WO2007069331A1

WO2007069331A1 - Method for detection of human immunodeficiency virus type 1

Info

Publication number: WO2007069331A1
Application number: PCT/JP2005/023120
Authority: WO
Inventors: Norimitsu Hosaka; Harumi Minekawa
Original assignee: Eiken Kagaku Kabushiki Kaisha
Priority date: 2005-12-16
Filing date: 2005-12-16
Publication date: 2007-06-21

Abstract

[PROBLEMS] To provide a method for detecting human immunodeficiency virus type 1 (which is a pathogenic virus) rapidly with high sensitivity for use in the diagnosis of the infection with human immunodeficiency virus type 1. [MEANS FOR SOLVING PROBLEMS] An oligonucleotide primer capable of specifically hybridizing with any nucleotide sequence which is designed based on the nucleotide sequence of the pol region of human immunodeficiency virus type 1; a method for the amplification of a nucleic acid using the primer; a method for the diagnosis of the infection with human immunodeficiency virus type 1 based on the detection of nucleic acid amplification; and a kit for the diagnosis of the infection with human immunodeficiency virus type 1.

Description

Specification

Method for detecting human immunodeficiency virus type 1

Technical field

The present invention relates to a method for detecting human immunodeficiency virus type 1 (hereinafter sometimes referred to as “HIV — 1”), and more specifically, an oligonucleotide primer for detecting HIV-1; The present invention relates to a method for detecting HIV-1 used, a method for diagnosing HIV-1 infection, and a kit for diagnosing HIV-1 infection.

Background art

[0002] HIV is increasing in the number of infected people worldwide, especially in sub-Saharan Africa, where the rate of infection is more than 15%, and in Southeast Asian drug users (IDU) more than 60%. There are two types of HIV: HIV-1 and HIV-2, but the latter is limited to detection in Africa, and the former is the former in Japan. In Japan, more than 10,000 people with HIV-1 infection and those with acquired immune deficiency syndrome (AIDS) are known. In Japan, the only number of infected people in developed countries is increasing. In recent years, about 1000 infected people and onset cases have been observed annually. AIDS caused by HIV develops after an asymptomatic period of about 10 years and develops opportunistic infections due to immunodeficiency.

[0003] Diagnosis of HIV infection is mainly performed by the presence or absence of HIV antibodies present in human serum. A nucleic acid detection method for measuring HIV RNA is also used as a highly sensitive method. Recently, both Amplicor ™ HIV detection and TaqMan ™ technology for real-time detection have been used to detect and quantify HIV RNA in a more sensitive PCR assay.

[0004] In blood for blood transfusion, a PCR assay, which is a nucleic acid detection method, is used for blood screening at the time of donation. However, even with these virus tests, it has become a major social problem that slip-through occurs and transfusion infection occurs.

[0005] Therefore, a test method that can detect HIV quickly and with high sensitivity has been desired.

[0006] Patent Document 1: JP 2002-330790 A

Patent Document 2: Special Table 2003-504077 Disclosure of the invention

Problems to be solved by the invention

[0007] As a result of intensive studies to solve the above problems, the present inventors hybridize with HIV-1 specific base sequences of the main subtypes (B, B—Thai type and C). It was found that HIV-1 infection can be detected with high sensitivity by preparing an oligonucleotide primer and amplifying a base sequence specific for HIV-1 by the loop-mediated isothermal amplification (LAMP) method. Was completed.

Means for solving the problem

That is, the present invention provides the following (1) to (8).

(1) An oligonucleotide primer designed from the human immunodeficiency virus type 1 (Genbank I. D. U43096) represented by SEQ ID NO: 1, 4208 to 4515 (pol region), or a complementary nucleotide sequence thereto.

(2) The oligonucleotide primer according to (1), comprising an oligonucleotide selected from the following (a) to (c):

(a) an oligonucleotide comprising at least 13 consecutive bases selected from the base sequences represented by SEQ ID NOs: 2 to 7 or a base sequence complementary thereto;

(b) An oligonucleotide that can hybridize with the oligonucleotide according to (a) under stringent conditions.

