIPCIÓN DE LAS FIGURAS DESCRIPTION OF THE FIGURES
Fig. 1. Representa el diseño del protocolo de retrotranscripcion y amplificación del gen pol partiendo de muestras de plasma con carga viral menor a 1.000 copias. El RNA extraído del plasma fue empleado como molde para la reacción de retrotranscripcion seguida de PCR (RT+PCR) utilizando la combinación de primers 5 -191 1 y 3'-3602 (previamente testada como la mejor combinación y especificada en los ejemplos descritos) con homología en los extremos 3' y 5' de la región pol, en distintas diluciones conteniendo diferentes concentraciones de RNA viral (D1 , D2, D3, D4, D5...). Fig. 2. Muestra el gel de electroforesis al 1% con las muestras resultantes de la reacción de retrotranscripcion seguida de una amplificación por PCR. PM: marcador de peso molecular en pares de bases. Fig. 1. Represents the design of the protocol for retrotranscription and amplification of the pol gene from plasma samples with viral load less than 1,000 copies. The RNA extracted from the plasma was used as a template for the back-transcription reaction followed by PCR (RT + PCR) using the combination of primers 5 -191 1 and 3 ' -3602 (previously tested as the best combination and specified in the described examples) with homology at the 3 ' and 5 ' ends of the pol region, in different dilutions containing different concentrations of viral RNA (D1, D2, D3, D4, D5 ...). Fig. 2. Shows the 1% electrophoresis gel with the samples resulting from the back transcription reaction followed by PCR amplification. PM: molecular weight marker in base pairs.
Fig. 3. Representa la amplificación del gen pol a partir de una muestra con una carga viral de 85 copias/ml_. El RNA extraído fue utilizado para la reacción de retrotranscripción-PCR (RT-PCR). Los productos de la reacción fueron cargados en un gel de electroforesis al 1 % y la banda de amplificación se muestra en la figura junto con el marcador de peso molecular (PM). Fig. 3. Represents the amplification of the pol gene from a sample with a viral load of 85 copies / ml_. The extracted RNA was used for the retrotranscription-PCR reaction (RT-PCR). The reaction products were loaded on a 1% electrophoresis gel and the amplification band is shown in the figure along with the molecular weight marker (PM).
EJEMPLOS EXAMPLES
A continuación se ilustrará la invención mediante unos ensayos realizados por los inventores, que ponen de manifiesto la efectividad de los métodos de detección, genotipado y detección de resistencias del VIH al tratamiento antirretroviral en muestras plasmáticas que presentan bajos niveles de carga viral (entre 20 y 1 .000 copias de ARN viral/mL de plasma). Estos ejemplos específicos que se proporcionan sirven para ilustrar la naturaleza de la presente invención y se incluyen solamente con fines ilustrativos, por lo que no han de ser interpretados como limitaciones a la invención que aquí se reivindica. Por tanto, los ejemplos descritos más adelante ilustran la invención sin limitar el campo de aplicación de la misma. Ejemplo 1. Amplificación del ARN del VIH desde muestras plasmáticas que presentan de 20 a 1.000 copias de ARN/mL The invention will now be illustrated by tests carried out by the inventors, which show the effectiveness of the methods of detection, genotyping and detection of HIV resistance to antiretroviral treatment in plasma samples that have low levels of viral load (between 20 and 1,000 copies of viral RNA / mL of plasma). These specific examples provided serve to illustrate the nature of the present invention and are included for illustrative purposes only, and therefore do not they must be interpreted as limitations on the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application. Example 1. Amplification of HIV RNA from plasma samples that have 20 to 1,000 copies of RNA / mL
1.1. Muestras biológicas. Las muestras analizadas han sido recogidas de pacientes que están recibiendo TAR y presentan fracaso del mismo con viremias plasmáticas bajas: entre 20 y 1 .000 copias RNA-VIH-1/mL. Se considera fracaso del TAR cuando la carga viral o copias de RNA-VIH-1/mL en plasma es superior a 20 copias/mL. A todos los candidatos se les solicita la participación en el estudio a través de consentimiento informado. 1.1. Biological samples. The samples analyzed have been collected from patients who are receiving ART and have a failure with low plasma viremias: between 20 and 1, 000 copies RNA-HIV-1 / mL. ART failure is considered when the viral load or copies of RNA-HIV-1 / mL in plasma is greater than 20 copies / mL. All candidates are requested to participate in the study through informed consent.
