WO2012019260A2 - A synthetic rna molecule of the hepatitis c virus, production method of same and use thereof as an internal control for the quantification and/or identification of the hepatitis c virus - Google Patents

Abstract

The present invention relates to an internal control for the detection and/or quantification of the hepatitis C virus using various methods. Said control may have one or two nucleotide sequence modifications: an insertion and/or substitution of one sequence for another, making the molecule polyvalent, and allowing it to be used in diverse quantification methods. The present invention relates to the method for developing said internal control. The present invention also relates to a detection and/or quantification kit for the hepatitis C virus.

Description

 Patent Invention Descriptive Report

HEPATITIS C VIRUS SYNTHETIC RNA Molecule, PROCESS FOR OBSERVATION AND USE AS A CONTROL

FOR THE QUANTIFICATION AND / OR IDENTIFICATION OF

 HEPATITIS C VIRUS

Field of the Invention

 The present invention pertains to healthcare, more precisely to the diagnosis of viral diseases, and describes an internal control produced from an unnatural synthetic RNA molecule, a virus such as Hepatitis C Virus (HCV) and useful as a tool for quantification and / or viral identification by different methodologies through the use of this internal control.

Background of the Invention

 In many infectious pathologies, quantification tests are important in monitoring them, in deciding to start or continue treatment, in response to therapy, in prognosis, among other factors relevant to clinical management.

A pathogen quantification test can be based on determining the number of genomes of this organism present in the biological sample. This quantification can be used as a parameter for treatment of virus diseases. The use of quantitative tests is important in the Flaviviridae family, since it encompasses, among others, the Hepatitis C Virus (HCV). HCV infection represents a public health problem in Brazil and worldwide with an estimated 170 to 200 million infected individuals worldwide, which corresponds to 3% of the world's population (World Health Organization, WHO, 2007). It is recognized as the largest cause of chronic viral hepatitis, leading the cases of cirrhosis and hepatocellular carcinoma (HCC), which is the sixth most common cancer in the world, with over 500,000 new cases per year. HCC is still the third cancer-related cause of death and the leading cause of death in cirrhotic patients (Llovet et al, 2008; WHO, 2007; Ministry of Health, MS, 2005; Kim, 2002; EASL International Consensus Conference on Hepatitis C, 1999).

 In addition to the hepatic effects, patients with Chronic Hepatitis C may have several extrahepatic manifestations, such as hematologic, renal, dermatological, endocrine, neuromuscular and joint diseases; changes in salivary and ocular glands; in addition to autoimmune and psychological disorders (Grossmann et al, 2008).

 The form of hepatitis C transmission is mainly parenteral, being considered at risk populations: individuals who received blood transfusion and / or blood products before 1993 when the first serological tests appeared, in addition to intravenous drug users. However, the cause of infection is undetermined in some patients (MS, 2005). Current treatment has not yet yielded satisfactory results for all treated patients. Only 50% of the treated exhibit viremia negativity six months after the end of treatment (sustained viral response - SVR) (Central for Disease Control and Prevention, CDC, 2008).

Conventional treatment is performed with Interferon (IFN 2a) and Ribavirin (RBV), which cause several adverse and severe effects, compromising the patient's quality of life, and are costly (Schackman et al, 2008). Thus, it has been of great value to establish predictive factors of treatment response, such as determining the rate of viral load drop (Lee et al, 2002). Thus, patients with high viral load at pretreatment are less likely to have SVR than those with low viral load. Twelve weeks after initiation of treatment, viral load should be re-evaluated to check for early viral response (PVR) (two log reduction units compared to pretreatment or non-detection of HCV RNA in serum) (Araújo et al. ai, 2007; Consensus on Conduct in Viral Hepatitis B and C, 2005; NIH Consensus Statement on Management of Hepatitis C, 2002; Lee et al., 2002). Another important type of response is rapid viral response (RVR), that is, negativity within the fourth week of treatment, allowing to know the chance of the patient being a responder before twelve weeks (Napoli et al, 2008; Lee et al, 2002) .

 IFN and RBV are part of the Brazilian Unified Health System (SUS) Exceptional Medicines Program. Data from 1999 showed annual spending on IFN alpha 2a / b of R $ 13,773,561.00 and RBV of R $ 884,668.00 (MS, available at: http://dtr2001.saude.gov.br/sas/relatorio /6.6%20Assistance.htm). In 2004, Pegylated IFN was the most expensive drug for the Brazilian public health system (Dornelles Picon et al, 2005).

 Hepatitis C, due to its magnitude, etiological diversity, forms of transmission, clinical evolution, in addition to its diagnostic and therapeutic complexity, demands from SUS managers specific public health policies (Federal Official Gazette n. 195, 09 / 10/2007).

 The diagnosis of hepatitis C is made by two test categories: indirect tests, which are serological assays for specific detection of anti-HCV antibody, and direct tests, which detect components of the viral particle, such as its genomic RNA (Pawlotsky, 2002). ).

The approach recommended for the laboratory diagnosis of hepatitis C is primarily performing serology ELISA method or ^the second generation to ^the third test for the presence of anti-HCV antibodies. Serologic tests, however, do not distinguish past infection from present infection or the phase of the disease, whether acute or chronic, and do not predict its progression (Grossmann et al, 2008). To verify if the infection is active, the presence of the viral particle is demonstrated by detection of viral RNA in the patient's serum (HCV RNA qualitative research). Thus, serological methods such as ELISA and RIBA (recombinant immunoblot assay), despite having adequate specificity, do not indicate presence of active infection. The ELISA test is low cost, but the immunoblot is high cost and less sensitive than the ELISA. Therefore, whether in diagnosis or in follow-up of infection, the most appropriate methods are those based on viral RNA detection and quantification (CDC, 2008; Consensus on Conduct of Viral Hepatitis B and C, 2005; WHO, 2000). Another limitation of immunoassays is related to the immunological window, as the average incubation period for antibody onset is 7 to 8 weeks, but this range can range from 2 to 26 weeks.

 Commercially available quantitative methods are based on target amplification (regular RT-PCR or real-time RT-PCR) or signal amplification (branched DNA - bDNA) (Pawlotsky, 2002). PCR (polymerase chain reaction) based methods are the most widespread and most practical. Ideally, the amplification of the nucleic acid being quantified and not the amplification of the hybridization signal, as in the case of bDNA.

 DE19814828 describes a generic method for identifying microorganisms based on amplifying a region of the target microorganism's genome and further hybridizing it to specific probes. Although it can be used with different microorganisms, the method does not provide for quantification.

 US2007 / 0141563 describes a method of detecting a target biomolecule (peptides, proteins, oligosaccharides, lipids, steroids, prostaglandins, prostacyclins and nucleic acids) in a biological sample suspected of contamination with a pathological agent. However, extra efforts will be required to quantify the target biomolecule.

In viral load measurement procedures, the accuracy of the method is known to be significantly increased when an internal control is used, ie an indicator (such as an RNA molecule) that accompanies the HCV genomic RNA present in the sample. , at all stages of its quantification. Thus, internal control monitors the methodological process as a whole, and losses during extraction, as well as the presence of inhibitors in the amplification reaction, will equally affect both targets, HCV RNA and internal control, producing more quantification. need. As is well known to those skilled in the art, this is valid for the quantification of any virus or microorganism.

 In addition, it is important that internal control has the same primer annealing sites as HCV RNA, giving rise to competitive PCR, which is not possible with external controls.

 The first quantitative methods of HCV were done semi-quantitatively, where successive dilutions of the serum to be quantified were made.

 In addition, a serial dilution of a known amount of standard was amplified in parallel with the samples of interest. However, one of the disadvantages of this method is the inability to evaluate sample-by-sample variations due to differences in amplification efficiency and the accuracy and reproducibility that may be affected (Kochanowski, B. & Reischl, U., 1999).

 With the emergence of commercial panels and sera containing known amounts of HCV, these were used for quantification through standard curves.

 However, none of the above methods necessarily uses an internal control and therefore the extraction process up to the quantification stage itself is not adequately monitored, such as the presence of reaction inhibitors, which may lead to less accurate quantification.

 With the advent of real-time PCR, quantification can be done using double stranded DNA interleavers, such as the SYBR Green method (Applied Biosystems; Invitrogen; among others). However, they are not target-specific interleaving. Still with real-time PCR, the use of specific probes allows the design of a probe for each target, HCV and internal control, making quantification more specific.

US2005 / 0202417 uses a method for detecting and quantifying HCV in biological samples, which employs a defined amount of an oligonucleotide labeled as a specific probe for internal control and a second oligonucleotide labeled as a probe. specific to the HCV genome. Although internal control presents a modification in its nucleotide sequence that differentiates it from HCV, this modification only allows the development of assays that use specific probes. And as is well known to those skilled in the art, methods using probes are costly.