(c) An oligonucleotide having a primer function, comprising a base sequence in which one to several bases are substituted, deleted, inserted or added among the oligonucleotides described in (a) or (b).

(3) Select the F3c, F2c, and Flc base sequence regions from the 3 'end side of the target nucleic acid in the pol region of human immunodeficiency virus, and select the base sequence regions B3, B2, and B1 from the 5' end side. When each complementary base sequence is F3, F2, Fl, and B3c, B2c, Blc, the base sequence is selected from the following (a) to (d): 1. The oligonucleotide primer according to (1) to (2).

(a) A base sequence having the F2 region of the target nucleic acid on the 3 ′ end side and the Fl c region of the target nucleic acid on the 5 ′ end side. (b) A base sequence having the F3 region of the target nucleic acid.

(c) A base sequence having the B2 region of the target nucleic acid on the 3 ′ end side and the Blc region of the target nucleic acid on the 5 ′ end side.

(d) A base sequence having a B3 region of the target nucleic acid.

(4) It is capable of amplifying 1J-specific nucleotide sequence specific to human immunodeficiency virus, and its 5 'end force is also directed to the 3' end, and consists of a base sequence selected from the following (a) to (b) Toss

The oligonucleotide primer according to (1) to (3).

(a) 5 ′ — (base sequence 1J complementary to the base sequence of SEQ ID NO: 2) — (any base sequence having 0 to 50 bases) — (base sequence of SEQ ID NO: 3) 1 3 ′.

(b) 5 '— (base sequence 1J of SEQ ID NO: 6)-(any base sequence 1J of 0 to 50 bases)-(base sequence IJ complementary to the base sequence of SEQ ID NO: 7) 3 '.

(5) A method for detecting HIV-1 comprising carrying out an amplification reaction of a target nucleic acid region of HIV-1 using the oligonucleotide primers according to (1) to (4).

(6) The method for detecting HIV-1 according to (5), wherein the amplification reaction of the target nucleic acid region of HIV-1 is the LAMP method.

(7) HIV1 characterized by diagnosing the presence or absence of HIV-1 infection by detecting amplification of the target nucleic acid region of human immunodeficiency virus using the oligonucleotide primers described in (1) to (4) How to diagnose infection.

(8) A kit comprising the oligonucleotide primer according to (1) to (4), for diagnosing HIV-1 infection.

The invention's effect

[0009] According to the present invention, an oligonucleotide primer that selectively hybridizes with a base sequence specific to HIV-1 is prepared, and a base sequence specific to HIV-1 is amplified by the LAMP method. It is possible to detect HIV-1 of major genotypes in Japan with high sensitivity and speed.

BEST MODE FOR CARRYING OUT THE INVENTION

[0010] Samples used in the present invention include specimens derived from living organisms of humans or other animals suspected of having HIV infection, such as blood, serum, plasma, spinal fluid, semen, sputum, gargle, saliva Urine, feces, tissue, and the like. In addition, a sample such as a cell or a culture solution thereof used for an infection experiment or a specimen containing a virus isolated from a biological specimen or a cultured cell can be used. These samples may be subjected to pretreatment such as separation, extraction, concentration and purification.

[0011] Such nucleic acid amplification is a new nucleic acid amplification method developed by Natomi et al. That does not require temperature control, which is indispensable for PCR method: Loop-mediated isothermal amplification method called LAMP method (International Publication No. 00/28082 No. brochure). In this method, a complementary nucleotide synthesis reaction is carried out by combining the primer that anneals with the loop formed at this time by annealing the 3 'end of the nucleotide itself to the caged nucleotide and using it as a starting point for the complementary strand synthesis. It is a nucleic acid amplification method that makes it possible. The LAMP method is a highly specific nucleic acid amplification method that uses four primers that recognize at least six regions.