Para el aislamiento del ARN viral se utilizan muestras de plasma aislado de la sangre en tubos de 5 mi K2-EDTA con separador. 1.2. Diseño de cebadores. For the isolation of viral RNA, plasma samples isolated from blood are used in 5 ml K2-EDTA tubes with separator. 1.2. Design of primers.
En primer lugar, utilizando alineamientos de secuencias de distintas estirpes de VIH y como referencia el provirus de VIH-1 pNL4.3 (número de acceso del genBank: M19921 ), se diseñaron distintos cebadores frente a regiones conservadas en los extremos 5' y 3' de gen pol que incluye los genes que codifican la proteasa y la retrotranscriptasa. En la tabla 1 se muestran algunos de los primers o cebadores diseñados y testados para ello. First, using sequence alignments of different strains of HIV and as reference the HIV-1 provirus pNL4.3 (GenBank accession number: M19921), different primers were designed against regions conserved at the 5 ' and 3 ends ' of pol gene that includes genes encoding protease and retrotranscriptase. Table 1 shows some of the primers or primers designed and tested for it.
Primer Secuencia First Sequence
5 -191 1 5 '-TACC AT AATG ATAC AG AAAG G C AA-3 ' 5 -191 1 5 ' -TACC AT AATG ATAC AG AAAG GC AA-3 '
5'-1941 5 '-G AACCAAAGAAAG ACTGTTAGTG-3 ' 5' -1931 5' -GCAATTTTAGGAACCAAAGAAAGAC-3 ' 5 ' -1941 5 ' -G AACCAAAGAAAG ACTGTTAGTG-3 ' 5 ' -1931 5 ' -GCAATTTTAGGAACCAAAGAAAGAC-3 '
5' -1986 5' -CACATAGCCAAAAATTGCAGGGCCCC-3 ' 5 ' -1986 5 ' -CACATAGCCAAAAATTGCAGGGCCCC-3 '
3' -3602 5' -TGGGCACCCTTCATTCTTGCATA-3' 3 ' -3602 5 ' -TGGGCACCCTTCATTCTTGCATA-3 '
3' -3514 5' -TTGATGGGTCATAATACACTCCATGT-3 ' 3 ' -3514 5 ' -TTGATGGGTCATAATACACTCCATGT-3 '
3' -3521 5' -TGCATCTGTATTTCTGCTATTAAG-3' 3 ' -3521 5 ' -TGCATCTGTATTTCTGCTATTAAG-3 '
3' -3577 3' -GATAAATTTGATATGTCCATTGGCCT-5' 3 ' -3577 3 ' -GATAAATTTGATATGTCCATTGGCCT-5 '
Tabla 1. Nombre de los primers, y sus secuencias, diseñados para la amplificación del gen pol. Table 1. Name of the primers, and their sequences, designed for the amplification of the pol gene.
1.3. Determinación del número de copias/mL mínimo necesario para la amplificación. 1.3. Determination of the minimum number of copies / mL necessary for amplification.