 US2002 / 0187488 describes a method for quantifying HCV using an internal control synthesized from bovine viral diarrhea virus (BVDV) and not from HCV itself. Thus, despite the high similarity between HCV and BVDV, the ideal is to use an internal control developed from the virus itself to be quantified, and not from a similar virus. This type of PCR is noncompetitive, and the ideal is to perform competitive PCR quantification, ie when the primer annealing sites are the same for HCV RNA and internal control.

 EP398748 describes oligomers capable of identifying the presence of HCV in biological samples due to its ability to hybridize to an HCV genome sequence. These oligomers can be employed as probes and primers in PCR reactions. One of the problems with this method is that extra efforts will be required for viral quantification as it only detects the virus.

 US 6,638,714 relates to a HCV detection method which, although it can be done in a single PCR step and with high sensitivity, unlike most methods that are done in two> PCR steps, does not allow viral quantification. Thus, extra efforts will be necessary to determine the viral levels in the biological sample.

 US 2005/0123901 describes a qualitative method for

HCV using primers preferably labeled with fluorescein, among others possible. Although allowing different types of detection according to the oligonucleotide marker, it does not allow viral quantification, requiring extra efforts.

US 5,580,718 and US 5,837,442 describe methods for HCV detection, without mentioning viral quantification, which will require extra effort. EP1746170 relates to a process of HCV genotyping from varied biological samples, without, however, referring to viral quantification. Therefore, extra efforts will be required to determine viral levels. A similar object is described in EP1746171 concerning a HCV genotyping process in biological samples, which also allows viral quantification in biological samples subjected to real time PCR. HCV genotyping is done by amplifying HCV 5'NTR (untranslated region) and NS5 (non-structural region 5) regions and further amplicon hybridization (amplification product) with specific probes capable of identifying existing polymorphism and identifying the viral subtype. Quantification is performed without the use of molecules that act as internal controls. Although quantitative, this method does not use an internal control that monitors the steps from viral RNA extraction to quantification, making quantification less accurate.

 US 7,462,709 describes a method for determining nucleic acid using a control, and as an example describes for HCV. Although the control has properties similar to the target nucleic acid, it has the disadvantage of providing a non-competitive PCR. Similar method is described in US 7,183,084.

 EP1026262 describes a sensitive method of detecting HCV in a sample based on amplification of the 5'NTR or 3'NTR region of the virus. This document, while describing a sensitive test, does not provide for viral quantification. One of the problems with the technique is that additional efforts will be needed to determine viral levels.

WO2004011612 provides a method and a kit for quantifying and identifying HCV present in a sample through an internal control. Transcribed HCV RNA is co-amplified with competitive PCR HCV RNA. Although this quantification occurs through internal control and competitive type PCR, this internal control is not polyvalent, that is, it can only be used in methods that use specific probes, which as the technicians in the field know, are expensive methods. WO / 2004/11612 describes a method for detection and quantification of HCV by capture methods, for example plaque. Although PCR is of the competitive type between HCV RNA and internal control, probe oligonucleotides for each of the targets are labeled and the detection method will depend on the marker type. As is well known to those skilled in the art, labeling allows this assay to be performed by some costly methodologies based on specific probes.

 US 7,250,506 describes a qualitative and quantitative method for HCV using an internal control that promotes competitive PCR. Despite this, the internal control was developed from an HCV-phage lambda hybrid, and ideally the internal control should be developed from the HCV itself.

The AMPLICOR HCV MONITOR TEST kit (Roche Diagnostics) utilizes an internal control designed in the HCV 5 ^* NTR region containing 351 nucleotides. Knowing that the HCV genome has approximately 9,600 nucleotides, we find that this internal control is very small, much smaller than HCV. Despite being a competitive-type RT-PCR, internal control has only one type of modification: one substitution of one HCV sequence for another, so the internal control and HCV amplicons ultimately have the same size. As a non-exhaustive example of this issue, it does not allow the individualization of internal control and HCV in agarose gel, only in methods with the use of target-specific probes, which are the most expensive methods.

Martell et al (1999) and Kleiber et al (2000) described quantitative HCV methods using internal control produced from the HCV 5'NTR region. For real-time RT-PCR a standard curve is made, constructed from samples with known viral levels, instead of making a direct correlation between the results obtained by HCV and internal control, which takes the most accurate quantification. One of the problems with these methods is that measurement is not guaranteed in the absence of any indication of reaction inhibition. Takeuchi et al. (1999) described a quantitative method of HCV by non-competitive RT-PCR, where the primers used for HCV amplification and internal control are different. The production of two amplicons (amplification products) has as disadvantage the lack of comparison between targets, as they can be amplified with different efficiencies, making quantification less accurate.

 Castelain et al. (2004) describes an internal control developed from the bovine viral diarrhea virus (BVDV). Despite the high similarity between HCV and BVDV, the internal control developed is from a different virus than one wants to quantify and therefore PCR is non-competitive. Similarly, Halfon et al. (2006) described an internal control for HCV derived from the hydroxypyruvate reductase gene from the Curcubita pepo and Villanova et al. (2007) from RNA from phage Qp of Escherichia coli. Thus, these methods, because they have different internal control than HCV, are not a competition reaction and are therefore subject to different amplification efficiencies.

 As can be seen, molecules that mimic HCV were already used as internal control, but in specific methodologies, not allowing the adaptation of the small laboratory or linked to entities, public research and health institutions to the technique most appropriate to their conditions. infrastructure and technical personnel, reagents and equipment. Furthermore, these described molecules lack the polyvalence of being able to be developed containing one or more modifications in their nucleotide sequence. And polyvalence is a potential target for constant improvement by altering its nucleotide sequence.

 To date, the state of the art has disadvantages and problems, such as the lack of polyvalent internal control that can be used by different methodologies.

The present invention aims to present solutions to the problems of the state of the art bringing as differential an internal control of RNA with high homology to the HCV genome, capable of being used as a control. internal viral quantification through different methodologies, with different laboratory infrastructure needs, different costs and use of reagents and equipment, ie it is multipurpose. Although internal controls developed from HCV are already part of the state of the art, this invention provides internal RNA control (Seq. Id 1, 2 and 3) which contains a sequence designed specifically to make the amplification product length of said control. distinct from the length of the HCV amplification product contained in the sample under analysis.

 The fact that internal control was developed from the genome of the virus itself to be detected and / or quantified, generating competitive amplification (competitive PCR) allows it to be used for both HCV quantification and detection, as well as also as a "control point" in purifications, reactions, subsequent steps, discarding different amplification efficiencies between HCV RNA and internal control.

Summary of the Invention

 The main object of the present invention is the process of producing internal control for the quantification and / or identification of a virus present in a biological sample, a process based on the synthesis of a synthetic, unnatural polynucleotide molecule with high homology to an untranslated sequence of the genome of a virus.

The second object deals with an internal control that can be used to quantify and / or identify viruses in a biological sample. This internal control is produced from a viral RNA molecule containing further modifications generated by the insertion and / or substitution of polynucleotide sequences. known. Said internal control is therefore a synthetic, unnatural, modified polynucleotide molecule that contains high homology to a specific viral genome sequence. This internal control enables the amplification, quantification and identification of the genome of a virus present in a biological sample. Also an object of this invention is an in vitro viral diagnostic method for the diagnosis of a virus causing a disease and / or dysfunction in a mammalian animal.

 An in vivo viral diagnostic method for diagnosing a virus causing disease and / or dysfunction in a mammalian animal is further object of this invention.

 Another object of this invention is a method of treatment wherein the patient is monitored by viral load comprising the steps of extracting viral RNA from biological sample, cDNA synthesis and amplification of an HCV genome region by chain reaction. of the quantitative polymerase at some stages before, during and after antiviral treatment.

 Finally, it is an object of this invention to use internal control for the preparation of compositions useful for viral diagnosis.

 Another object of this invention is a stabilizing composition containing internal control, stabilizing substances and chemical adjuvants.

 It is a further object of this invention a kit for viral quantification in a biological sample comprising an internal control molecule and a stabilizing composition thereof.

 Another object of this invention is a kit for identifying a virus in a biological sample comprising an internal control molecule and a stabilizing composition thereof.

Description of the Figures

Fig. 1: Schematic of the development of internal control through modifications in the 5 ^' untranslated region (NTR) of the hepatitis C virus (HCV) genome.

 Fig. 2: Schematic of internal control development containing the following modifications: insertion only; replacement only; both modifications (insertion and replacement).

Fig. 3: Electrophoretic analysis of 5 ^' NTR region fragments containing insertion and / or substitution obtained by HCV RT-PCR. Fig. 4: Autoradiography of in vitro transcribed HCV RNA containing insertion and replacement, showing that it is in the expected size of 1.1 Kb.