[0012] The oligonucleotide primer used in the LAMP method is a method for calculating the base sequence of a cage nucleic acid.

At least 4 types of primers that recognize the base sequences of 6 regions, namely the region F3c, F2c, Flc from the 3 ′ end side and the region B3, B2, B1 from the 5 ′ end side, each inner primer F And B and outer primers F and B. The complementary sequences of Flc, F2c, and F3c are called Fl, F2, and F3, and the complementary strands of Bl, B2, and B3 are called Blc, B2c, and B3c, respectively. The inner primer recognizes a “specific nucleotide sequence region” on the target base sequence, and has a base sequence that gives the origin of synthesis at the 3 ′ end, and at the same time generates a nucleic acid synthesis reaction starting from this primer. An oligonucleotide having a base sequence complementary to an arbitrary region of the product at the 5 ′ end. Here, a primer containing “base sequence U selected from F2” and “base sequence IJ selected from F lc” is designated as inner primer F (hereinafter abbreviated as FIP), and “prime selected from B2”. A primer containing “base sequence 1J” and “base sequence 1J selected from Blc” is referred to as inner primer B (hereinafter abbreviated as BIP). On the other hand, an outer primer is an oligonucleotide having a base sequence that recognizes "a specific nucleotide sequence region existing 3 'from the region recognized by the inner primer" on the target base sequence and provides a starting point for synthesis. is there. Here, “a primer containing base sequence 1 selected from F3 is referred to as outer primer F (hereinafter abbreviated as F3”), and a primer including “base sequence IJ selected from B3” is used as outer primer B (hereinafter referred to as “F1”). Abbreviated as B3). Where in each primer F is a primer display that complementarily binds to the sense strand of the target base sequence and provides a synthetic origin, while B is complementary to the antisense strand of the target base sequence and binds the synthetic origin. It is a primer indication to provide. Here, the length of the oligonucleotide used as a primer is 10 bases or more, preferably 15 bases or more. Either chemically synthesized or natural primers may be used as a single oligonucleotide. It may be a mixture of oligonucleotides.

[0013] In the LAMP method, in addition to the inner primer and the outer primer, another primer, that is, a loop primer can be used. When the complementary sequences generated on the same strand of the amplified product by the LAMP method are mutually annealed to form a loop, the loop primer is a base sequence complementary to the sequence in the loop. Two types of primers included at the ends (one for each of the duplexes). When this primer is used, the starting point of nucleic acid synthesis is increased, and the reaction time can be shortened and the detection sensitivity can be increased (WO 02/24902 pamphlet).

[0014] Oligonucleotides can be produced by known methods, and can be chemically synthesized, for example. Alternatively, a natural nucleic acid can be cleaved with a restriction enzyme or the like, modified so as to have a desired base sequence, or ligated. Specifically, it can be synthesized using an oligonucleotide synthesizer or the like. In addition, as a method for synthesizing an oligonucleotide having a base sequence in which one to several bases are substituted, deleted, inserted or added, a method known per se can be used. For example, it is possible to synthesize powerful oligonucleotides by site-directed mutagenesis, gene homologous recombination, primer extension, or PCR alone or in combination as appropriate.

[0015] As the "stringent hybridization conditions" in the present specification, generally known ones can be selected. Stringent conditions include, for example, 50% honremamide, 5 X SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 X Denhart's solution, 10% dextran sulfate, and 20 _β solution containing gZ ml of DNA, after hybrida I See Chillon 42 ° C De晚, 2 X SS C at room temperature - a primary lavage in 0. l ⁰ / oSDS in, in the following Rere, Itoshaku 65 . C (This is the condition that secondary cleaning is performed with 0.1 X SSC-0.1% SD s. [0016] As a result of intensive studies on the base sequence of LAMP primer and its combination that can rapidly amplify a base sequence specific to HIV-1, the present inventors have determined that the base sequence (sequence) of the pol region of HIV-1 From the base arrangement 1J) indicated by No. 1, the following one set was selected as a primer set.

