Oligonucleotide primer pair of primers, the primer set (options), the manufacture method for nucleic acid amplification of hiv-1

 

(57) Abstract:

The invention relates to the field of molecular biology and can be used for the detection of human immunodeficiency virus type 1 (HIV-1). For the diagnosis of HIV-1 use the method of amplification involving the use of improved oligonucleotide primers SKCC1 and SKCC3, and their combination with primer SK145, on the basis of these primers generated sets, one of which in addition to the pairs of primers primer contains SK145M2, and the other one primer, a pair of primers or the above set of primers. Of a nucleotide sequence listed primers given in the text description. The proposed primers able to amplify new, newly discovered isolates of group M HIV-1, as well as all known isolates of group M HIV-1 with approximately the same efficiency. 5 C. and 4 h.p. f-crystals, 4 PL.

The present invention relates to molecular biology and chemistry of nucleic acids. In particular, it relates to methods and reagents designed for detection of human immunodeficiency virus type 1 (HIV-1). Therefore, the scope of the invention is to medicine in General and more specifically medievalista nucleic acids, in particular polymerase chain reaction (PCR), makes possible the rapid detection of nucleic acids present in the sample in such small quantities that they previously were unable to detect (see U.S. patent 4683195, 4683202 and 4965188). In the literature there are numerous data on the improvement and application of PCR. For example, a wide range associated with PCR problems presented in such publications as PCR Technology - principles and applications for DNA amplification, 1989 (as amended H. A. Erlich), Stockton Press, New York, NY; PCR Protocols: A guide to methods and applications, 1990 (as amended M. A. Innis and others), Academic Press, San Diego, CA; and in PCR Strategies, 1995 (as amended M. A. Innis and others), Academic Press, San Diego, CA. Commercial suppliers such as the company Perkin Elmer (Norwalk, CT), put on the market the reagents for PCR and post-PCR protocols.

Review data on the use of PCR and hybridization using probes for amplification and detection of nucleic acids of HIV-1 presents the Kwok, 1992, Ann. Med. 24: 211-214; and Coutlee and others, 1991, Mol. Cell. Probes 5: 241-259. Based on the use of PCR methods for the detection of HIV-1 are described, for example, in U.S. patents 5008182 and 5176775; Kellogg and Kwok, 1990, PCR Protocols: A Guide to Methods and Applications, (edited Innis and others), Academic Press, San Diego, CA, 337-347; Holodniy and others, 1991, J. Inf. Dis. 163: 802-865; Jackson and others, 1991, AIDS 5: 1463-1467; and Mulder and others, 1994, J. Clin. Environ. 32(2): 292-300.

The commercial is intended for the detection in vitro of proviral DNA of HIV-1. Set AMPLICOR HIV-1 MONITORTMThe Test is intended for the quantitative assessment of in vitro HIV-1 RNA. In both test sets Amplicor for nucleic acid amplification of HIV-1 uses a pair of primers SK462 (SEQ ID NO: 5) and SK431 (SEQ ID NO: 6), described by Mulder and others, 1994, J. Clin. Environ. 32(2): 292-300, referred to in the present description Amplicor-primers for HIV-1.

HIV-1 has significant variability in the genomic sequence. Phylogenetic analysis of sequences of nucleic acids of genes gag and env HIV-1 described in the work of Myers and others, 1993, Human Retrovirus and AIDS, 1993, Los Alamos National Laboratory, Los Alamos, NM, included in the present description by reference. Within group M were identified subtypes A-j

The literature describes the common methods of molecular biology and chemistry of nucleic acids related to the present invention (see, for example, Sambrook and others, 1989, Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; Oligonucleotide Synthesis (Ed. by M. J. Gait, 1984); Nucleic Acid Hybridization (editor B. D. Hames and S. J. Higgins, 1984); and the series Methods in Enzymology (Academic Press, Inc.)).

Identified numerous isolates of group M of human immunodeficiency virus type 1 (HIV-1) that or not, it provided amplification, or not effectively, it provided amplification with previously described primers for the gene ga is annoy previously variability of the sequences in the field, including the binding sites of primers Amplicor-primers for HIV-1.

The present invention relates to improved primers able to amplify these newly discovered isolates, in addition to all of the isolates that are capable of amplificates using Amplicor-primers for HIV-1. In addition, the primers according to the invention is able to amplify all known isolates of group M HIV-1 with approximately the same efficiency.