 Fig. 5: Analysis of the presence of plasmid DNA from the clone containing the insertion and amplifiable substitution after treatment of the internal control preparation with digestive enzymes.

 Fig. 6: Agarose gel electrophoresis evaluation of internal control stability in extraction solution for up to 60 days at 4 ° C.

Fig. 7: Standardization of internal control content to be used as a competitor in RT-PCR assay. Raiai: Serum without internal control. Lanes 2 to 6: successive dilutions of a pool of HCV genotype 3a-infected sera and constant amount (5,000 molecules) of internal control. Lane 7: Internal control in HCV negative serum. Lane 8: M = 100 bp molecular weight marker. Lane 9: Negative reaction control (H ₂ 0). RT-PCR products were analyzed by 2% agarose gel electrophoresis on 1x TBE stained with ethidium bromide.

 Fig. 8: Probe specificity test specific for internal control. Fluorescence versus number of PCR cycles show that HCV RNA-containing serum was not detected and found to be specific.

 Fig. 9: HCV RNA specific probe specificity test.

 Fluorescence versus number of PCR cycles show no detection of internal control, showing to be specific.

 Fig. 10: Evaluation of specificity of HCV RNA probes and internal control present in the same reaction. Reaction 5 containing both probes was made. QRT-PCR analysis by TaqMan® system.

Amplification curves of the different serum dilutions and 5,000 internal control molecules shown in module Rn (reporter molecule signal measurement) versus cycle. Samples are indicated on the right. The lines with different colors represent the different samples: 1 = Pure Internal Control 0 (no serum), 2 = 1,000x Diluted Serum, 3 = 100x Diluted Serum, 4 = 10x Diluted Serum and 5 = Serum without Internal Control. Fig. 1 1: Amplification of HCV-containing serum and internal control by real-time RT-PCR using the two specific probes, for internal control and for HCV RNA. The amplification curves in this fluorescence versus number of PCR cycles plot show that HCV-containing serum is at different concentrations and internal control at constant concentration. As a control, serum containing HCV without internal control. Serum = 1.8x10 IU / mL.

 Fig. 12: Determination of the sensitivity limit of detection of internal control in serum by qRT-PCR. Amplification curves of the different amounts of internal control tested shown in module Rn (measurement of Γβροιΐβή molecule versus cycle signal. Samples are indicated on the right.

 Fig. 13: Determination of detection limit of internal control detection by nested RT-PCR and agarose gel electrophoresis.

 Fig. 14: Determination of HCV levels by agarose gel electrophoresis using internal control produced on previously known high, medium and low viral load sera. Analysis by 2% agarose gel electrophoresis in 1x TBE stained with ethidium bromide.

 Fig. 15 Correspondence between number of internal control molecules in International Units (Ul). 5,000 internal control molecules and a commercial panel of real-time PCR quantified sera based on IU / mL were used.

Detailed Description of the Invention

 For purposes of this invention we will make some definitions that will assist in further describing the claimed objects.

 Any virus with the ribonucleic acid (RNA) genome may be employed in this invention, more preferably, the virus employed in this invention is HCV and all its types and subtypes should be considered.

We consider as a region with a high degree of conservation of a genome, that nucleotide sequence that presents a homology greater than 90% when alignment of nucleotide sequences of different viral types and subtypes is observed. Preferably, said homologous region exhibits greater than 92% homology when an alignment of nucleotide sequences of different HCV viral types and subtypes is observed.

 Quantitative PCR is defined as PCR capable of defining the amount of a microorganism present in a biological sample, as well as being used for the in vitro diagnosis of viral contaminants contained in cell, tissue and replicon cultures.

 For purposes of this invention, replicons are molecules containing part of the genome of eukaryotic or prokaryotic cells or viruses and sequences that make them self-replicating.

 It is further understood that the sample of biological material may be derived from any human or non-human mammalian animal and may belong to the group consisting of: serum, biopsies, lymphocytes, whole blood and its fractions, blood products, saliva, semen, hair and skin. Preferably, the biological material sample of this invention is blood taken from human and non-human mammals; even more preferably, the biological sample is human serum obtained from whole blood.

 After making the appropriate definitions, we will describe in more detail the objects that will be claimed.

 The present invention relates to the process of producing an internal control useful for viral quantification and identification in a biological sample, which is comprised of any of Identifier Sequences 1, 2 and 3.

The beginning of the production process of said internal control begins by producing a copy of a region with a high degree of conservation of the viral genome and obtaining a cDNA of the said with a high degree of conservation. This process step begins by extracting viral RNA followed by reverse transcription (RT) reaction using any method as is known to those skilled in the art. Expansion of viral RNA requires the design of an oligonucleotide primer which can be any of the identifying sequences 4, 5, 6, 7, 10,11, 12 and 13.

 After obtaining a copy of said conserved region of the viral genome, two primers, one sense and one antisense, are designed to make modifications to said conserved sequence. One skilled in the art will appreciate that the location of modifications is not essential and can be performed in any position and in any order.

 Modifications are made by employing primers designed to contain a random nucleotide sequence that is not similar to any other sequence deposited in public databases so that this sequence is inserted into the PCR product called an "insert fragment" ( Seq. Id .: 5 and 8); as well as by designing the primer containing a random nucleotide sequence and not resembling any other sequence deposited in public databases to be replaced by the HCV sequence called the "replacement fragment" (Seq. Id .: 4 and 9 ).

 Preferably, a modification should be made by the addition, insertion, and / or both modifications. The sequence to be added contains between 30 and 100 nucleotides while substitution alters the highly conserved viral cDNA sequence between 10 and 100 nucleotides. Most preferably, the insert comprises a sequence containing from 45 to 60 nucleotides in length and the substitution of from 20 to 30 nucleotides in length.

The inserted sequence, hereafter referred to as insertion only, as well as the substituted sequence, hereafter referred to as substitution only, may not show significant similarity to any other viral genome or even to any other human polynucleotide sequence, which can be confirmed by searching. in databases, some audiences, such as GenBank (Entrez Nucleotide NCBI). One skilled in the art will appreciate that the inserted or substituted nucleotide sequence may be variable in size as mentioned above as well as in the order of nucleotides, provided that there is no significant similarity to database sequences, such as public ones.

 For the purposes of this invention, significant similarity means high similarity coverage, low "E" values (probability of similarity being by chance) and high percentage of identity.

 The primers used for making the modifications are extensive; SED ID: 4 and 5 should be used as external primers and SEQ ID 8 and 9 as internal primers.

 Preferably, the primer employed for performing the insertion is described in SEQ ID NO: 8 and the primer employed for performing the substitution is described in SEQ ID NO: 9; In addition, both modifications (insertion and substitution) may be introduced in the promotion of the modifications, both primers represented by Seq.ld 8 and 9 being employed.

 Insertion enables the development of quantification and / or identification methods by size difference between amplified fragments, ie insertion increases the amplicon molecular weight and can be used, for example, for quantification and / or identification by techniques such as agarose gel electrophoresis and double stranded DNA intercalating dye staining such as ethidium bromide and SYBR Green. These examples are not limiting of the present invention. This size difference between the ampicons (amplification product) of HCV and the internal control makes it possible to distinguish in agarose gel the products obtained by PCR amplification of the HCV genome from that of the artificial polynucleotide molecule of this invention. It also allows the design of specific probes and as a non-limiting example is the RT-PCR method followed by plaque capture.

The substitution allows the development of quantification and / or identification methods by specific probes, and as non-exhaustive examples of this invention we have the quantification and / or identification of amplified products, HCV RNA and internal control by RT-PCR capture by plate and real-time RT-PCR (qRT-PCR). Three possible modification strategies containing insertion and / or substitution can be performed at this stage in order to produce the 3 different PCR products:

 1) to form an internal control containing the 2 proposed modifications: insertion and substitution the external primers SEQ are used

ID NO: 4 and SEQ ID NO: 5 and the inner primers SEQ ID NO: 8 and SEQ ID NO: 9;

 2) to form an internal control containing only insertion modification, external primers SEQ ID NO: 4 and SEQ ID NO: 5 and internal primer SEQ ID NO: 8 are used;

 3) to form a fragment containing only substitution modification, the external primers SEQ ID NO: 4 and SEQ ID NO: 5 and the internal primer SEQ ID NO: 9 are used.

 For qRT-PCR it is important that probes are designed in the replacement region, as there is no size increase in the resulting sequence, unlike insertion. Thus, both the HCV and internal control qRT-PCR products are the same size and small as recommended by the method. This is because the specific probes should hybridize near the annealing site of the primers.