(Primer set) -3 '(SEQ ID NO: 10) -3' (SEQ ID NO: 11)

F3: 5 -CCCCAAAGTCAAGGAGTAGT-3 '(SEQ ID NO: 4)

B3: 5'-TTTGCTGGTCCTTTCCAA-3 '(SEQ ID NO: 8)

LF: 5'-TGGATGAATACTGCCAT-3 '(SEQ ID NO: 5)

LB: 5'- AACAGACATACAAACTAGAGAATTAC-3 '(SEQ ID NO: 9)

[0017] The enzyme used in the nucleic acid synthesis is not particularly limited as long as it is a cage-dependent nucleic acid synthase having strand displacement activity. Examples of such an enzyme include Bst DNA polymerase (large fragment), Bca (exo_) DNA polymerase, Talenow fragment of E. coli DNA polymerase I, and preferably Bst DNA polymerase (large fragment).

[0018] The reverse transcriptase used in the RT-LAMP method is not particularly limited as long as it has an activity of synthesizing cDNA using RNA as a cage. Examples of such enzymes include AMV, Cloned AMV, MMLV reverse transcriptase, Superscript II, ReverTraAce, Thermoscript and the like, and preferably AMV or Cloned AMV reverse transcriptase. If an enzyme having both reverse transcriptase activity and DNA polymerase activity is used, such as Bca DNA polymerase, the RT-LAMP reaction can be performed with a single enzyme.

[0019] The enzyme or reverse transcriptase used in the nucleic acid synthesis may be purified from a virus or a bacterium, or may be produced by a gene recombination technique. These enzymes may be modified by fragmentation or amino acid substitution.

[0020] A known technique can be applied to the detection of the nucleic acid amplification product after the LAMP reaction. For example, increase It can be detected using a labeled oligonucleotide that specifically recognizes the widened base sequence or the fluorescent intercalator method (Japanese Patent Laid-Open No. 2001-242169), and the reaction solution after the reaction is completed It can be easily detected by subjecting it to agarose gel electrophoresis as it is. In agarose gel electrophoresis, the LAMP amplification product detects a number of bands with different base lengths in a ladder form. In addition, in the LAMP method, a large amount of substrate is consumed by nucleic acid synthesis, and pyrophosphate ions, which are by-products, react with coexisting magnesium ions to become magnesium pyrophosphate, and the reaction solution becomes cloudy enough to be confirmed with the naked eye. Therefore, this cloudiness can be confirmed by measuring the change in absorbance at 400 nm using a normal spectrophotometer, which can optically observe the increase in turbidity over time after completion of the reaction or during the reaction. It is also possible to detect nucleic acid amplification reactions (WO 01Z83817 pamphlet).

[0021] Various reagents necessary for detection of nucleic acid amplification using the primer of the present invention can be prepackaged and kited. Specifically, various oligonucleotides necessary as the primer or loop primer of the present invention, four types of dNTPs that serve as substrates for nucleic acid synthesis, DNA polymerase that performs nucleic acid synthesis, an enzyme having reverse transcription activity, and suitable for enzymatic reactions Buffers and salts that give appropriate conditions, protective agents that stabilize enzymes and molds, and reagents necessary for detection of reaction products, if necessary, are provided as kits.