The present invention further relates to improved the oligonucleotide primers capable of amplification using the polymerase chain reaction (PCR) of the region of the gag gene of isolates of subtypes A-G group M HIV-1 with approximately the same efficiency and without simultaneous amplification of sequences that were not targeted.

In particular, the present invention relates to the oligonucleotide primers designed for amplification of nucleic acid of human immunodeficiency virus type 1 (HIV-1), with the indicated oligonucleotide primer selected from the group consisting of SKCC1 (SEQ ID NO: 3) and SKCC3 (SEQ ID NO: 4).

Preferably each of these primers paired oligonucleotide PR: 1) and SKCC3 (SEQ ID NO: 4).

In another embodiment, these pairs of primers can be combined with the primer SK145M2 (SEQ ID NO: 2) into a set of oligonucleotide primers consisting of oligonucleotide primers SK145 (SEQ ID NO: 1), SKCC1 (SEQ ID NO: 3) and SK145M2 (SEQ ID NO: 2), or a set of oligonucleotide primers consisting of oligonucleotide primers SK145 (SEQ ID NO: 1), SKCC3 (SEQ ID NO: 4) and SK145M2 (SEQ ID NO: 2).

The invention relates also to improved methods of amplification region of the gag gene of subtypes of group M HIV-1, including the implementation of PCR using primers according to the invention.

The present invention further relates to kits containing amplificating primer of the present invention. These kits can include additional reagents, such as probes for detecting or one or more reagents for amplification, such as polymerase, buffers, and nucleosidases.

For a better understanding of the invention listed below are some concepts.

"Nucleic acid" and "oligonucleotide" refer to polyethoxylated (containing 2-deoxy-D-ribose), to polyribonucleotides (containing D-ribose), and to any other type of polynucleotide representing N-glycosides of a purine or pyrimidine base PII" do not imply differences in the chain length and can be used interchangeably. These concepts apply only to the primary structure of the molecule. Thus, these terms include double - and single-stranded DNA, as well as two - and single-stranded RNA.

Oligonucleotides can be obtained by any suitable for this purpose way, including, for example, cloning and restriction of appropriate sequences and direct chemical synthesis by using a method such as phosphocreatine method described by Narang and others, 1979, Meth. Enzymol. 68: 90-99; fosfodiesterzy method described by Brown and others, 1979, Meth. Enzymol. 68: 109-151; diethylphosphoramidite method described by Beaucage and others, 1981, Tetrahedron Lett. 22: 1859-1862; and a method using a solid substrate, is described in U.S. patent 4458066. Overview of synthesis methods presented in Goodchild, 1990, Bioconjugate Chemistry, 1(3): 165-187.

The term "hybridization" refers to the formation of a duplex structure by two single-stranded nucleic acids in the complementary pairing of bases. Hybridization may occur between fully complementary chains of nucleic acid or between "almost complementary" sequences of nucleic acids that contain minor field of incorrect mating. Under which conditions can hybridisierung only fully cialiseli, specific for the sequence". Stable duplexes almost complementary sequences can be obtained with less stringent hybridization conditions; the extent permissible erroneous pairings can be controlled by proper adjustment of the hybridization conditions. Experts in the field of technology of nucleic acids can determine the stability of the duplex empirically, taking into account various parameters, including, for example, the length and concentration of base pairs of oligonucleotides, ionic strength and the frequency of erroneous mating grounds, using for this purpose are known in the art guide (see, for example, Sambrook and others, 1989, Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; and Wetmur, 1991, Critical Reviews in Biochem. and Mol. Biol., 26(3/4): 227-259).

The term "primer" refers to a natural or synthetic oligonucleotide capable of acting as a point of initiation of DNA synthesis under conditions which induced the synthesis of the product of the elongation of the primer, the complementary chain nucleic acid, i.e., in the presence of four different nucleosidases and agent, providing for the polymerization (i.e., DNA polymerase or reverse transcriptase) in the ochechnogo oligodeoxyribonucleotide. The appropriate length of a primer depends on the intended use of the primer but typically ranges from 15 to 35 nucleotides. Short primer molecules generally require lower temperatures for the formation of a sufficiently stable hybrid complexes with the matrix. You do not want the primer reflect the exact sequence of the nucleic acid-matrix, but must be sufficiently complementary to hybridisierung with the matrix. The primers can include additional structure that can detect or be immobilized primer, but do not change the basic property of primers, namely the ability to act as a point of initiation of DNA synthesis. For example, the primers may contain additional nucleotide sequence at the 5'-end, which does not's hybrid with nucleic acid-target, but which facilitates the cloning of the amplified product. Region primer, complementary to the matrix in sufficient for hybridization extent, in the context of the present description is referred to as "the region of hybridization".