 The ideal PCR conditions in any strategy used are preferably between 0.1 to 0.3 mM dNTPs and between 0.5 to 2.5 units of the desired polymerase enzyme; a concentration of 10 to 30 pmoles of each primer, with about 1 unit of the enzyme. The buffer solution should be employed as described by the polymerase enzyme supplier.

 The cycling should be between 22 and 30 cycles, with denaturation between 90 and 95 ° C for 15 to 20 seconds; annealing between 50 and 65 ° C for 14 to 20 seconds; and the extension phase between 68 and 72 ° C for 15 to 20 seconds; and final extension between 70 and 72 ° C for 09 to 15 minutes.

The PCR products obtained should be cloned into any vector that enables the identification of colonies containing the insert. For the purposes of this invention, a vector is any vector that serves for cloning, transcription and expression. Non-limiting examples of this invention are: pGEX (GE Healthcare Life Sciences), pET (Novagen) and pMOS (Amersham).

 State-of-the-art vectors, such as vectors containing LacZ gene fragment and / or an antibiotic resistance gene, may be employed, but the use of vectors with these types of identification strategy should not be limited.

 Next, vectors containing the inserted PCR product, with one or both modifications, must be inserted into microorganisms to enable maintenance and multiplication of the transformed vector inserted by techniques belonging to the state of the art. Preferably employed microorganisms are Escherichia coli bacteria.

 Subsequently, plasmid DNA isolation should be performed by any protocol as known to those skilled in the art, such as by alkaline lysis, followed by sequencing of colonies for clone identification. The insert should be in the correct orientation for the subsequent in vitro transcription step.

 The next step requires clone DNA linearization with the use of some restriction enzyme according to methodologies known to those of skill in molecular biology. The restriction enzyme should preferably leave a large fragment to undergo the in vitro transcription process.

 Confirmation of clonal DNA linearization is performed on a 0.6 to 1.2% 5 agarose gel and the fragment of interest should be removed by any known agarose gel fragment extraction method.

The in vitro transcription reaction is then performed using a methodology known to those skilled in the art and using a tracer for subsequent calculation of transcription reaction yield, such as [ ³² P] -UTP . RNA purification, hereafter referred to as internal control, obtained by in vitro transcription should be performed in order to promote the removal of unincorporated nucleotides, as well as to eliminate in vitro synthesized template plasmid DNA by techniques known to the of the subject, such as using chromatographic columns.

 The internal control obtained according to the process described above is a polyvalent molecule that can be used for both viral quantification and HCV identification in a biological sample taken from a human or non-human primate. Preferably, the biological sample is a human serum sample.

 Thus, this internal control can be used in quantification and / or viral identification processes via RT-PCR product analysis by different methods, and as non-exhaustive examples we have agarose gel, plaque capture and real-time PCR, these at different cost-effectiveness.

 Thus, it is a further object of this invention a polyvalent internal control, comprising any of Identifying Sequences 1, 2 and 3, composed of a non-natural synthetic genomic polynucleotide molecule based on the genome of an RNA virus; more preferably, based on the HCV genome containing modifications by insertion, substitution and / or insertion and substitution of nucleotides.

 The internal control of this invention therefore comprises the nucleotide sequence similar to a particular highly conserved HCV sequence to be quantified and / or identified. Preferably, the internal control should comprise the nucleotide sequence similar to a particular highly conserved sequence, such as the 5'NTR region of the HCV genome.

Modifications to the polynucleotide sequence obtained from the virus allow the development of viral quantification and / or identification methods by employing standard amplification techniques of viruses. polynucleotide molecules, such as the various PCR techniques known to those skilled in molecular biology.

 This is because insertion increases the molecular weight of amplicon generated by PCR and can be used, for example, for quantification and / or identification of amplicons by visualization techniques of state-of-the-art amplified polynucleotide molecules, such as gel electrophoresis. agarose and staining with double stranded DNA intercalating dyes such as ethidium bromide, SYBR Green and others.

 This size difference between the HCV and internal control amplicons makes it possible to distinguish on agarose gel the products obtained by PCR amplification of the HCV genome from that of the artificial polynucleotide molecule of this invention. It also allows the development of specific probes for viral quantitation. In this case, specific probes may be developed for use in prior art methods such as RT-PCR followed by plaque capture, but not limited to this viral quantitation method alone.

 Replacement allows the development of quantification and / or identification methods by specific probes of amplified products (viral RNA and internal control), using state-of-the-art methods, such as RT-PCR by plate capture, by RT- Real-time PCR (qRT-PCR) and others.

 The in vitro viral diagnostic method aimed at obtaining viral quantification and / or identification in a mammalian animal is based on the promotion of the quantification and / or identification of a disease-causing virus and / or dysfunction in a mammalian animal.

Quantification and / or viral identification may be performed using said polyvalent internal control as a mimetic marker in laboratory analysis of biological samples made by state of the art techniques such as: RT-PCR followed by gel electrophoresis. agarose; RT-PCR with plate capture followed by colorimetric detection; and RT-PCR in real time, but not limited to these techniques. According to the definitions previously made in this report, we understand that a sample of biological material may be derived from any human or non-human mammalian animal and may belong to the group consisting of: serum, biopsies, lymphocytes, whole blood and its fractions, blood products, saliva, semen, hair and skin. Preferably, the sample of biological material employed in this object is blood collected from human and non-human mammals; even more preferably, the biological sample is human serum obtained from whole blood.

 This diagnostic method has as its main objective to quantify and / or identify viruses whose genome is formed by one or more RNA molecules, preferably, this diagnostic method is aimed at the quantification and / or detection of RNA viruses. Even more preferably, this diagnostic method is directed to the quantification and / or detection of HCV.

 Optionally, tests may be performed to verify the sensitivity and specificity of the quantitation and / or identification methods using the internal control of this invention. In addition, the number of molecules of said internal control can be matched with International Units (Ul). International Unit (UL) is the definition made by the World Health Organization (WHO) through the establishment of standard sera containing known viral levels (Saldanha et al, 2003; Saldanha et al, 1999).

 This invention further relates to an in vivo viral diagnostic method for the diagnosis of a virus in a mammalian animal based on the quantification and / or identification of a virus causing a disease and / or dysfunction in a mammalian animal.

This diagnostic method can be applied to a human or non-human mammalian animal and its main purpose is to quantify and / or identify viruses whose genome is formed by one or more RNA molecules. Preferably, this diagnostic method is aimed at quantifying and / or detection of RNA genome viruses. Even more preferably, this diagnostic method is directed to the quantification and / or detection of HCV. Quantification and / or viral identification may be performed using said polyvalent internal control as a mimetic marker in laboratory analysis of biological samples made by state of the art techniques such as: RT-PCR followed by gel electrophoresis. 5 agarose; RT-PCR with plate capture followed by colorimetric detection;

 Real-time RT-PCR (quantitative PCR, qRT-PCR), but not limited to these techniques.

 The method of treatment in which the patient is monitored for viral levels in his body comprises the steps of extracting viral RNA from a biological sample, cDNA synthesis, and amplification of a region of the HCV genome by chain reaction. quantitative polymerase in some phases before, during and after antiviral treatment.

 The present invention relates to the use of internal control for preparation of compositions useful in diagnosing disorders and disorders caused by viruses.

 The compositions to which this object relates are compositions containing a known effective amount of internal control, an enzymatic compound, deoxypolinucleotides and chemical adjuvants.

The effective amount of said internal control in this composition should be from 1 to 10 ⁶ copies of the internal control / ml of the solution; preferably the internal control copy number present in the diagnostic composition should be from 5 to 5x10 ⁵ internal control copies / ml of the solution.

 The enzyme compound should contain DNA polymerase enzymes, and any prior art polymerases should be employed. Preferably, the polymerases contained in said container are high temperature resistant DNA polymerases having a high replication fidelity standard.

Among the chemical adjuvants that may be employed in this object we can mention diluents, preservatives, stabilizers, pH stabilizers, osmolarity stabilizers and mineral salts, all of these components are known to those skilled in the art. The diagnostic composition of this invention has several uses; for example, this composition may be employed in quantitative HCV testing on a biological sample; as a false negative control in HCV diagnostic tests and in qualitative HCV tests. In addition, it can also be used as an internal control in nucleic acid detection amplification (NAT) testing in blood centers in donor blood samples.

 The kit for viral quantification and / or detection in a biological sample described hereinafter consists of a container containing an effective amount of an internal control for viral quantification and / or identification, as well as containers containing either alone or in combination dNTPs, solution stabilizer and polymerases.

 The effective amount of internal control in this object of the invention is from 1 to 100 internal control units / μΙ. Preferably, the number of copies of the internal control contained in this kit should be between 5 and 50 units.

 Any prior art polymerases should be employed, preferably the polymerases contained in said container are high temperature resistant DNA polymerases which have a high standard of replication fidelity.

