WO2007069331A1 - Méthode de détection du virus de l'immunodéficience humaine de type 1 - Google Patents

Méthode de détection du virus de l'immunodéficience humaine de type 1 Download PDF

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Publication number
WO2007069331A1
WO2007069331A1 PCT/JP2005/023120 JP2005023120W WO2007069331A1 WO 2007069331 A1 WO2007069331 A1 WO 2007069331A1 JP 2005023120 W JP2005023120 W JP 2005023120W WO 2007069331 A1 WO2007069331 A1 WO 2007069331A1
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WO
WIPO (PCT)
Prior art keywords
base sequence
human immunodeficiency
immunodeficiency virus
nucleic acid
region
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PCT/JP2005/023120
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English (en)
Japanese (ja)
Inventor
Norimitsu Hosaka
Harumi Minekawa
Original Assignee
Eiken Kagaku Kabushiki Kaisha
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Publication date
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Priority to PCT/JP2005/023120 priority Critical patent/WO2007069331A1/fr
Publication of WO2007069331A1 publication Critical patent/WO2007069331A1/fr

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

Definitions

  • the present invention 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.
  • 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%.
  • IDU Southeast Asian drug users
  • HIV-1 and HIV-2 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.
  • HIV-1 more than 10,000 people with HIV-1 infection and those with acquired immune deficiency syndrome (AIDS) are known.
  • AIDS acquired immune deficiency syndrome
  • 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.
  • 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.
  • Amplicor TM HIV detection and TaqMan TM technology for real-time detection have been used to detect and quantify HIV RNA in a more sensitive PCR assay.
  • PCR assay which is a nucleic acid detection method, is used for blood screening at the time of donation.
  • virus tests it has become a major social problem that slip-through occurs and transfusion infection occurs.
  • Patent Document 1 JP 2002-330790 A
  • Patent Document 2 Special Table 2003-504077 Disclosure of the invention
  • 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.
  • LAMP loop-mediated isothermal amplification
  • the present invention provides the following (1) to (8).
  • 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.
  • oligonucleotide primer according to (1) comprising an oligonucleotide selected from the following (a) to (c):
  • 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).
  • 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).
  • 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.
  • a kit comprising the oligonucleotide primer according to (1) to (4), for diagnosing HIV-1 infection.
  • 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.
  • 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.
  • 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.
  • 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).
  • LAMP method International Publication No. 00/28082 No. brochure.
  • 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.
  • the LAMP method is a highly specific nucleic acid amplification method that uses four primers that recognize at least six regions.
  • 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 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).
  • 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.
  • each primer F is a primer display that complementarily binds to the sense strand of the target base sequence and provides a synthetic origin
  • B is complementary to the antisense strand of the target base sequence and binds the synthetic origin.
  • 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.
  • the LAMP method in addition to the inner primer and the outer primer, another primer, that is, a loop primer can be used.
  • a loop primer 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).
  • Oligonucleotides can be produced by known methods, and can be chemically synthesized, for example.
  • 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.
  • it can be synthesized using an oligonucleotide synthesizer or the like.
  • 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.
  • Stringent hybridization conditions 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 ⁇ solution containing gZ ml of DNA, after hybrida I See Chillon 42 ° C De ⁇ , 2 X SS C at room temperature - a primary lavage in 0. l 0 / oSDS in, in the following Rere, Itoshaku 65 .
  • 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).
  • 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.
  • 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.
  • 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.
  • reagents necessary for detection of nucleic acid amplification using the primer of the present invention can be prepackaged and kited.
  • 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.
  • FIG. 1 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.
  • FIG. 3 shows the results of electrophoresis. Lane 1 is HIV positive sample, Lane 2 is D
  • Lane 3 is an electropherogram of the 100b marker. In Lane 3, a ladder pattern unique to LAMP products was confirmed.
  • HIV RNA in the specimen 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 ⁇ L of Pellet Paint (Novagen) to 100 ⁇ L 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 ⁇ L of this solution was brought into the LAMP reaction.
  • 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.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
  • FIG. 3 is a diagram showing a result of electrophoresis of a sample after LAMP reaction of an HIV positive specimen.
  • 3 ⁇ 4 paper (mm Garden 4 is a graph representing the results of specificity tests using HIV positive samples c

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Virology (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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  • AIDS & HIV (AREA)
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  • General Engineering & Computer Science (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne une méthode de détection du virus de l'immunodéficience humaine de type 1 (qui est un virus pathogène) d'une façon rapide et avec une sensibilité élevée, pour utilisation dans le diagnostic de l'infection par le virus de l'immunodéficience humaine de type 1. La présente invention concerne spécifiquement une amorce oligonucléotidique susceptible de s'hybrider de façon spécifique avec toute autre séquence de nucléotides conçue à partir de la séquence de nucléotides de la région pol du virus de l'immunodéficience humaine de type 1 ; une méthode d'amplification d'un acide nucléique utilisant ladite amorce ; une méthode de diagnostic de l'infection par le virus de l'immunodéficience humaine de type 1 basée sur la détection de l'amplification de l'acide nucléique ; et un kit pour le diagnostic de l'infection par le virus de l'immunodéficience humaine de type 1.
PCT/JP2005/023120 2005-12-16 2005-12-16 Méthode de détection du virus de l'immunodéficience humaine de type 1 WO2007069331A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012515539A (ja) * 2009-01-27 2012-07-12 クレティス・アクチェンゲゼルシャフト 粘稠液生体試料の処理と分析

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002024902A1 (fr) * 2000-09-19 2002-03-28 Eiken Kagaku Kabushiki Kaisha Procede permettant de synthetiser un polynucleotide
JP2002330790A (ja) * 2001-01-09 2002-11-19 Becton Dickinson & Co Ｈｉｖ−１の配列と検出方法
JP2004500014A (ja) * 1998-10-30 2004-01-08 ロシュ　ダイアグノスティックス　ゲーエムベーハー Ｈｉｖを検出するための新規のプライマーおよびプローブ

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004500014A (ja) * 1998-10-30 2004-01-08 ロシュ　ダイアグノスティックス　ゲーエムベーハー Ｈｉｖを検出するための新規のプライマーおよびプローブ
WO2002024902A1 (fr) * 2000-09-19 2002-03-28 Eiken Kagaku Kabushiki Kaisha Procede permettant de synthetiser un polynucleotide
JP2002330790A (ja) * 2001-01-09 2002-11-19 Becton Dickinson & Co Ｈｉｖ−１の配列と検出方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HOSAKA N. ET AL.: "Loop-Mediated Isothermal Amplification (LAMP)-ho o Mochiita HIV-I RNA no Kenshutsu", THE JOURNAL OF AIDS RESEARCH, vol. 6, no. 4, 2004, pages 530, XP003014449 *
NOTOMI T. ET AL.: "Loop-mediated isothermal amplification of DNA", NUCLEIC ACIDS RES., vol. 28, no. 12, 2000, pages E63 (I-VII), XP003014450 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012515539A (ja) * 2009-01-27 2012-07-12 クレティス・アクチェンゲゼルシャフト 粘稠液生体試料の処理と分析

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