In the context of the present description, the term "primer, initiating the synthesis of progress against transcription" refers to the primer, the product onlinenow transcription" refers to the primer, product extension which is suppositionally, complementary non-coding chain.

The concept of "sequence-target", "area-target" and "nucleic acid target" refers to the field of nucleic acid to be amplificatio, detection or other analysis.

In the context of the present description primer is "specific" for a target sequence, if the number of erroneous pairings formed between the primer and the sequence of the target is less than the number of erroneous pairing formed between the primer and sequences that are not targets that may be present in the sample. Can be selected such hybridization conditions under which stable duplexes are formed only in those cases if the number of present erroneous pairings not more than the number of erroneous pairings formed between the primer and the sequence of the target. Under such conditions, primer specific to the target can form stable duplex only with sequence-target. Thus, the use of primers specific to the target, in terms of amplification of the corresponding severity pauses targets for primer. Similarly, the use of probes specific for the target, under conditions of hybridization of the corresponding severity has the ability to detect a specific sequence of a target.

The term "mixture for amplification reaction" refers to a solution which contains the reagents necessary for the implementation of the amplification reaction, and which typically contains primers, a thermostable DNA polymerase, dNTP and divalent cation metal in a suitable buffer. The reaction mixture is called complete if it contains all reagents necessary for the implementation of the reaction, and is called incomplete if it contains an incomplete set of necessary reagents. To a person skilled in the art it is obvious that for convenience, stability during storage, or to be able to regulate the concentration of the components depending on the destination components of the reaction is usually stored in separate solutions, each of which contains a non-exhaustive set of all components and the components themselves reactions to unite the reaction to produce the complete reaction mixture. In addition, those skilled in the art it is obvious that for sale components of the reaction are packaged separately and that p according to the present invention.

Primers for the amplification of HIV-1

The primers of the present invention have the ability to amplify nucleic acid subtypes of group M HIV-1. Primers are significantly improved compared with previously described primers in the sense that they have the ability to amplify with almost equal efficiency of nucleic acid from the region of the gag gene in all isolates of subtypes A-G belonging to the group M, including the recently opened isolates. The nucleotide sequences of the primers are shown below in orientation from left to right 5'-->3'.

The primers that initiate the synthesis of progress against transcription:

SK145 (SEQ ID NO: 1) AGTGGGGGGACATCAAGCAGCCATGCAAAT

SK145M2 (SEQ ID NO: 2) AGTGGGGGGACACCAGGCAGCAATGCAAAT

The primers that initiate synthesis during transcription:

SKCC1 (SEQ ID NO: 3) TACTAGTAGTTCCTGCTATGTCACTTCC

SKCC3 (SEQ ID NO: 4) TGAAGGGTACTAGTAGTTCCTGCTAT

The primers of the present invention, initiating synthesis during transcription, can be applied to any given in the present description primer, initiating the synthesis of progress against transcription. The primers of the present invention, initiating synthesis during transcription, preferably used with initiating the synthesis of progress against transcription primer . nationwide synthesis progress against transcription primer SK145 (SEQ ID NO: 1) described by Kellogg and Kwok, 1990, PCR Protocols: A Guide to Methods and Applications (edited Innis and others ), Academic Press, San Diego, CA: 337-347. The second initiating the synthesis of progress against transcription primer SK145M2 (SEQ ID NO: 2) 's hybrid with the same area that SK145 (SEQ ID NO: 1), but it is designed for more precise pairing with nucleotide sequence of certain isolates of subtype a and E of HIV-1. As indicated below in the examples, the use of both initiating synthesis of progress against transcription primers can contribute to the alignment of the efficiency of amplification of certain subtypes.