En primer lugar se determinó el número mínimo de copias de ARN del virus por ml_ de plasma necesario para obtener producto de amplificación susceptible de ser secuenciado. En la figura 1 se muestra el protocolo de diseñado. Partiendo de una muestra de plasma con carga viral y genotipo conocidos, se realizó la extracción de RNA vírico utilizando el protocolo de extracción de RNA viral QlAamp Viral RNA Mini Kit (QIAGEN, Valencia, CA, USA). Partiendo de las 1 .743 copias de RNA vírico aislado, se realizaron diluciones seriadas en base dos (mezclando 15 μΙ_ del RNA y 15μΙ_ de H20 libre de RNasa) hasta llegar a una dilución aproximada de 5 copias/mL. De cada una de las diluciones, 10 μΙ_ fueron utilizados como molde junto con los primers específicos en una reacción de retrotranscripción seguida de una amplificación por PCR utilizando enzimas (retrotranscriptasa y polimerasa) de alta fidelidad (Invitrogen). First, the minimum number of RNA copies of the virus was determined per ml_ of plasma needed to obtain amplification product that could be sequenced. Figure 1 shows the design protocol. Starting from a plasma sample with known viral load and genotype, viral RNA extraction was performed using the QlAamp Viral RNA Mini Kit viral RNA extraction protocol (QIAGEN, Valencia, CA, USA). Starting from the 1,743 copies of isolated viral RNA, serial dilutions were made in base two (mixing 15 μΙ_ of the RNA and 15μΙ_ of H 2 0 free of RNase) until reaching an approximate dilution of 5 copies / mL. Of each of the dilutions, 10 μΙ_ were used as a template together with the specific primers in a retrotranscription reaction followed by PCR amplification using high fidelity enzymes (retrotranscriptase and polymerase) (Invitrogen).
La reacción de retrotranscripción se incubó a 55°C durante 30 minutos, seguida de la amplificación por PCR con las condiciones de amplificación siguientes: 1 ciclo de 2 minutos a 94°C, 40 ciclos de 15 segundos a 94°C; 30 segundos a 55°C; 90 segundos a 68°C y un último ciclo de 5 minutos a 68°C, tras el cual las muestras se mantuvieron a 4°C hasta su utilización. Los productos de la amplificación fueron separados por electroforesis en un gel de agarosa al 1 %. The retrotranscription reaction was incubated at 55 ° C for 30 minutes, followed by PCR amplification with the following amplification conditions: 1 cycle of 2 minutes at 94 ° C, 40 cycles of 15 seconds at 94 ° C; 30 seconds at 55 ° C; 90 seconds at 68 ° C and a last 5 minute cycle at 68 ° C, after which the samples were kept at 4 ° C until use. The amplification products were separated by electrophoresis on a 1% agarose gel.
Los resultados obtenidos con las distintas combinaciones de primers utilizadas mostraron el producto de amplificación esperado correspondiente al tamaño del gel pol únicamente para la combinación de primers: 5 -191 1 v 3'-3602 (TablaThe results obtained with the different combinations of primers used showed the expected amplification product corresponding to the size of the pol gel only for the combination of primers: 5-191 1 v 3 ' -3602 (Table
1 ). one ).
La dilución más pequeña en la que se obtuvo producto de amplificación corresponde al número mínimo de copias de RNA vírico/mL que deberá ser usado posteriormente para la amplificación de las muestras en estudio. Como se muestra en la figura 2 la dilución 7 (D7) fue la dilución más pequeña en la que se obtuvo producto de amplificación, que corresponde a 14 copias totales de RNA vírico. The smallest dilution in which the amplification product was obtained corresponds to the minimum number of viral RNA copies / mL that should be used later for the amplification of the samples under study. As shown in Figure 2, dilution 7 (D7) was the smallest dilution at which amplification product was obtained, which corresponds to 14 total copies of viral RNA.
1. 4. Protocolo de amplificación. 1. 4. Amplification protocol.