 Stabilizing solution is an aqueous solution containing labeled salts, pH stabilizing compounds, osmolarity stabilizing compounds and dideoxynucleotides.

 Preferably, this kit is directed to the quantification and / or detection of HCV in a biological sample from a human or non-human mammalian animal.

To better illustrate, the diagnostic kit useful for both HCV quantification and identification as well as this molecule itself, we demonstrate the use of the HCV quantification tool, in which this polynucleotide molecule acts as an internal control. Said internal control developed by the process described above may be used in different methods of quantification and / or viral detection. This internal control was developed 5 'NTR from the HCV viral genome. This region has a high degree of conservation among the different HCV types and subtypes and is therefore ideal for use in quantifying and / or identifying the different HCV types and subtypes found in the Brazilian population.

 Preferably, this biological sample is human serum.

 Those skilled in the art will immediately appreciate the benefits of using the present invention as factors capable of providing improved diagnostic processes and more efficient clinical treatments.

 The following examples are illustrative only and should not be employed to limit the rights of the holders of the present invention.

Example 1: Internal Control Production Process:

1.1 Selection of the conserved sequence, obtaining the copy of the conserved sequence and making the modifications:

The HCV genome region used for internal control production was 5 ^* NTR (Figure 1), as it is the region with the highest conservation in the nucleotide sequence among the different virus genotypes and subtypes.

 HCV RNA used as a template for the generation of amplification products with the characteristics described above was extracted from a pool of HCV genotype 1 b infected sera. This material was collected and maintained in the laboratory prior to June 30, 2000.

 The HCV sequence used for primer design is the product of the alignment of 29 sequences from the 5'NTR region with 7 referring to genotype 1 (1a, 1b, 1c), 8 to genotype 2 (2a, 2b, 2c, 2k) , 5 to genotype 3 (3a, 3b), 2 to genotype 4 (4a), 1 to genotype 5 (5a) and 6 to genotype 6 (6b, 6d, 6g, 6h and 6k) taken from the NCBI database (Entrez Nucleotide NCBI).

Primers were designed with the aid of the SciTools OligoAnalyzer 3.0 IDT (Integrated DNA Technologies) program and a primer design guide, the PCR Primer Design Guidelines (Premier Biosoft International). The inserted 50 nucleotide sequence and 25 substituted nucleotide sequence showed no significant similarity to any other microorganism and not even with any human sequence, confirmed by GenBank database searches (Entrez Nudeotide NCBI). Because these primers are large in size, prediction analyzes of secondary structure formation and annealing were performed between them.

5 (SEQ ID NO: 8 and SEQ ID NO: 9). Primers were purified by SDS-PAGE by the manufacturer Dialabs Diagnostics, SA.

As mentioned above, template HCV RNA was obtained from a pool of HCV genotype 1b infected sera properly stored at -70 ° C. Genotyping of this serum was taken for sequencing e automatic RT-PCR product generated by primer set SEQ ID NO: 4 and SEQ ID NO: 5 (first ^the PCR step), SEQ ID NO: 6 and SEQ ID NO : 7 ^(the second PCR step) shown in table 1, using for the sequencing primer SEQ ID NO: 7 and DYEnamic ET ™ Dye Terminator Cycle sequencing Kit for MegaBACE (Amersham Pharmacia Biotech Inc.). Genotyping was done

15 by comparing the sequence produced with sequences from public databases: The Los Alamos Sequence Database (Los Alamos National Library) and nucleotide-nucleotide BLAST (NCBI).

TABLE 1

 0 Nested PCR Primers

SEQ ID NO: 7 5 'CAC TCT CgA gCA 70 26 214 bp

(antisense) CCC TAT CAg gCA gT

 3 '

The HCV RNA extraction method used was that of isothiocyanate-phenol, based on the protocol established by Chomzynski & Sacchi (1987). This extraction method was used for all sera in this work. The extraction solution (GT) is composed of: phenol saturated in 1 M Tris-HCl pH 8.0, with final pH between 7 and 8, was added equal volume of isothiocyanate solution (2.5 M guanidine isothiocyanate; 50mM Tris -HCI pH 8.0, 25mM EDTA pH 8.0 and H ₂ 0) and 1/100 volume of glycogen 10 mg / ml ortho-toluidine dye was added until the solution became light blue. At the end, the solution was aliquoted and stored at -20 ° C under light protection.

 To 100μΙ_ of serum was added 300μΙ_ of GT solution described above, followed by vortexing for 1 minute. A 50µl volume of pure chloroform was added for phase separation, followed by vortexing for 1 minute and centrifugation for 5 minutes at 12,000 rpm in Eppendorf microcentrifuge. The supernatant was placed in a tube containing 300μΙ of isopropanol for nucleic acid precipitation followed by vortexing for 10 seconds and centrifugation for 10 minutes at 8,000 rpm. The supernatant was discarded and 300μΙ of absolute ethanol added to wash the precipitate and centrifuged for 5 minutes at 8,000 rpm. The supernatant was removed by inversion of the tube and dried for 2 minutes at 65 ° C in heating block. Then the RT solution was added directly to the precipitate.

1,2 Nested type RT-PCR of the HCV qenoma 5'NTR region cDNA for introduction of insertion and / or substitution modifications.

RT-PCR was performed according to the protocol of the reverse transcriptase enzyme manufacturer M-MLV RT (Moloney Murine Leukemia Virus Reverse Transcriptase) and recombinant Taq DNA Polymerase (Invitrogen), with the modifications described below. The RNA precipitate extracted from 10ΌμΌ serum was resuspended in 25μΙ of the RT reaction [1 x reverse transcriptase reaction buffer (5x first strand buffer: 250mM Tris-HCI pH 8.3; 375mM KCI and 15mM MgCI ₂ ), 0 .01 M DTT, 0.5 mM dNTPs (dATP, dTTP, dCTP and dGTP), 30 pmoles NCR2C primer and 200 units (U) of the enzyme M-MLV RT (Invitrogen)]. This reaction was performed by incubating for 1 hour at 37 ° C and 15 minutes at 95 ° C, followed by centrifugation in microcentrifuge at 8,000 rpm for 1 minute. The product was stored at -20 ° C.

As mentioned above, the reaction in two stages of PCR (nested) was done using for step 1 primers SEQ ID NO: 4 and SEQ ID NO: 5 (Table 2), forming a sequence of 261 bp. For the second step were performed three combinations described primers SEQ ID NO: 8 / SEQ ID NO: 9, SEQ ID NO: 8 / SEQ ID NO: 5 and SEQ ID NO: 4 / SEQ ID NO: 9, generating 311 bp, 311 bp and 261 bp fragments, respectively.

TABLE 2

 Initiators employed in making the modifications

 Initiators Size

 Sequences (nt)

SEQ ID NO: 8 5 'CgT TAg TAT gAg TgT CgT gCA 115

(sense) gcc TCC Agg ACC CCC TCT Agg TTA

 I I I gCg TTT Cgg TCg gCg gTT TCT

 TgA gCg gCg gTg TTA gCC CTC CCg

 ggA gAg CCA TAg Tgg TCT gCg g 3 '

 SEQ ID NO: 9 5 'gTg CTC ATg gTg gAC ggT CTA 108

(antisense) CgA gAC CTC CCg ggg CAC TCg CAA

 gCA CCC TAT CAg gCA gTA CCA

 CAg TTC TTC Agg ACg gTT gTC ACG

CAg Cgg CTA gCA gTC TTg 3 ' SEQ ID NO: 5 5 'gTg CTC ATg gTg gAC ggT CTA 29 (antisense) CgA gAC CT 3'

 SEQ ID NO: 4 5 'CgT Tag TAT gAg TgT CgT gC 3' 20

(sense)

PCR reactions were performed using 0.15mM dNTPs (d ATP, dTTP, dCTP and dGTP), 2mM MgCl ₂ , 15 pmoles of each primer and 1 unit (U) of the recombinant Taq DNA Polymerase enzyme ( Invitrogen) and its 1x buffer (10x buffer: 200mM Tris-HCI pH 8.4 and 500mM KCI) in a total volume of 50μΙ_. In the first stage of PCR, 10μΙ_ of cDNA sample and 40μί of the reaction mixture were used, and in the second stage of PCR, 5μΙ_ of first stage product and 45μΙ_ of the reaction mixture were used. Cycling was 25 cycles, denaturation at 94 ° C for 15 seconds, annealing at 53 ° C for 15 seconds and extension at 72 ° C for 15 seconds, 10 minutes at 72 ° C. After synthesis, an aliquot of the RT-PCR products were visualized by 2% agarose gel electrophoresis stained with ethidium bromide at a concentration of 1 pg / mL (Figure 3). The PCR products from the 03 reactions which 2 ^the PCR step was done with primer combinations SEQ ID NO: 8 / SEQ ID NO: 9, SEQ ID NO: 8 / SEQ ID NO: 5 and SEQ ID NO: 4 / SEQ ID NO: 9 were stored at -20 ° C until cloning as described below.