Amplification

Amplification is carried out in conditions that allow to amplify all of the subtypes of group M HIV-1, but which have sufficient rigor in order to avoid amplification of sequences that are not targets. Preferred reaction conditions for amplification are described in the examples Mulder and others, 1994, J. Clin. Environ. 32(2): 292-300, and the instructions included in the test set AMPLICOR HIV-1 MONITOR Test. The exact conditions not of crucial importance in the practical implementation of the invention. Optimization of amplification conditions can be carried out in the usual way, based on the guide, pie or proviral DNA of HIV-1, or HIV-1 RNA. Amplification of RNA using polymerase chain reaction with reverse transcriptase (RT-PCR) are well known in the art and are described in U.S. patents 5322770 and 5310652; Myers and Gelfand, 1991, Biochemistry, 30(31): 7661-7666; and Young and others, 1993, J. Clin. Environ. , 31(4): 882-886. Amplification of HIV-1 RNA using RT-PCR described by Mulder and others, 1994, J. Clin. Environ. 32(2): 292-300 and Holodniy and others, 1991, J. Inf. Dis. 163: 802-865.

Methods sample preparation suitable for amplification of DNA and RNA HIV-1, described in the literature. The choice of a particular method is not significant in the practical implementation of the present invention. Specialist in the art can select and optimize suitable methods of obtaining the sample, based on the guidance given in this description. Preferred methods of sample preparation used for the detection of proviral DNA of HIV-1, described in Casareale and others, 1992, PCR Methods and Applications, 2: 149-153, and the Butcher and Spadoro, 1992, Clin. Immunol. Newsletter, 12: 73-76. A preferred kit for sample preparation for the detection of proviral DNA of HIV-1 is marketed as part of a set of Amplicor HIV-1 Test. Preferred methods of sample preparation used for the detection of HIV-1 RNA in plasma, described by Mulder and others, 1994, J. Clin. Msgic-1 available on the market as part of a set of AMPLICOR HIV-1 MONITOR Test.

Analysis addititional product

Primers for the amplification and methods of the present invention is suitable for any application, based on the use of amplified nucleic acid. For example, cloning and/or sequencing sequences of HIV-1 is facilitated by using the primers of the present invention. Methods of detecting nucleic acids, amplified by PCR, are well known in the art. The method that is used for analysis of the amplified nucleic acid, is not significant in the practical implementation of the invention and therefore can be used by any suitable method. Preferably used to amplify HIV-1 RNA, as described in the examples related to the measurement of the content of the virus.

Examples of methods of detection of amplified nucleic acids include analysis of the amplification product using gel electrophoresis and detection by hybridization with complementary oligonucleotide probes.

Suitable methods of analysis for the study of hybridization of the target-probe are well known in the art and include methods such as dot-blot is ery immobilized on a solid substrate, such as a nylon membrane. Complex membrane-targeted incubated with the labeled probe under appropriate hybridization conditions, legibility probe is removed by washing under conditions of appropriate stringency and the membrane is examined for the presence of bound probe. Detection by dot-blotting of PCR products amplification are described, for example, Saiki and others , 1986, Nature, 324: 163-166, and in U.S. patent 5468613.

When using the reverse dot blot probes immobilized on a solid substrate, such as a nylon membrane, and mark amplificare DNA target. DNA target usually mark in the process of amplification by the inclusion of labeled primers. One or both of the primer can be labeled. Complex membrane-probe incubated with labeled amplificare DNA target under appropriate hybridization conditions, dehybridization DNA target is removed by washing under conditions of appropriate stringency and the filter is then examined for the presence of bound DNA targets. Detection using the methods of reverse dot blot described, for example, Saiki and others, 1989, Proc. Natl. Acad. Sci. USA, 86: 6230, and in U.S. patent 5468613.

Alternatively, the analysis on the basis of the reverse dot blot may be performed with use is most suitable for large-scale clinical applications of the methods of the present invention. The probes can be immobilized on microwell tablet or by passive binding, or through an intermediate protein, such as bovine serum albumin (BSA), which sticks to the microwell plates (see Tung and others, 1991, Bioconjugate Chem., 2: 464-465). Methods based on reverse dot blot using microwell tablet is described in U.S. patent 5232829; Loeffelholz and others, 1992, J. Clin. Environ. , 30(11): 2847-2851; Jackson and others, 1991, AIDS 5: 1463-1467; Mulder and others, 1994, J. Clin. Environ. 32(2): 292-300; instructions included in the commercial kit Amplicor HIV-1 Test; and instructions included in the commercial kit AMPLICOR HIV-1 MONITOR Test.