Una vez conocido el umbral mínimo de copias/mL necesario para conseguir una amplificación exitosa, se llevó a cabo la amplificación y genotipado de diferentes muestras plasmáticas con niveles de carga viral variables. Se realizó para ello la extracción de RNA vírico a partir de plasma aislado de pacientes utilizando el protocolo de extracción de ARN viral (Qiagen). En los pacientes con un número de copias de RNA mL de sangre muy bajo, se concentró la muestra pasando el volumen de plasma necesario para obtener la carga viral mínima por una columna de afinidad por RNA del sistema de QlAamp Viral RNA Mini Kit (QIAGEN, Valencia, CA, USA). Once the minimum threshold of copies / mL needed to achieve a successful amplification was known, the amplification and genotyping of different plasma samples with varying viral load levels was carried out. For this purpose, viral RNA was extracted from plasma isolated from patients using the viral RNA extraction protocol (Qiagen). In patients with a very low copy of RNA mL of blood, the sample was concentrated by passing the volume of plasma necessary to obtain the minimum viral load through an RNA affinity column of the QlAamp Viral RNA Mini Kit system (QIAGEN, Valencia, CA, USA).
A continuación, se llevó a cabo una reacción de retrotranscripción utilizando como molde el RNA vírico aislado junto con distintas combinaciones de los primers específicos diseñados (Tabla 1 ). El cDNA obtenido fue utilizado a continuación para la amplificación por PCR utilizando una enzima polimerasa de alta afinidad de SuperScriptII I One-Step RT-PCR System con la polimerasa Platinum Taq (Invitrogen Corporation, Carlsbad, CA, USA). Next, a retrotranscription reaction was carried out using the isolated viral RNA as a template together with different combinations of the specific primers designed (Table 1). The cDNA obtained was then used for PCR amplification using an enzyme polymerase. High-affinity of SuperScriptII I One-Step RT-PCR System with Platinum Taq polymerase (Invitrogen Corporation, Carlsbad, CA, USA).
Los resultados obtenidos con las distintas combinaciones de primers utilizadas mostraron el producto de amplificación esperado correspondiente al tamaño del gel pol únicamente para la combinación de primers: 5 -191 1 y 3'-3602 (TablaThe results obtained with the different combinations of primers used showed the expected amplification product corresponding to the size of the pol gel only for the combination of primers: 5-191 1 and 3 ' -3602 (Table
1 ). one ).
La reacción de retrotranscripción se incubó a 55°C durante 30 minutos, seguida de la amplificación por PCR con las condiciones de amplificación siguientes: 1 ciclo de 2 minutos a 94°C, 40 ciclos de 15 segundos a 94°C; 30 segundos a 55°C; 90 segundos a 68°C y un último ciclo de 5 minutos a 68°C, tras el cual las muestras se mantuvieron a 4°C hasta su utilización. Los productos de la amplificación fueron separados por electroforesis en un gel de agarosa al 1 %. The retrotranscription reaction was incubated at 55 ° C for 30 minutes, followed by PCR amplification with the following amplification conditions: 1 cycle of 2 minutes at 94 ° C, 40 cycles of 15 seconds at 94 ° C; 30 seconds at 55 ° C; 90 seconds at 68 ° C and a last cycle of 5 minutes at 68 ° C, after which the samples were kept at 4 ° C until use. The amplification products were separated by electrophoresis on a 1% agarose gel.
El protocolo de amplificación se llevó a cabo con éxito en un total de 50 muestras con carga viral por debajo de 1 .000 copias/mL. En la figura 3 se muestra un ejemplo utilizando plasma de un paciente con carga viral de 85 copias/mL. The amplification protocol was successfully carried out in a total of 50 samples with viral load below 1 000 copies / mL. An example using plasma from a patient with viral load of 85 copies / mL is shown in Figure 3.
Se obtuvo producto de amplificación en todos los casos mostrados en la tabla 2, en la que se representa la utilización de este protocolo en un total de 14 muestras de pacientes con carga viral menor a 200 copias/mL. El protocolo diseñado permite, por tanto, obtener un límite de detección de la amplificación de 20 copias de RNA totales por mL de plasma. Amplification product was obtained in all cases shown in Table 2, which represents the use of this protocol in a total of 14 samples of patients with viral load less than 200 copies / mL. The designed protocol allows, therefore, to obtain an amplification detection limit of 20 copies of total RNA per mL of plasma.