1.3 Cloning of RT-PCR products containing the 50 insert, 25 nucleotide substitution or both, in the 5'NTR region of HCV.

The three distinct PCR products obtained from the different primer combinations were cloned with the TOPO Cloning Kit (Invitrogen) using the pCR ^® 2.1 - TOPO ^® vector. This vector contains, among others, a LacZa fragment which, in the presence of Xgal (5-bromo-4-chloro-3-indol-pD-galactosidase), allows the identification of colonies containing the insert; an amplicillin resistance gene; and a T7 promoter. The binding reaction with the PCR product was performed according to the manufacturer's instructions. Then it was competent bacteria Escherichia coli DH5a were transformed with efficiency of 10 ⁶ transformants / g DNA. Competence induction was chemical based on the calcium chloride method (CaCl ₂ ), using an adapted protocol from Sambrook & Russel (2001). Identification of recombinant clones 5 was performed on plates containing 0.8mg X-gal. Transformed cells were plated in LB (Lysogenic Broth) medium containing 100μg / mL ampicillin. The plates were incubated for 18 hours at 37 ° C. Plates containing the white transformant colonies were stored at 4 ° C until proceeding to the isolation of plasmid DNA.

1.4 Isolation of plasmid DNA.

 The protocol used was based on that described by Sambrook & Russel (2001), with the modifications described below.

A bacterial colony was grown in 3mL of LB medium with 100μg / ml ampicillin for 16 hours at 37 ° C under agitation. The cultures were centrifuged for 1 minute at 13,200 rpm in microcentrifuge and the cell pellet resuspended in 50mM glucose solution, 25mM Tris-HCl pH 8.0 and ice cold 10mM EDTA. Bacteria were lysed in 0.2 N NaOH solution and 1% SDS and incubated on ice for 10 minutes. Finally, the pH of the solution was neutralized with the 3M potassium acetate solution pH 4.8 followed by incubation on ice for 5 minutes and centrifugation for 5 minutes at 13,200 rpm. The supernatant was transferred to another tube and treated with 20ng / µl RNase A for 1 hour at 37 ° C and then extracted with saturated phenol-chloroform 5 in 0.1 M Tris-HCl pH 8.0. After vortex homogenization and incubation at room temperature for 2 minutes, it was centrifuged for 2 minutes at 13,200 rpm. The aqueous phase was transferred to another tube and plasmid DNA precipitated with two volumes of ice cold absolute ethanol followed by vortex homogenization and incubations at room temperature for 20 minutes and -70 ° C for 15 minutes. After centrifugation for 5 minutes at 13,200 rpm, the supernatant was discarded and 1 mL of ice cold 70% ethanol was added for washing and centrifugation for 2 minutes at 13,200 rpm. THE The supernatant was discarded again and the precipitate left for a few minutes at room temperature to dry. The plasmid DNA pellet was resuspended in 30μΙ TE (10mM Tris-HCl pH 8.0; 1mM EDTA pH 8.0) and stored at -20 ° C. After obtaining the plasmid DNA, the samples were submitted to 1% agarose gel electrophoresis in 1x TBE and stained with 1 g / mL ethidium bromide for visualization in UV light transilluminator.

1.5 Automatic sequencing of transformants containing insertion / substitution, insertion only and substitution only.

 Automatic sequencing of plasmid DNAs was performed using the universal primers T7 (sense) and M13 reverse (reverse) and the DYEnamic ™ ET Dye Terminator Cycle Sequencing Kit for MegaBACE (Amersham Pharmacia Biotech Inc.). Confirmation of the correct orientation of the insert (towards the T7 vector promoter) was analyzed considering that these plasmids would be subjected to in vitro transcription. The three clone types (insertion / substitution, insertion and substitution) were obtained. For purposes of non-limiting exemplification of the invention, the development of the internal control object of this invention from the recombinant plasmid containing insertion of the 50 nucleotide sequence and substitution of the 25 nucleotide sequence (insertion / substitution clone) will be briefly described.

1.6 Linearization of insertion / replacement clone DNA.

The chosen enzyme, ApaU, has two restriction sites on the pCR ^® -2.1-TOPO (3,931 bp) vector used for cloning. The recombinant plasmid containing the insert (31 bp) totals 4,242 nucleotides. After digestion with ApaU, 1,246 bp is removed, leaving 2,996 bp containing the insert, as shown in Figure 2. The 2,996 bp fragment was used as a template for the production of internal control of 1,120 nucleotides by in vitro transcription. Since HCV RNA has 9,600 nucleotides, this was the option to obtain a larger transcript than the insert to facilitate its extraction as well as to approximate the size of the HCV genome. Clone Digestion was performed using 10pg of the plasmids as instructed by the manufacturer, in 50μΙ_ reaction for 2 hours at 37 ° C.

 To make sure that there is no internal restriction site for the ApaU restriction enzyme in the HCV genome region, the restriction map (by the NEBcutter - New England Biolabs program) of the HCV genotype 1 b amplified region was previously mapped.

 To the digestion product was added 0.3M sodium acetate pH 5.6 followed by precipitation with two volumes of ice cold absolute ethanol, vortex homogenization and incubation for 30 minutes at -70 ° C. It was then centrifuged for 5 minutes at 13,200 rpm, the supernatant discarded and 1 ml of ice cold 70% ethanol added to wash the precipitate. After centrifugation for 2 minutes at 13,200 rpm, the supernatant was discarded and the precipitate resuspended in 22μΙ_ of 1x TE. The material was stored at -20 ° C. Digestion was confirmed by 1.5% agarose gel electrophoresis in 1 x TBE, stained with 1 pg / mL ethidium bromide.

 1.7 Extraction of ApaU-digested insert / replacement clone DNA fragment from agarose gel.

 The total volume of the ApaU digestion product was analyzed by 1% preparative agarose gel electrophoresis in ethidium bromide stained 1x TBE. Using hand-held UV, the gel was rapidly irradiated to locate the fragment of interest (2,996 bp). Extraction of the DNA fragments from the agarose gel was done using the adapted protocol of Tautz & Renz, 1983. In this protocol, fragments of interest are cut from the agarose gel and these pieces are equilibrated in a buffer (0.3M acetate. pH 7.0 and 1 mM EDTA), followed by freezing and centrifugation with siliconized glass wool filtration by which the DNA contained in the buffer is eluted. At the end, the precipitate is resuspended in 30μΙ of 0mM Tris-HCl pH 8.0. The extracted fragments were then analyzed in 1% agarose gel electrophoresis in 1x TBE and the DNA content measured in a spectrophotometer (Eppendorf BioPhotometer).

1.8 Production of internal control by in vitro transcription reaction from the insertion / replacement clone. The in vitro transcription reaction for internal control production was performed using 0.2 pmoles of the purified approximately 3kb ApaU digested fragment DNA. The reaction contained 0.5mM of each NTP, 10mM DTT, 1x transcription buffer [5x Buffer: 0.2M Tris-HCl pH 8.0; 40mM MgCl ₂ ; 10mM spermidine- (HCI) ₃ and 125mM NaCl], 0.1 mg / ml BSA, 20 units (U) of Rnasin (Gibco BRL), 25 units (U) of T7 RNA polymerase enzyme (Gibco BRL), 20μΟί of [ ³² P] -UTP and Rnase free H ₂ 0, in a total volume of 45,5μΙ_. Incubate for 2 hours at 37 ° C. The product was then filtered through Sephadex G-50 column. Incorporated radioactivity was counted in a TRI-CARB 1,600 TR scintillator (PACKARD) using 2μΙ of the transcription product in 4mL of scintillating solution.

Aiming at eliminating the plasmid DNA template from the in vitro synthesized internal control, it was subjected to DNAse treatment as described below: 10μΙ_ of the in vitro transcript product were treated with RQ1 Dnase Rnase-free (Promega), in the digestion buffer of enzyme containing 75 units (U) of the enzyme RQ1 Dnase Rnase-free and H ₂ 0 Rnase-free, for a final volume of 300μΙ_. Incubated for 60 minutes at 37 ° C. Then 30 L of RQ1 Dnase Stop Solution was added, following a 10 minute incubation at 65 ° C. An aliquot of 100μΙ_ of solution containing the in vitro synthesized internal control in Dnase buffer was extracted with isothiocyanate-phenol as described above.

Example 2. Analysis of the in vitro synthesized internal control.