Example

[0022] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0023] Example 1: Primer design

In order to detect the major subtypes of HIV-1, we performed alignment analysis by referring to the gene sequence from Genbank (Fig. 1). As a result, pol regions with relatively high conservation of all gene sequences were selected and primers were prepared. The reactivity of the primer was confirmed by the following method. The composition of the reaction solution for performing nucleic acid amplification by the LAMP method is as follows. Primer synthesis was requested from QIAGEN and purified by HPLC (high performance liquid chromatography). 20 mM Tris-HCl pH 8.8

lOmM KC1

8 mM MgSO

1.9mM dNTPs

lOmM (NH) SO

0. 8M Betaine (SIGMA)

0. 1% Tween20

1. 6μΜ FIP

1. 6μΜ

0.6μΜ F3

0.6μΜ Β3

1. 6μΜ LF

1. 6μΜ LB

AMV Reverse Transcriptase 2U (Finnzyme)

Bst DNA polymerase 16U (NEB)

[0024] A dilution series of 10 to 1000 copies of HIV RNA (produced by Eiken Chemical) was added to the above reaction solution, and a LAMP reaction was performed at 60.0 ° C for 40 minutes. The reaction was detected in real time using a real-time turbidity measurement device LA-200 (Eiken Chemical). Figure 2 is a graph showing the results of detection sensitivity when HIV RNA is used. As shown in Figure 2, amplification of up to 10 copies of HIV RNA was confirmed for this set of primers.

[0025] Example 2: Confirmation of amplification products

The LAMP product amplified with the above primer set was confirmed by electrophoresis. FIG. 3 shows the results of electrophoresis. Lane 1 is HIV positive sample, Lane 2 is D

W, lane 3 is an electropherogram of the 100b marker. In Lane 3, a ladder pattern unique to LAMP products was confirmed.

[0026] Example 3: Evaluation of primers (specificity test)

Specificity was evaluated using commercially available HIV plasma or serum samples. Details of the samples are shown in Table 1. [0027] [Table 1]

INPATH-BCP, Inc.

BCP Lot No IHatr i x PCR HIV Ab

0009-C37-00484 Serum Pos. Pos.

0009-037-00486 Serum Pos. Pos.

0009-037-00508 Serum Pos. Pos. Pos .: Positive

0009-037-00511 Serum Pos. Pos, Neg .: Negat i ve

0009-037-00526 Serum Pos. Pos.

Teraeeni, Inc,

MBI # atr i x PCR * Subtype HIV A 1/2 HIV As

0000010731.0022 P I asma-CP -A Pos.

0000012416.0011 Plasma-CP -A Pos. One Pos. Neg.

0000017498.0029 Plasma-EDTA (K3) Pos. AG ― One

0000022748.0013 Plasma-Sodium Citrate PDS. ― Pos. Pos.

0000026067.0027 Serum Pos .-- Pos.Pos.

0000027210.0030 Plasma-CPD-A PDS. AG Pos. Neg.

0000044887.0026 P I asma-CPD-A Pos. AG Pos.

0000058306.0070 P I asma-CPD-A PDS. AE Pos.

0000059 & 72.0023 PI asma-Sodium Citrate Pos. I Nee. Neg.

* PCR: Amp 1 ί cor

[0028] In order to isolate HIV RNA in the specimen, it was extracted by the following method. Add 300 zL of lysis reagent [5.76M guanidine thiocyanate, 194 mM DTT, 10 mM Tris—HCl (pH8.8)] and 1 μL of Pellet Paint (Novagen) to 100 μL of the positive sample. Incubated for 10 minutes at room temperature. Next, add 400 a L isopropanol, mix, and centrifuge at 15000 i "pm for 15 minutes. After removing the supernatant, add 70% ethanol, mix, and centrifuge at 1500 Orpm for 10 minutes. The supernatant was removed, allowed to stand at room temperature for 5 minutes, dissolved in 10 M sterile water, and 5 μL of this solution was brought into the LAMP reaction.

[0029] [Table 2]

Replacement ^ Paper (Rule 26) INPATH-BCP, Inc.

BCP Lot No PCR * copy / test Posit ive / test

0009-037-00484 35, 000 1,750 2/2

0009-037HX) 486 19, 000 950 2/2

0009-037-00508 180,000 9,000 2/2

0009-037-00511 130,000 6,500 2/2

0009-037-00526 250, 000 12, 500 2/2

Teragen i, Inc.