Preferably the detection and/or quantification of the amplified product is performed using hybridization with oligonucleotide probe immobilized on microwell tablet, using reagents and protocols Amplicor HIV-1 Test or AMPLICOR HIV-1 MONITOR Test. The methods of the present invention to quantify HIV-1 RNA described below in the examples.

In another embodiment, the probes conjugated with BSA, associated with magnetic microparticles. Related probes hybridizing in solution with the labeled amplification product and the resulting product is removed from the process is Obama.

Another suitable method of analysis, called 5'-nuclease the method described in U.S. patent 5210015 and 5487972 and Holland and others, 1988, Proc. Natl. Acad. Sci. USA, 88: 7276-7280. 5'-nuclease method detectable labeled probes that have been modified so that they could not act as primers in DNA synthesis, is added to the reaction mixture during amplification. Any probe that hybridizes with DNA targets for each stage of the synthesis, i.e. in the process of lengthening primers, digested using 5'-->3' ectonucleoside activity of DNA polymerase, for example DNA polymerase Taq. Then identify the product decomposition of the probe. Thus, the presence of the product of cleavage of the probe is also evidence of what happened hybridization between the probe and DNA target, and about what happened reaction amplification. In U.S. patent 5491063 and 5571673 described improved methods of detection of cleavage of the probe, which is accompanied by amplification.

In the above-described analysis methods to facilitate detection of hybrid duplexes usually use labeled oligonucleotides. Oligonucleotides can be labeled by incorporating a label detectable by spectroscopic, photochemical, biochemical, immunochemical, or, armenti (for example, those which are used in the method ELISAS), Biotin, or haptens and proteins for which available anticavity or monoclonal antibodies. Labeled oligonucleotides according to the invention can be synthesized and labeled using the methods described above for synthesizing oligonucleotides.

An alternative method of detecting nucleic acid amplification of HIV-1 by estimating the increase in the total amount of double-stranded DNA in the reaction mixture described by Higuchi and others, 1992, Bio/Technology 10: 413-417; Higuchi and others , 1993, Bio/Technology 11: 1026-1030; and in the publication of the European application 512334. Detection of double-stranded DNA targets based on vozrastnoi fluorescence, which occurs when ethidiumbromid (EtBr) and the other to bind to the DNA of the label associated with double-stranded DNA. Tag to bind to the DNA added to the reaction mixture for amplification. Amplification of a target sequence leads to an increase in the number of double-stranded DNA, which in turn leads to a detectable increase in fluorescence.

The present invention also relates to kits, megacontinent sets containing the corresponding components for the practical implementation of the method according to the H-1. The kit may also contain means for detecting amplified nucleic acids of HIV-1, such as oligonucleotide probes. In some cases, the probes fixed on the corresponding substrate-membrane. Other optional components of the kit include, for example, an agent, suitable as catalyst for the synthesis of products of elongation of the primer, nucleosidases as a substrate, means for tagging (e.g., conjugate, avidin-enzyme, or an enzyme substrate and Chromogen in the case when the label is a Biotin), appropriate buffers for PCR, or hybridization reactions, and instructions for implementation of this method.

Below the invention is more illustrated in the examples which do not limit the scope of the invention. Numerous embodiments of the invention within the scope of the claims, which follow from the examples, it should be evident to experts in the art based on the above description and the examples that follow.

Example 1

Designing quantitative standard

To quantify the content of the virus is performed using the AMPLICOR HIV-1 MONITOR Test, using quantitative with is the use of another probe. KS added to the test sample at a known concentration to obtain the known reference signal. For a wide range of concentrations of the target signal generated by the amplified target or amplified KS, proportional to the existing number. The number of copies of the target are estimated by comparing the signal generated during the amplification of the unknown target signal generated by a known CA.

The primers of the present invention amplified region, which is not completely covered by the COP included in the set AMPLICOR HIV-1 MONITOR Test (KC-Amplicor). Therefore, for the application of primers of the present invention designed a new COP. New KS designed from KS-Amplicor by lengthening the sequence KC-Amplicor to include the binding sites SKCC1 (SEQ ID NO: 3) and SKCC3 (SEQ ID NO: 4). The resulting KS can be found using the CC-specific probe used in the AMPLICOR HIV-1 MONITOR Test. Construction of plasmids containing the sequence of the new COP, from which transcribing RNA KS, can be carried out using standard methods, as described below.

