EP2809784A1 - Amorces d'amplification et sondes pour détecter le vih-1 - Google Patents

Amorces d'amplification et sondes pour détecter le vih-1

Info

Publication number
EP2809784A1
EP2809784A1 EP13742978.3A EP13742978A EP2809784A1 EP 2809784 A1 EP2809784 A1 EP 2809784A1 EP 13742978 A EP13742978 A EP 13742978A EP 2809784 A1 EP2809784 A1 EP 2809784A1
Authority
EP
European Patent Office
Prior art keywords
hiv
seq
probes
genome
oligonucleotide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP13742978.3A
Other languages
German (de)
English (en)
Other versions
EP2809784A4 (fr
Inventor
Thomas Leitner
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
LEITNER, THOMAS
Advanced Liquid Logic Inc
Original Assignee
Advanced Liquid Logic Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Advanced Liquid Logic Inc filed Critical Advanced Liquid Logic Inc
Publication of EP2809784A1 publication Critical patent/EP2809784A1/fr
Publication of EP2809784A4 publication Critical patent/EP2809784A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification

Definitions

  • HIV-1 Human immunodeficiency virus type 1
  • AIDS acquired immunodeficiency syndrome
  • HIV- 1 shows considerable genetic variability among different isolates. A combination of a rapid replication cycle, replication errors, high viral loads and in vivo selective pressure all contribute to the genetic diversity of HIV- 1.
  • M, O, N and P Four groups (M, O, N and P) of HIV-1 have been described based on their genetic relatedness. Group M is further divided into 9 subtypes (A, B, C, D, F, G, H, J and K) and circulating recombinant forms (CRFs).
  • nucleic acid-based assays such as reverse transcriptase polymerase chain reaction (RT-PCR) assays have been used to amplify HIV- 1 nucleic acid for early detection of HIV- 1 infection.
  • RT-PCR reverse transcriptase polymerase chain reaction
  • the genetic variability of HIV- 1 presents particular challenges for diagnostic RT-PCR assays which may be limited by their inability to detect all genotypes with equal efficiency.
  • the selection of oligonucleotides to be used as primers and probes in the amplification and detection of nucleic acid sequences is critical for the sensitivity and specificity of the assay. Consequently, there is a need for oligonucleotide primer sets and detection probes for specifically and sensitively amplifying and detecting all HIV-1 groups as wells the various subtypes within or derived from these groups.
  • Figure 1 shows an example of a plot of genetic variation across the HIV genome
  • Figures 2A and 2B show examples of a plot of entropy in region 3000-4000 and a schematic diagram of the landmarks of the HIV- 1 genome, respectively;
  • Figures 3A and 3B show examples of a plot of entropy in region 5000-6000 and a schematic diagram of the landmarks of the HIV- 1 genome, respectively;
  • Figures 4A and 4B show examples of a plot of entropy in region 9000-10000 and a schematic diagram of the landmarks of the HIV- 1 genome, respectively;
  • Figure 5 illustrates a table of PCR forward and reverse primer sequences for sub-regions poll . l, pol2.1, env 1.1, and env 1.3;
  • Figure 6 illustrates a table of probe constructs for sub-regions poll . l, poll .2, pol2.1, and envl . l .
  • the present invention provides amplification oligonucleotides (primers) and detection probes for the detection of human immunodeficiency virus type-1 (HIV- 1) in a test sample.
  • the primers and probes may be used in an assay for the detection and/or quantitation of HIV- 1 nucleic acid.
  • the primers and detection probes may be used for real-time reverse transcription polymerase chain reaction (RT-PCR) assays for detection of HIV- 1 nucleic acid.
  • the detection probes may, for example, be fluorescently-labeled TaqMan® probes.
  • amplification primers and probes may be specific for the pol region of the HIV- 1 genome.
  • the amplification primers and probes may be specific for the env region of the HIV- 1 genome.
  • Amplification primers and probes of the invention may be used to detect all subtypes of HIV-1, including groups M, N, and O, as well as major circulating recombinant forms. "Cocktails" of multiple amplification primers and/or probes may be used to achieve maximal coverage of different HIV- 1 subtypes.
  • HIV- 1 is one of the most variable organisms known to man. HIV- 1 evolves at a rate a million times faster than humans and other higher organisms. This high rate of evolution is determined by a combination of replication errors, large population size within a human host, a rapid replication cycle, and strong selection pressures. As the selection pressure varies in mode and strength across the genome, the resulting genetic variation varies significantly across HIV- l 's genome.
  • Figure 1 shows an example of a plot 100 of genetic variation across the HIV genome. The Shannon entropy for all HIV-1/CPZ full genome sequences in the 2009 'web alignment' (www.hiv.lanl.gov) was calculated by a bias-corrected maximum likelihood method 1 .
  • Each class (subtype, group, or recombinant form) with >10 sequence representatives has its own line, and the remaining sequences are combined into "others".
  • the entropy was calculated as the mean in a window of 25 nt (i.e., the approximate size of a primer) spaced 100 nt apart.
  • the following HIV-1 classes (groups, subtypes, and recombinants) were considered in this analysis:
  • region 3000-4000 start of the pol gene
  • region 5000-6000 end of the pol gene
  • region 9000-10000 at the junction of gpl20 and gp41 in the env gene.
  • SIV CPZ was removed from each regional alignment, and sequences with big indels (i.e., insertions or deletions), uncalled nucleotide positions (N's), and otherwise suspected sequencing artifacts were removed. Subsequently, each regional alignment was manually checked and unnecessary gaps were removed before further analysis.