2.1 Auto-radiographic analysis.

For the autoradiographic analysis of the transcription product in vitro, an aliquot containing 18,816 counts per minute (cpm) of the transcription product was used. Denaturing agarose gel electrophoresis at 1.5% containing formaldehyde and 1x MOPS [3- (n-morpholino) propanesulfonic acid] as described by Sambrook & Russel, 2001 was performed. After the run, overnight transfer ( 16 hours) at room temperature in 20x SSC for nylon membrane (Amersham Biosciences). The membrane was cassette exposed using intensifier at -70 ° C (Figure 4) for three different times: 8; 15.5; and 74 hours. They were used as a quality reference of material migration the Trypanosoma cruzi rRNAs (ribosomal RNAs) (1.8kb; 2.1kb and 2.4kb) obtained from 5pg total epimastigote RNA extracted with Rneasy Mini Kit (Qiagen).

 2.2 Determination of the in vitro synthesized internal control mass:

 Calculations were made to determine the in vitro synthesized internal control mass by the specific activity of the isotope (3,000Ci / mmol) used and the amount of radioactivity incorporated into RNA (235,231cpm).

 2.3 Determination of the purity of in vitro synthesized RNA:

To ensure that the material contained no contaminating plasmid DNA in the internal control, RT-PCR testing was performed in the presence and absence of reverse transcriptase (Superscript ™ III Reverse Transcriptase, Invitrogen Corporation) in duplicates of four different amounts of internal control (6x10 ⁸ , 6x10 ⁷ , 6x10 ⁶ and 6x10 ⁵ molecules) (Figure 5) Initially RNA extraction was performed, as described in item 1.1. The extracted RNA precipitate was resuspended in 25μί of the reverse transcriptase solution containing 0.01 M DTT, 0.5mM dNTPs (dATP, dTTP, dCTP, and dGTP), 3 pmoles of primer SEQ ID NO: 5 (Table 1 ) and 8 units (U) of the enzyme Superscript ™ III Reverse Transcriptase (Invitrogen) in 1x first strand buffer (5x first strand buffer: 250mM Tris-HCI pH 8.3; 375 mM KCI and 15mM MgCl ₂ ). This reaction was performed by incubating for 1 hour at 55 ° C and 15 minutes at 95 ° C, followed by centrifugation in microcentrifuge at 8,000 rpm for 1 minute. The product was stored at -20 ° C. The PCR reaction was in two steps (nested PCR), using two outermost primers (SEQ ID NO: 4 and SEQ ID NO: 5 in 1 step as described in table 1) plus two internal primers (SEQ ID NO: 6 and SEQ ID NO: 7 in ^the step 2 described in table 1). Both PCR reactions were performed using 0.15mM dNTPs (dATP, dTTP, dCTP and dGTP), 2mM MgCl ₂ and 0.025 units (U) of Platinum® Taq DNA Polymerase enzyme (Invitrogen) and its buffer (10x PCR Buffer - 200mM Tris-HCI pH 8.4 and 500mM KCI) at 1x final concentration in a total volume of 50μΙ .. The difference between the two PCR steps is the concentration of the primers, in the first reaction 5 pmoles were used. while on Monday, 30 pmoles. In the first stage, 10μΙ_ cDNA and 40μΙ_ reaction mixture were used. In the second stage 5μΙ_ of reaction of the first stage in final 50μΙ_ were used. The reaction was initially incubated at 95 ° C for 3 minutes, 25 cycles at 94 ° C for 15 seconds; 53 ° C for 15 seconds; 72 ° C for 15 seconds. At the end, an extension step of 10 minutes at 72 ° C.

 This test is necessary to ensure that serum HCV RNA quantification by the following methods is only products generated from RNA and not plasmid DNA.

 2.4 Determination of internal control stability in extraction solution.

 Since we aim to establish conditions to enable the use of the tool developed for commercial production, we tested the stability of the internal control in RNA extraction solution (GT) at 4 ° C since most commercial kits of this nature are transported. and stored at 4 ° C in Brazil. The times of zero, 07, 15, 30, 45 and 60 days of sample storage (vials containing 5,000 internal control molecules in 300μΙ_ of extraction solution) in this medium and at this temperature were tested. The samples were submitted to nested RT-PCR (Figure 6) and qRT-PCR.

 Example 3: Utilization of internal control in human serum HCV quantification: analysis by agarose gel electrophoresis and real time PCR.

3.1 Standardization of internal control content to be used as a competitor in HCV quantification by aqarose gel electrophoresis and real-time PCR:

Sera containing 10 ³ and 5x10 ³ internal control molecules were used. Viral RNA was extracted from these sera, RT reaction and PCR nested as described above. The analysis was performed with 5x10 ³ internal control molecules by ethidium bromide stained agarose gel electrophoresis (Fig. 7).

 3.2 Determination of detection sensitivity of internal control by electrophoretic analysis and agarose gel staining of nested RT-PCR products:

Different amounts of internal control (5x10 ⁵ , 5x10 ⁴ , 10 ⁴ , 5x10 ³ , 10 ³ and 10 ² copies / mL) were added to the control serum, followed by extraction in GT solution to define the RNA detection sensitivity limit following the protocol mentioned. RT reaction and nested PCR were performed as described.

 Sensitivity limit was determined by checking the last dilution whose RT-PCR fragment was visible on ethidium bromide stained 2% agarose gel (Figure 13).

 3.3 Determination of HCV viral load in human serum by agarose gel electrophoresis of RT-PCR products.

For HCV RNA quantification, serial dilutions of known sera were used in the presence of a constant amount of internal control. To show the viability of the method in different commonly encountered situations, this was done with serum containing known viral load (low, medium and high) (Figure 14). Low-load 1.8x10 ⁴ IU / mL (4.25 logarithmic units) low-load viral serum, 6.7x10 ⁵ IU / mL average viral load (5.82 logarithmic units) and high viral load 2.2 x 10 ⁶ IU / mL (6.34 logarithmic units).

Serum RNA extraction was done as described above. To the extraction solution (GT) was added a known number of internal control molecules, 5x10 ³ molecules. Nested RT-PCR reactions were performed as described. PCR products were analyzed by 2% agarose gel electrophoresis, stained with 1 g / mL ethidium bromide and visualized under UV light. The relative intensity of staining of the obtained fragments was analyzed with the Kodak Digital Science ™ ID Image Analysis Software program. For the quantification, two serum dilutions were chosen, where, preferably, for one of them the predominant PCR product was derived from HCV RNA (most intense fragment) and, for the other, the majority PCR product was derived from internal control. .

Quantitation was done using the formula C = dx R x M x \ 0, where C = amplified genome copy number / mL; d = serum dilution; R = ratio of relative fragment intensity (HCV RNA / internal control); M = number of internal control molecules added to serum (5x10 ³ ); 10 = conversion from 100 µl to 1 ml serum. The calculation was made for each of the selected dilutions, where a viral load range was obtained, whose arithmetic mean of these values were transformed into logarithmic units.

 3.3 Design of primers and probes for HCV RNA quantification by real-time RT-PCR (gRT-PCR).

To quantify HCV RNA by qRT-PCR, the Applied Biosystems TaqMan ^® probe detection system was used. The probes were designed in the PCR modified 25 nucleotide region, referred to as a "replacement fragment".

 A primer pair was designed which, as a prerequisite, annuls both HCV RNA and internal control and two specific probes, one for HCV RNA and the other for internal control (Table 3). The generated PCR product is 66bp. Primer Express v.2.0 software (Applied Biosystems) was used for primer and probe design.

TABLE 3

 Detector System Primers and Probes Characteristics

 TaqMan® for HCV RNA quantification

 Taq System Size Tm Marking Marking

 Sequence

 Man (NT) (° C) reporter quencher

SEQ ID NO: 10 5 '

 (Initiator CCGCAAGACT 18 59 ° No MGB sense) GCTAGCCG 3 '

 5 '

 SEQ ID NO: 11

 GCACCCTATC

 (Anti-primer 21 59 ° No MGB

 AGGCAGTACC

 sense)

 3 '

 SEQ ID NO: 12 5 '

 (RNA probe TGTTGGGTCG 16 70 ° FAM MGB

HCV) 3 'CGAAAG

 SEQ ID NO: 13 5 '

(Probe TGCGTGACAA 15 70 ° VIC MGB Control CCGTC 3 ' internal)

The HCV RNA probe was labeled with the FAM ™ reporter fluorophore (6-carboxyfluorescein), while the one for the internal control was labeled with VIC ^® (6-carboxyrhodamine). The quencher fluorophore was non-fluorescent and coupled to an MGB (minorgroove binder) molecule.