KC-Amplicor obtained from the AMPLICOR HIV-1 MONITOR Test, amplified using primers SK145 (SEQ ID NO: 1) and SK151 (SEQ ID NO: 7) in what was alocale DNA product, contains the binding sites for SK145 (SEQ ID NO: 1) and SK151 (SEQ ID NO: 7) and retaining internal sequence containing the binding site KS-specific probe.

Next, the formed amplificatory product lengthened to enable the binding site of primer SKCC1 (SEQ ID NO: 3) and add the linker for cloning of the product. This is achieved by re-amplification product using primer SK145 (SEQ ID NO: 1), extended at the 5'-end with a view to enable the linker HindIII and SKCC1 (SEQ ID NO: 3). Appropriate amplification conditions described by Kellogg and Kwok, 1990, PCR Protocols: A Guide to Methods and Applications, (edited Innis and others), Academic Press, San Diego, CA: 337-347, except that they use a lower temperature annealing/extension (for example, the 42o(C) for hybridization SKCC1 (SEQ ID NO: 3).

After that, the formed amplificatory product again lengthened to enable the binding site of primer SKCC3 (SEQ ID NO: 4) and add a second linker to the other end to the possibility of cloning product. This is achieved through amplification product using primer SK145 (SEQ ID NO: 1), extended at the 5'-end with a view to enable the HindIII linker, as described above, along with primer SKCC3 (SEQ ID NO: 4), extended to politizirovanny product is inserted into the plasmid. Amplified DNA and plasmid pSP64 (firm Promega, Madison, WI) separately digested with HindIII and HVA, and then are ligated using standard methods. Competent cells transformed with recombinant plasmids and get a clone that contains the correct box. The cloned insert in the resulting recombinant plasmids need to be sequenced to determine that the binding sites of the primer or probe is not introduced mutations.

RNA quantitative standard Transcriber from recombinant plasmids containing KS-sequence, using the MEGAscript kitTMSP6 (firm Ambion, Inc., Austin, TX).

Example 2

Quantifying HIV-1 RNA

This example describes the method of estimating the relative efficiency of amplification of different isolates of HIV-1. For comparison amplification was carried out using a set of AMPLICOR HIV-1 MONITOR Test.

Isolates of HIV-1 were obtained from HIV-1-positive clinical specimens. The region of the gag gene was cloned and sequenced by standard methods, and the subtype of cloned HIV-1 was identified based on sequence analysis. Was discovered a number of new isolates of HIV-1. For this purpose, on the basis of the nucleotide is the training set AMPLICOR HIV-1 MONITOR Test. In addition, used the isolates, which was inherent variability of the sequence, characteristic of subtypes of group M

Nucleic acid-target

Designed plasmid containing the region of the gag gene of HIV-1 from each isolate, and the RNA matrix HIV-1 transcriptional almost in accordance with the method described in Holodniy and others, 1991, J. Inf. Dis., 163: 802-865. Prepared uterine solutions for each matrix and determined the concentration matrix. Royal solutions were diluted based on the determination of relative concentrations, so that the concentration of the matrices to be added to each reaction mixture was the same. The absolute number of copies of the matrix is added to the reaction mixture was approximately 4000-8000.

Quantitative standard

The COP described in example 1 was introduced into each reaction mixture in a known concentration, usually about 100 copies per reaction mixture.

Primers

Reactions a-E was performed using combinations of the primers are presented in table. A.

All primers were biotinilated at the 5'-end detection method, the reverse dot-blot on the microwell plate. Sequence, additional, NIV progress transcription:

SK462 (SEQ ID NO: 5) AGTTGGAGGACATCAAGCAGCCATGCAAAT

The primers that initiate synthesis during transcription:

SK431 (SEQ ID NO: 6) TGCTATGTCAGTTCCCCTTGGTTCTCT

SK151 (SEQ ID NO: 7) TGCTATGTCACTTCCCCTTGGTTCTCT

Amplification

Reaction amplification And was performed using reagents and conditions set AMPLICOR HIV-1 MONITOR Test.

Reaction amplification, and B were carried out in volumes of 100 μl containing the following reagents:

RNA-matrix HIV-1

RNA KS

50 mm bicin, pH 8.3

111 mm(SLA)

3.6 mm MP(SLA)2< / BR>
500 μm dUTP

300 μm each of dATP, dCTP and DSTF

50 μm dTTP

15% glycerine

0.2 μm of each biotinylated primer

2 units of uracil-DNA glycosylase (UDG)*

10 units of DNA polymerase, rTth*

*Production and development company Hoffmann-La Roche and sale by the company Perkin Elmer (Norwalk, CT).