Carga viral Volumen plasma Viral load Plasma volume
Paciente  Patient
(copias RNA/mL) (ML)  (RNA / mL copies) (ML)
G0 85 470  G0 85 470
G5 98 560  G5 98 560
G6 130 560  G6 130 560
G8 72 560  G8 72 560
G10 20 560 G12 20 560 G10 20 560 G12 20 560
G14 161 420  G14 161 420
G23 122 420  G23 122 420
G25 104 420  G25 104 420
G27 154 420  G27 154 420
G56 89 560  G56 89 560
G59 96 560  G59 96 560
G64 124 420  G64 124 420
G66 92 560  G66 92 560
Tabla 2. Características de los pacientes con carga viral inferior a 200 copias/mL en los que se ha aplicado el protocolo de amplificación y genotipado optimizado. Se destaca para cada paciente la carga viral (copias RNA/mL) de partida y el volumen de plasma necesario para el aislamiento de RNA. Table 2. Characteristics of patients with viral load less than 200 copies / mL in which the optimized amplification and genotyping protocol has been applied. The starting viral load (RNA / mL copies) and the volume of plasma necessary for RNA isolation are highlighted for each patient.
Ejemplo 2. Genotipado del VIH a partir de muestras plasmáticas que presentan de 20 a 1.000 copias de ARN/mL. Para verificar la calidad de las secuencias del gen pol, obtenidas tras la amplificación, se procedió a la secuenciación de los productos obtenidos. Para ello, las bandas obtenidas y separadas en el gel de electroforesis como se explica en el apartado anterior fueron cortadas y purificadas utilizando el kit de purificación de bandas en gel QIAquick gel extraction (QIAGEN, Valencia, CA, USA). En todos los casos la concentración de las bandas purificadas osciló entre 35 y 40 ng^L. Para la secuenciación (Secugen, España) de cada producto de amplificación fue necesario un volumen mínimo de 15 μΙ_. Example 2. Genotyping of HIV from plasma samples that have 20 to 1,000 copies of RNA / mL. To verify the quality of the pol gene sequences, obtained after amplification, the products obtained were sequenced. For this, the bands obtained and separated in the electrophoresis gel as explained in the previous section were cut and purified using the QIAquick gel extraction gel band purification kit (QIAGEN, Valencia, CA, USA). In all cases the concentration of the purified bands ranged between 35 and 40 ng ^ L. For the sequencing (Secugen, Spain) of each amplification product a minimum volume of 15 μΙ_ was necessary.
Ejemplo 3. Determinación precoz de mutaciones de resistencia a partir de muestras plasmáticas que presentan de 20 a 1.000 copias de ARN/mL. Example 3. Early determination of resistance mutations from plasma samples that have 20 to 1,000 copies of RNA / mL.
Las secuencias obtenidas correspondientes al gen pol fueron analizadas utilizando la base de datos gratuita y disponible a través de la Universidad de Stanford {HIV Drug Resistance Datábase: http://hivdb.stanford.edu/) para obtener el patrón de resistencias. La calidad de las secuencias obtenidas fue comparada con las obtenidas previamente para el mismo paciente utilizando el programa vector NTI 10.3.0 (Invitrogen Corporation). El alineamiento de las secuencias obtenidas con las disponibles con anterioridad mostraron una identidad del 100%, demostrando que las secuencias obtenidas en los productos de amplificación, no difieren del genotipo obtenido utilizando métodos convencionales, obteniendo así una alta fidelidad en el genotipado final y, por tanto, que se pueden obtener secuencias desde muestras con un número mínimo de 20 copias del genoma viral, demostrando la optimización del método. The sequences obtained corresponding to the pol gene were analyzed using the free database and available through Stanford University {HIV Drug Resistance Datábase: http://hivdb.stanford.edu/) to obtain the resistance pattern. The quality of the sequences obtained was compared with those previously obtained for the same patient using the NTI 10.3.0 vector program (Invitrogen Corporation). The alignment of the sequences obtained with those previously available showed a 100% identity, demonstrating that the sequences obtained in the amplification products do not differ from the genotype obtained using conventional methods, thus obtaining high fidelity in the final genotyping and, by so much so that sequences can be obtained from samples with a minimum number of 20 copies of the viral genome, demonstrating the optimization of the method.