 3.4 Standardization of the human serum HCV RNA quantification test by qRT-PCR.

The cDNAs used originated from reverse transcription reaction (item 1.2) from samples whose HCV RNA had been previously extracted according to the protocol described above (item 1.1). The reaction conditions, primer concentration and probes used were those recommended by the manufacturer (Applied Biosystems). The reaction mixture was composed of 900nM from each primer (SEQ ID NO: 10 and SEQ ID NO: 11, table 3), 250nM from each probe (SEQ ID NO: 12 and SEQ ID NO: 13, table 3 ), Taq Man ^® Universal Master Mix at final concentration 1x and H ₂ O qsp 25μΙ_. The reaction volume used was 25μΙ_, with 22μΙ_ of reaction mixture and 3μ! _ Of cDNA.

 Taq Man® Universal Master Mix contains in its composition dNTPs with dUTP, producing amplicons containing U (uracil), unlike the genomic HCV RNA containing T (thiamine). In addition, it contains the enzyme AmpErase® UNG (uracil DNA glycosylase) that digests dUTPs during the initial stage of the PCR reaction, thus preventing contamination with amplicons present in the environment (Watzinger et al, 2006).

 The equipment used was the 7500 Real Time PCR System from Applied Biosystems. The PCR reaction was performed for 2 minutes at 50 ° C (UNG action), 10 minutes at 95 ° C, 45x (15 seconds at 95 ° C and 1 minute at 60 ° C). At the end of the race the baseline was determined manually according to Applied Biosystems protocol.

In order to determine the specificity of the probes to be able to use them simultaneously it was necessary to perform three different reactions: 1. Containing only the probe for internal control (Figure 8).

 2. Containing only the probe for HCV RNA (Figure 9).

 3. Containing probes for HCV RNA and internal control simultaneously (Figure 10).

3.5 Determination of detection sensitivity of internal control by qRT-PCR:

To verify the minimum amount of internal control that the qRT-PCR system was capable of detecting, a curve with 5x10 ⁴ , 5x10 ³ , 5x10 ² , 50 and 5 copies of internal control / mL of serum was performed (Figure 12). . These samples were subjected to all internal control extraction steps in the presence of HCV negative control serum as described. Reverse transcription was performed with the enzyme Superscript ™ III Reverse Transcriptase using the methodology already cited in example 1.2. Real-time PCR was performed using the conditions defined by the test standardization protocol.

 3.6 Determination of serum HCV viral load by qRT-PCR:

 To demonstrate the usefulness of the qRT-PCR system for quantifying different viral loads, the two sera described previously in the agarose gel electrophoresis quantification method and low viral load ethidium bromide staining (4.25 log units) (Figure 11) and high (6.34 logarithmic units) so, with a difference of two logarithmic units between them, were also used in this analysis.

The quantification was done through the formula C =

xc / x10, where C = amplified genome copy number / mL; M = number of internal control molecules added to serum (5,000); y = conversion of the number of duplicates to logio (3.33, since 3.33 CTs = 1 log unit); A ^ CT = X internal control CT - X CT HCV RNA; d = serum dilution; 10 = conversion from 100 µl to 1 ml serum. For quantification, two serum dilutions were chosen in which the internal control CTs were closest to the internal control CT alone in the reaction. 3.7 Correspondence between number of internal control molecules in International Units (Ul):

 5,000 internal control molecules and a commercial panel of real-time PCR quantified sera based on IU / mL according to WHO were used. A plot was made correlating the CTs resulting from panel samples amplified by real-time PCR with IU / mL (Figure 15). Linear regression analysis showed that 1 internal control molecule equals 1 IU / mL.

Claims

Claims:

1) Synthetic Hepatitis C Virus RNA molecule characterized by being used as internal control for quantification and / or identification of the HCV genome.

 Synthetic hepatitis C virus RNA molecule genotype 1b according to claim 1, characterized in that it consists of SEQ ID NO 1 containing an insert at any position of the molecule having a size of 30 to 100 base pairs.

Synthetic hepatitis C virus RNA molecule genotype 1b according to claim 1, characterized in that it consists of SEQ ID NO 2 containing a substitution at any position of the molecule having a size of 10 to 100 base pairs.

 Genetic 1b Hepatitis C Virus RNA RNA molecule according to claim 1, characterized in that it consists of SEQ ID NO 3 containing an insert at any position of the molecule having a size of 30 to 100 base pairs and a substitution at any position. of the molecule having a size of 10 to 100 base pairs.

 5) Artificial synthetic DNA molecule complementary to the untranslated part of the Hepatitis C Virus 5 'sequence consisting of SEQ ID

NO 4, 5, 8, 9, 10, 11, 12 or 13.

 An artificial synthetic DNA molecule complementary to the untranslated part of the Hepatitis C Virus 5 'sequence according to claim 5, characterized in that it consists of SEQ ID NO 4.

An artificial synthetic DNA molecule complementary to the untranslated part of the Hepatitis C Virus 5 'sequence according to claim 5, characterized in that it consists of SEQ ID NO 5.

 An artificial synthetic DNA molecule complementary to the untranslated part of the Hepatitis C Virus 5 'sequence according to claim 5, characterized in that it consists of SEQ ID NO 8.

An artificial synthetic DNA molecule complementary to the untranslated part of the Hepatitis C Virus 5 'sequence according to claim 5, characterized in that it consists of SEQ ID NO 9. An artificial synthetic DNA molecule complementary to the untranslated part of the Hepatitis C Virus 5 'sequence according to claim 5, characterized in that it consists of SEQ ID NO 10.

 An artificial synthetic DNA molecule complementary to the untranslated part of the Hepatitis C Virus 5 'sequence according to claim 5, characterized in that it consists of SEQ ID NO 11.

 An artificial synthetic DNA molecule complementary to the untranslated part of the Hepatitis C Virus 5 'sequence according to claim 5, characterized in that it consists of SEQ ID NO 12.

An artificial synthetic DNA molecule complementary to the untranslated part of the Hepatitis C Virus 5 'sequence according to claim 5, characterized in that it consists of SEQ ID NO 13.

 A cloning and expression vector comprising one of the molecules as described in any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13.

 A composition comprising any of the molecules as defined in claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 in a non-inert carrier containing salts and stabilizing compounds. pH and osmolarity, where the molecule is not merely diluted.

A composition comprising any of the vectors as defined in claim 14 in a vehicle.

 A process for obtaining an artificial synthetic hepatitis C virus RNA molecule as defined in any one of claims 2, 3 and 4.

Process for obtaining artificial synthetic Hepatitis C Virus RNA molecule according to claim 17 as defined in claim 2 characterized by chemical synthesis or in vitro transcription.

 Process according to Claim 17, characterized in that the in vitro transcription takes place in the following steps:

(a) extraction of viral RNA from serum, biopsies, lymphocytes, whole blood and its fractions, blood products, saliva, semen, cell culture, replicons and other body fluids in human and non-human primates;

b) cDNA synthesis; c) Amplification of the untranslated 5 'region using primers as defined in claims 6, 7 and 8 or 6, 7 and 9 or 6, 7, 8 and 9.

d) Cloning of the amplified fragment in vector.

e) In vitro transcription reaction.

20) In vitro quality control method for false negative hepatitis C virus samples characterized by the steps:

The introduction of known number of artificial synthetic RNA molecules as defined in claims 2, 3 or 4 into a biological sample.

b) Extraction of viral RNA.

c) cDNA synthesis.

d) Amplification by polymerase chain reaction.

e) Detection.

 21) Qualitative in vitro HCV detection method characterized by the steps:

The introduction of known number of artificial synthetic RNA molecules as defined in claims 2, 3 or 4 into a biological sample.

b) Extraction of viral RNA.

c) cDNA synthesis.

d) Amplification by polymerase chain reaction.

e) Detection.

 22) In vitro quantitative HCV method characterized by fluorescence measurement in quantitative PCR by the steps:

The introduction of known number of artificial synthetic RNA molecules as defined in claims 2, 3 or 4 into a biological sample.

b) Extraction of viral RNA.

c) cDNA synthesis.

d) Amplification by polymerase chain reaction.

e) Detection.

 23) In vitro quantitative method of HCV characterized by immobilization of artificial synthetic RNA molecule on a surface for detection by steps:

The introduction of known number of artificial synthetic RNA molecules as defined in claims 2, 3 or 4 into a biological sample.

b) Extraction of viral RNA. c) cDNA synthesis.

d) Amplification by polymerase chain reaction.

e) Detection.

 HCV diagnostic kit comprising an artificial synthetic RNA molecule as claimed in claim 2, 3 or 4.

 Use of the synthetic hepatitis C virus RNA molecule according to claim 24, characterized in that it is used for the qualitative and / or quantitative detection of the HCV genome.

 Use of the Hepatitis C Virus synthetic RNA molecule as defined in one of claims 2, 3 and 4 characterized in that it is employed as an internal control for preparing a composition as defined in claim 15 for the purpose of qualitative and / or quantitative detection of the HCV genome.