Amplification reaction and D was carried out under almost the same conditions that were used in the reaction of B, but with the following changes:

100 mm(SLA)

500 μm each of dATP, dCTP and DSTF

7,5% glycerine

10 units of UDG

Amplification reaction D and E perform almost the same conditions that were used in the reactions C and D, but except that koncentrisanje conditions of amplification for each pair of primers.

Amplification reaction B-E were carried out in DNA-thermoacetica type TS (firm Perkin Elmer, Norwalk, CT) using the following temperature profile:

incubation before the reaction: - 50oC for 2 min

reverse transcription: - 60oC for 30 min

4 cycles: denaturation: - 95oC for 10 s

annealing: - 55oC for 10 s

elongation: - 72oC for 10 s

26 cycles: denaturation: - 90oC for 10 s

annealing: - 60oC for 10 s

elongation: - 72oC for 10 s

final elongation: - 72oC for 15 min

Detection of the amplified product by hybridization with probe

Amplificatoare products were detected using reagents and protocols from a set of AMPLICOR HIV-1 MONITOR Test. The estimated initial concentration of the target was calculated in accordance with the present description.

Results

When using a quantitative method AMPLICOR HIV-1 MONITOR Test initial concentration of the target are estimated by comparing the signal generated after amplification of the target, with the generated signal after amplification COP known concentration. As is known, the concentration of CA is konzentrationen in the relative efficiency of amplification should influence the assessment of the initial concentration of the unknown target. A quantitative assessment using the AMPLICOR HIV-1 MONITOR Test using a calibration based on the efficiency of amplification subtypes In HIV-1. It is known that other subtypes of HIV-1 can amplificates with lower efficiency and, consequently, the estimated concentration of the target can be lower than the true.

In this experiment, each reaction mixture was added to the RNA target in a known concentration. Thus, the relative efficiency of amplification for each isolate can be determined by comparing the measured concentrations of the target. Because when it comes to quantifying the set of AMPLICOR HIV-1 MONITOR Test using a calibration based on the efficiency of amplification subtypes In HIV-1, as the standard used to assess the concentration of the target subtype (clone 105-1). For each isolate estimate the concentration of the target subtype (clone 105-1) correlated with estimated concentration of the target protein isolate, getting the measure of the relative efficiency of amplification. These relative efficiency of amplification are given in table. 1. The symbol "---" indicates that the reaction is not conducted.

The results show that when using a set of AMPLICOR HIV-1 MONITOR Test (reactions is including the isolate of subtype E, clone 110-5, amplificadores with an efficiency of at least about 500 times lower than the efficiency of amplification of clone 105-1 subtype Century

Although not all isolates were investigated, using pairs of primers SK145 (SEQ ID NO: 1) and SK151 (SEQ ID NO: 7) (reaction B) allowed to substantially align the effectiveness of amplification. However, the clone 110-5 subtype E was still amplificatoare with an efficiency of at least about 500 times lower than the efficiency of amplification of clone 105-1 subtype Century

The use of primers SK145 (SEQ ID NO: 1) and SKCC1 (SEQ ID NO: 3) (reaction) allowed to amplify all isolates, including isolates of subtype E, clone 110-5, with an efficiency of approximately 3 times lower than the efficiency of amplification of clone 105-1 subtype C. Adding primer SK145M2 (SEQ ID NO: 2) (reaction G) further improved the efficiency of amplification of the isolate 110-5, making it almost equal to the reference strain of subtype C.

Similarly, the use of primers SK145 (SEQ ID NO: 1) and SKCC3 (SEQ ID NO: 4) (reaction D) allowed to amplify all isolates, including isolates of subtype E, clone 110-5, with an efficiency of approximately 5 times less than the efficiency of amplification of clone 105-1 subtype C. Adding primevel that of the reference strain of subtype C.

Example 3

Quantification of HIV-1 in clinical samples

In this example, 30 clinical samples obtained from patients from Senegal with positive serological reaction, were analyzed for the presence of HIV-1 RNA. Subtypes present in clinical samples was not determined. However, subtypes a and D are typical for this region of Africa, so it was expected that some of these clinical samples will or will not be amplificates at all, or will amplificates inefficient when using the AMPLICOR HIV-1 MONITOR Test.