Ejemplo 4. Optimización del tratamiento antirretroviral en base a la detección precoz de mutaciones de resistencia. Example 4. Optimization of antiretroviral treatment based on the early detection of resistance mutations.
4. 1. Hipótesis. 4. 1. Hypothesis.
La optimización del tratamiento antirretroviral de manera precoz en pacientes con fracaso virológico, en base a la determinación del genotipo viral es esencial para seleccionar nuevas combinaciones de fármacos a las que el virus sea sensible, y conseguir suprimir de nuevo la replicación viral evitando la acumulación de nuevas mutaciones que puedan comprometer futuros tratamientos. The optimization of early antiretroviral treatment in patients with virological failure, based on the determination of the viral genotype is essential to select new combinations of drugs to which the virus is sensitive, and to suppress viral replication again avoiding the accumulation of new mutations that may compromise future treatments.
4. 2. Objetivo General. Optimizar el tratamiento antirretroviral de forma precoz en pacientes con fracaso virológico y con viremias bajas y en pacientes con fracaso virológico asociado al uso del inhibidor de la integrasa, adaptando su prescripción a las mutaciones de resistencia observadas y a las concentraciones de fármaco valle observadas. 4. 3. Objetivos específicos. 4. 2. General Objective. Optimize antiretroviral treatment early in patients with virological failure and with low viremias and in patients with virological failure associated with the use of the integrase inhibitor, adapting its prescription to the resistance mutations observed and the observed valley drug concentrations. 4. 3. Specific objectives.
1. Amplificar los genes de la retrotranscriptasa y proteasa en pacientes en fracaso virológico con carga viral plasmática por debajo de 1 .000 copias/ml. 1. Amplify the retrotranscriptase and protease genes in virological failure patients with plasma viral load below 1 000 copies / ml.
2. Determinar las mutaciones de resistencias asociadas a los genes de la retrotranscriptasa y la proteasa amplificados. 2. Determine resistance mutations associated with amplified retrotranscriptase and protease genes.
3. Adaptar la dosificación de los fármacos en función de las mutaciones de resistencia observadas. 3. Adapt the dosage of the drugs according to the resistance mutations observed.
4. 4. Diseño. 4. 4. Design.
Para ello se recogieron muestras de plasma de pacientes que presentaban fracaso virológico con cargas virales inferiores a 1 .000 copias/ml. A partir de las muestras de plasma, se llevó a cabo por RT-PCR, la retrotranscripción del ARN de VIH aislado y la posterior amplificación de los genes de la retrotranscriptasa y la proteasa para su secuenciación y obtención del genotipo viral. En base a la historia previa de tratamiento y a los resultados del ensayo genotípico de resistencias se modificó el TAR de forma que el nuevo régimen contase con > 2 fármacos completamente activos. Finalmente, se evaluó la eficacia del programa de optimización del tratamiento antirretroviral en pacientes con fracaso virológico con baja carga viral valorando el porcentaje de pacientes incluidos que consiguen una CV indetectable tras el cambio de tratamiento. A todos los candidatos se les solicitó la participación en el estudio a través de consentimiento informado. For this, plasma samples were collected from patients who presented virological failure with viral loads of less than 1,000 copies / ml. From the plasma samples, it was carried out by RT-PCR, the retrotranscription of the isolated HIV RNA and the subsequent amplification of the retrotranscriptase and protease genes for sequencing and obtaining the viral genotype. Based on the previous history of treatment and the results of the genotypic resistance test, ART was modified so that the new regimen had> 2 fully active drugs. Finally, the efficacy of the antiretroviral treatment optimization program in patients with virological failure with low viral load was evaluated, assessing the percentage of patients included who achieve undetectable CV after the change of treatment. All candidates were asked to participate in the study through informed consent.