  • a full resolution entropy analysis was performed (i.e., moving the 25 nt primer window 1 nt across each regional alignment).
  • the full resolution entropy analysis was used to identify smaller sub-regions (designated poll .1, poll .2, pol2.1, pol2.2, envl . l, env 1.2, and env 1.3) for primer and probe design.
  • Figures 2A and 2B show examples of a plot 200 of entropy in region 3000-4000 and a schematic diagram 250 of the landmarks of the HIV-1 genome, respectively.
  • schematic diagram 250 shows the gene locations and coordinates for the HIV-1 reference strain HXB2.
  • the HXB2 genome is used as a universal HIV-1 coordinate system (the standard in the HIV database www.hiv.lanl.gov). Open reading frames (e.g., pol, env) are shown as rectangles.
  • a target region 252 for primer/probe design corresponds to HXB2 coordinates 2238-3138.
  • Figures 3A and 3B show an example of a plot 300 of entropy in region 5000-6000 and another view of schematic diagram 250 of the landmarks of the HIV- 1 genome, respectively.
  • target region 252 for primer/probe design corresponds to HXB2 coordinates 4127-5060.
  • Figures 4A and 4B show an example of a plot 400 of entropy in region 9000-10000 and yet another view of schematic diagram 250 of the landmarks of the HIV-1 genome, respectively.
  • target region 252 for primer/probe design corresponds to HXB2 coordinates 7308-81 15.
  • Figure 5 illustrates a table 500 of PCR forward and reverse primer sequences for sub-regions poll . l, pol2.1, envl . l, and envl .3. Individual primer constructs were selected for genetic variation, complexity, and melting temperature (Tm).
  • Figure 6 illustrates a table 600 of probe constructs for sub-regions poll . l, poll .2, pol2.1, and ⁇ 0 envl .1. Individual probe constructs were selected for complexity, melting temperature (Tm), and the distance from the forward primer 3'-end to the start (5'-end) of the probe (F dist).
  • F 10.2329.2355 is a forward primer designed to detect sequences with nucleotide variation per site that has been empirically observed at or above 10% in the underlying alignment. This primer spans positions 2329 through 2355 in the HXB2 genome. The fact that the first coordinate is smaller than the second also indicates that this is a forward primer. Conversely, reverse primers will have the larger coordinate first, e.g., R5.2424.2397. Probes follow the same labeling system. All amplicon lengths are ⁇ 150 bp. Because HIV-1 is genetically very diverse, HIV-1 targets amplified using the same primer pairs may not yield amplicons of the same length.
  • the genetic variation per primer/probe is indicated by the estimated Shannon entropy. Lower entropy values indicate less genetic variation.
  • the complexity is the number of individual sequences that a primer/probe may be deconvoluted into. Primers/probes were selected for a complexity equal to or less than 64. Too great a complexity means many different primers, possible primer dilution and increased risk of non-specific amplification. Complexity and entropy are closely linked; in general, higher entropy means higher complexity. Thus, at a lower detection level (5% vs 10%), complexity usually increases, but not always (e.g., pol2.1 primers).
  • the melting temperature (Tm) was calculated as a mean of all deconvoluted individual sequences as they would appear on the plus-strand.
  • the Tm was calculated according to the thermodynamic nearest neighbor model 2 .
  • the Tm indicates a target annealing temperature and may be used as an initial start-point for determining reaction annealing temperatures. Note that for the reverse primers this is a further approximation.
  • primer pair Tm's were designed to be within 4° C of each other in a 60-64° C annealing temperature range. Because all Tms are approximations, small deviations from this range may occur. Probes were designed to have a Tm >5° C higher than matching primers.
  • primers were designed for each sub-genomic region (i.e., poll . l, pol2.1, envl . l, envl .3). Some primers may be included in several sets. Bolded sequences overlap with other sequences in table 500. In a preferred embodiment, pol primers were constructed with G/C at the 3 '-end to improve extension. For env primers, this constraint was relaxed due to more genetic variation in this part of the genome. Risk of primer dimerization was assessed through a sliding alignment procedure. Primers and probes within sets selected to have ⁇ 10 consecutive matches. Only primers in sets 3-5 of envl. l are shown.
  • Primers in sets 1 and 2 involved a rare length variation at the forward primer 3 '-end.
  • Tm's for envl . l primer sets 3-5 may be further adjusted (i.e., lowered) by removing the 5' nucleotides of the primers.
  • column "F dist" lists the distance from the forward primer 3'-end to the start (5'-end) of the probe.
  • detection probes are TaqMan® probes. To avoid quenching of the fluorophore, the first 5'-base of the probe is not a "G". Probes were designed to be located 2-10 bp from one of the primers, on the same strand. Probes were designed to have a Tm >5° C higher than matching primers.
  • Primers and probes with different detection levels may be used in combination. For example, primers with a 5% detection level may be combined with a probe with a 10% detection level. Alternatively, primers and probes with the same detection level (e.g., 5%) may be used. Primers at lower detection level may be used to detect more HIV-1 classes and at lower template concentrations, and once detected the probes will find the amplicons with less degeneracy.
  • the annealing temperature may be selected such that all forms of HIV- 1 , including those that have not yet been sequenced, may be detected.
  • primers and probes may be selected at 10% detection level and the Tm adjusted to detect all HIV-1 forms. Primers and probes at 10% detection level may have less misannealing and less effects of primer dilution.