[0030] All specimens could be detected.

Brief Description of Drawings

[0031] [Fig. LA] Shows the results of a homology search based on the results of 14 cases of HIV-1 sequences searched from GenBank and 4 cases of sequence analysis from HIV positive specimens.

[Fig.lB] The results of homology searches based on the results of 14 cases of HIV-1 sequences and 4 cases of HIV positive specimens retrieved from GenBank (continued).

[Fig.lC] Shows the results of homology searches based on the results of 14 cases of HIV-1 sequences and 4 cases of sequence analysis from HIV positive specimens retrieved from GenBank (continued).

FIG. 2 is a graph showing the results of a sensitivity test. 10 ~: 1000 is HIVRN per Atsey

Represents the number of copies of A.

FIG. 3 is a diagram showing a result of electrophoresis of a sample after LAMP reaction of an HIV positive specimen.

¾ paper (mm Garden 4 is a graph representing the results of specificity tests using HIV positive samples _c

Claims

The scope of the claims

Arbitrary base arrangement IJ selected from the nucleotide sequence of 42 08 to 4515 (pol region) of human immunodeficiency virus type 1 (Genbank ID U43096) represented by SEQ ID NO: 1, or a base complementary thereto An oligonucleotide primer designed from IJ.

The oligonucleotide primer according to claim 1, comprising an oligonucleotide selected from the following (a) to (c).

(a) an oligonucleotide comprising at least 13 consecutive bases selected from the base sequences represented by SEQ ID NOs: 2 to 9 or a base sequence complementary thereto;

Select the base sequence regions F3c, F2c, Flc from the 3 'end of the target nucleic acid of the pol region of human immunodeficiency virus type 1 and the base sequence regions B3, B2, B1 from the 5' end. 2. Each of the complementary base sequences is F3, F2, Fl, and B3c, B2c, Blc, and comprises a base sequence selected from the following (a) to (d): The oligonucleotide primer described in 2 above.

(a) A base sequence having the F2 region of the target nucleic acid on the 3 ′ end side and the Fl c region of the target nucleic acid on the 5 ′ end side.

(b) A base sequence having the F3 region of the target nucleic acid.

(d) A base sequence having a B3 region of the target nucleic acid.

A base sequence specific to human immunodeficiency virus type 1 can be amplified, and consists of a base sequence selected from (a) to (b) below from the 5 'end to the 3' end: The oligonucleotide primer as described in 1-3.

(a) 5 ′ (base sequence lj complementary to the base sequence of SEQ ID NO: 2) (arbitrary 0-50 bases (Base sequence) (base sequence of SEQ ID NO: 3) 3 '.

(b) 5 ′ (base sequence IJ of SEQ ID NO: 6) (arbitrary base sequence IJ having 0 to 50 bases) (base sequence 3 ′ complementary to the base sequence of SEQ ID NO: 7)

[5] A human immunodeficiency virus using the oligonucleotide primer according to claims 1-4

A method for detecting human immunodeficiency virus type 1, which comprises carrying out an amplification reaction of a target nucleic acid region of type 1.

6. The method for detecting human immunodeficiency virus type 1 according to claim 5, wherein the amplification reaction of the target nucleic acid region of human immunodeficiency virus type 1 is the LAMP method.

[7] The presence or absence of human immunodeficiency virus type 1 infection is diagnosed by detecting amplification of a target nucleic acid region of human immunodeficiency virus using the oligonucleotide primers according to claims 1 to 4. To diagnose human immunodeficiency virus type 1 infection.

[8] A kit comprising the oligonucleotide primer according to claim 1 for diagnosing human immunodeficiency virus type 1 infection.

[9] An oligonucleotide designed from any nucleotide sequence selected from nucleotide sequences 1 to 422 of the pol region of human immunodeficiency virus 1 represented by SEQ ID NO: 1, or a complementary nucleotide sequence thereto Primer.