Samples were prepared as follows. Plasma samples (80-250 μl) were combined in a conical centrifuge tube with a volume of 1.5 ml with 20 μl of 0.25% (wt. /about.) red polystyrene microspheres type Estapor (firm Bangs Laboratories, Inc., Carmel, IN) and centrifuged for 1 h at 25300xg at 4oC. the Supernatant was aspirated and debris resuspendable 250 ál lyse buffer (50 ál lyse buffer contains 6,7 ál of 30 u/ml RNasin, Promega, Madison, WI); 0,67 ál of 100 mm dithiothreitol (DTT); 2 μl of 10% NP40 (firm Pierce, Rockford, IL); 0,25 µl of 4 mg/ml poly rA RNA; and 40.4 μl of Depc-treated H2About). Debris incubated at room temperature for at least 15 min and intensively Paramecium, containing 50 μl of a solution of viral lysate and 50 μl of 2-fold amplified mixture of reagents prepared so that the final concentration of the reagent is consistent with the above. In each reaction mixture in the form of part of the mixture of reagents was added approximately 100 copies of the corresponding CC. Detection of the amplified product was performed using reagents and protocols AMPLICOR HIV-1 MONITOR Test, as described above.

Amplification of each sample was performed with combinations of primers are presented in table. Century

The results, expressed as the number of copies of HIV-1 matrix of 1 ml of plasma are summarized in table. 2.

The results indicate that the combination of primers B and C of the present invention allow to amplify a larger number of clinical samples. Combinations of primers B and C allow to amplify all but one of the 30 samples. In contrast, when using the AMPLICOR HIV-1 MONITOR Test failed to amplify 6 of 29 samples. The amplification of the sample MIH 053 using AMPLICOR HIV-1 MONITOR Test has led to a strong signal from the target, but failed to generate the signal KS. So I was not able to quantify this icesto copies identified using combinations of primers B and was significantly higher than that detected using the AMPLICOR HIV-1 MONITOR Test. Given the variability of the efficiency of amplification, which is shown above in example 2, it can be assumed that the lower the detected concentration of the matrix, obtained using the AMPLICOR HIV-1 MONITOR Test, are probably a result of the significantly lower efficiency of amplification.

Viral RNA was not detected in the sample MIN 067. However, it is unknown whether this is a consequence of undetected variation in the target sequence that serves as a hindrance for hybridization of the primer or probe, or due to any other cause.

1. Oligonucleotide primer used for amplification of nucleic acid of human immunodeficiency virus type 1 (HIV-1), and oligonucleotide primer chosen from the group comprising SKCC1 (SEQ ID NO: 3) and SKCC3 (SEQ ID NO: 4).

2. A pair of oligonucleotide primers designed for amplification of nucleic acid of human immunodeficiency virus type 1 (HIV-1) and consisting of SK145 (SEQ ID NO: 1) and SKCC1 (SEQ ID NO: 3) or SK145 (SEQ ID NO: 1) and SRCC3 (SEQ ID NO: 4).

3. A set of oligonucleotide primers, the second of the pair of oligonucleotide primers for p. 2 and SK145M2 (SEQ ID NO: 2).

4. The set, designed for amplification of nucleic acid of human immunodeficiency virus type 1 (HIV-1), which includes oligonucleotide primer under item 1, or a pair of primers for p. 2, or a set of primers for p. 3.

5. The method of amplification of nucleic acid of human immunodeficiency virus type 1 (HIV-1), including the implementation of polymerase chain reaction using at least one primer SKCC1 (SEQ ID NO: 3) or SKCC3 (SEQ ID NO: 4).

6. The method according to p. 5, where polymerase chain reaction carried out using primers SK145 (SEQ ID NO: 1) and SKCC1 (SEQ ID NO: 3).

7. The method according to p. 6, where polymerase chain reaction carried out using primer SK145M2 (SEQ ID NO: 2).

8. The method according to p. 5, where polymerase chain reaction carried out using primers SK145 (SEQ ID NO: 1) and SKCC3 (SEQ ID NO: 4).

9. The method according to p. 8, wherein the polymerase chain reaction carried out using primer SK145M2 (SEQ ID NO: 2).

 

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