4. 5. Resultados. Hasta la fecha han sido incluidos 47 pacientes que presentaban fracaso viral con cargas virales por debajo de 1 .000 copias/ml. Se recogieron muestras de plasma de las que se aisló el ARN vírico que se utilizó como molde para amplificar por RT-PCR los genes de la retrotranscriptasa y la proteasa. Los productos amplificados fueron secuenciados y se obtuvo el genotipo viral. 4. 5. Results. To date, 47 patients who had viral failure with viral loads below 1,000 copies / ml have been included. Plasma samples were collected from which viral RNA was used which was used as a template for amplify the retrotranscriptase and protease genes by RT-PCR. The amplified products were sequenced and the viral genotype was obtained.
En 21 de los casos (44%) se optó por cambiar el tratamiento en base al genotipo obtenido y a la historia previa de tratamiento. Como consecuencia, en 19 (91 %) de los pacientes la carga viral se hizo indetectable tras el cambio de tratamiento. En los dos pacientes restantes aún no ha pasado el tiempo necesario para su evaluación. In 21 of the cases (44%) it was decided to change the treatment based on the genotype obtained and the previous history of treatment. As a consequence, in 19 (91%) of the patients the viral load became undetectable after the change of treatment. In the two remaining patients, the time necessary for their evaluation has not yet passed.
4. 6. Conclusión. 4. 6. Conclusion.
Como consecuencia de la optimización del tratamiento, en base a la determinación precoz del genotipo viral en pacientes con fracaso virológico con cargas virales por debajo de 1 .000 copias/ml, se consigue alcanzar una carga viral plasmática indetectable que es el objetivo actual del tratamiento antirretroviral. As a consequence of the optimization of the treatment, based on the early determination of the viral genotype in patients with virological failure with viral loads below 1,000 copies / ml, an undetectable plasma viral load is achieved, which is the current goal of the treatment. antiretroviral

Claims

REIVINDICACIONES
Método de detección precoz de VIH que comprende: a. obtener una muestra biológica aislada, Method of early detection of HIV comprising: a. obtain an isolated biological sample,
b. extraer el ARN del VIH presente en la muestra del paso (a) mediante una columna de afinidad por ARN,  b. extracting the HIV RNA present in the sample from step (a) using an RNA affinity column,
c. poner en contacto el ARN obtenido en el paso (b) con una mezcla de reacción que contiene los cebadores SEQ ID NO: 1 y SEQ ID NO: 2,  C. contacting the RNA obtained in step (b) with a reaction mixture containing primers SEQ ID NO: 1 and SEQ ID NO: 2,
d. retrotranscribir el gen pol del VIH y amplificar el ADNc obtenido mediante RT-PCR, y  d. retrotranscribe the pol gene of HIV and amplify the cDNA obtained by RT-PCR, and
e. detectar los productos de la RT-PCR obtenidos en el paso (d).  and. Detect the RT-PCR products obtained in step (d).
Método de genotipado del VIH que comprende todos los pasos según la reivindicación 1 y además: f. secuenciar los productos detectados en el paso (e). HIV genotyping method comprising all the steps according to claim 1 and further: f. sequence the products detected in step (e).
Método de detección precoz de mutaciones de resistencia del VIH al tratamiento antirretroviral que comprende todos los pasos del método según la reivindicación 1 y del método según la reivindicación 2, y además: g. comparar las secuencias obtenidas en el paso (f) con una base de datos de secuencias del VIH. Method of early detection of resistance mutations of HIV to antiretroviral treatment comprising all the steps of the method according to claim 1 and the method according to claim 2, and in addition: g. compare the sequences obtained in step (f) with an HIV sequence database.
Método según cualquiera de las reivindicaciones 1 a 3 donde la muestra biológica del paso (a) presenta entre 10 y 1 .000 copias de ARN de VIH por ml_ de muestra. Method according to any one of claims 1 to 3 wherein the biological sample from step (a) has between 10 and 1,000 copies of HIV RNA per ml of sample.
5. Método según cualquiera de las reivindicaciones 1 a 4 donde la muestra biológica del paso (a) presenta entre 20 y 1 .000 copias de ARN de VIH por ml_ de muestra. 6. Método según cualquiera de las reivindicaciones 1 a 5 donde la muestra biológica del paso (a) presenta entre 20 y 100 copias de ARN de VIH por ml_ de muestra. 5. Method according to any one of claims 1 to 4 wherein the biological sample from step (a) has between 20 and 1,000 copies of HIV RNA per ml of sample. 6. A method according to any one of claims 1 to 5 wherein the biological sample from step (a) has between 20 and 100 copies of HIV RNA per ml of sample.
7. Método según cualquiera de las reivindicaciones 1 a 6 donde la muestra biológica aislada del paso (a) es plasma sanguíneo. 7. Method according to any of claims 1 to 6 wherein the biological sample isolated from step (a) is blood plasma.
8. Método según cualquiera de las reivindicaciones 1 a 7 donde la polimerasa empleada en cualquiera de los pasos (c) y (d) es de alta afinidad. 9. Método según cualquiera de las reivindicaciones 1 a 8 donde la detección de los productos de la RT-PCR del paso (e) se realiza mediante electroforesis. 8. Method according to any of claims 1 to 7 wherein the polymerase used in any of steps (c) and (d) is of high affinity. 9. Method according to any of claims 1 to 8 wherein the detection of the RT-PCR products of step (e) is carried out by electrophoresis.
10. Cebadores de secuencia nucleotídica SEQ ID NO: 1 y SEQ ID NO: 2. 10. Nucleotide sequence primers SEQ ID NO: 1 and SEQ ID NO: 2.
1 1 . Uso de los cebadores según la reivindicación 10 para amplificar el gen pol del VIH en una muestra de plasma que presenta entre 10 y 1 .000 copias de ARN de VIH por ml_ de plasma. 12. Uso de los cebadores según la reivindicación 1 1 donde la muestra de plasma presenta entre 20 y 1 .000 copias de ARN de VIH por ml_ de plasma. eleven . Use of the primers according to claim 10 to amplify the HIV pol gene in a plasma sample having between 10 and 1,000 copies of HIV RNA per ml_ of plasma. 12. Use of the primers according to claim 1 wherein the plasma sample has between 20 and 1,000 copies of HIV RNA per ml_ of plasma.
13. Uso de los cebadores según cualquiera de las reivindicaciones 1 1 ó 12 donde la muestra de plasma presenta entre 20 y 100 copias de ARN de13. Use of the primers according to any one of claims 1 or 12 wherein the plasma sample has between 20 and 100 copies of RNA from
VIH por ml_ de plasma. HIV per ml_ of plasma.
14. Kit de detección y/o genotipado del VIH que comprende los cebadores según la reivindicación 10. 14. HIV detection and / or genotyping kit comprising the primers according to claim 10.
15. Kit de detección y/o genotipado del VIH según la reivindicación 14 que además comprende una polimerasa de alta afinidad. 15. HIV detection and / or genotyping kit according to claim 14 further comprising a high affinity polymerase.
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CN111560480A (en) * 2020-06-01 2020-08-21 昆明市官渡区妇幼保健计划生育服务中心 Method for detecting HIV-1 drug resistance of pregnant and lying-in women

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