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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)
  • Immunology (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • AIDS & HIV (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des oligonucléotides d'amplification (amorces) et des sondes de détection permettant de détecter le virus de l'immunodéficience humaine de type 1 (VIH-1) dans un échantillon testé. Les amorces et les sondes peuvent être utilisées dans un essai permettant de détecter et/ou de quantifier un acide nucléique du VIH-1. Dans divers modes de réalisation, les amorces et les sondes de détection peuvent être utilisées dans des essais faisant appel à une réaction d'amplification en chaîne par polymérase par transcription inverse en temps réel (RT-PCR) pour détecter un acide nucléique de VIH-1. Les sondes de détection peuvent être, par exemple, des ondes TaqMan® à marquage fluorescent. Dans un mode de réalisation, les amorces d'amplification et les sondes peuvent être spécifiques de la région pol du génome du VIH-1. Dans un autre mode de réalisation, les amorces d'amplification et les sondes peuvent être spécifiques de la région env du génome du VIH-1. Des « cocktails » contenant de multiples amorces d'amplification et/ou sondes peuvent être utilisés pour couvrir au maximum les différents sous-types de VIH-1.
EP13742978.3A 2012-01-31 2013-01-22 Amorces d'amplification et sondes pour détecter le vih-1 Withdrawn EP2809784A4 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201261592989P 2012-01-31 2012-01-31
PCT/US2013/022503 WO2013116039A1 (fr) 2012-01-31 2013-01-22 Amorces d'amplification et sondes pour détecter le vih-1

Publications (2)

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EP2809784A1 true EP2809784A1 (fr) 2014-12-10
EP2809784A4 EP2809784A4 (fr) 2015-07-15

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US10464067B2 (en) 2015-06-05 2019-11-05 Miroculus Inc. Air-matrix digital microfluidics apparatuses and methods for limiting evaporation and surface fouling
JP2020501107A (ja) 2016-08-22 2020-01-16 ミロキュラス インコーポレイテッド デジタルマイクロ流体デバイスにおける並行小滴制御のためのフィードバックシステム
WO2018126082A1 (fr) 2016-12-28 2018-07-05 Miroculis Inc. Dispositifs microfluidiques numériques et procédés
US11623219B2 (en) 2017-04-04 2023-04-11 Miroculus Inc. Digital microfluidics apparatuses and methods for manipulating and processing encapsulated droplets
WO2019023133A1 (fr) 2017-07-24 2019-01-31 Miroculus Inc. Systèmes microfluidiques numériques et procédés à dispositif de collecte de plasma intégré
JP7341124B2 (ja) 2017-09-01 2023-09-08 ミロキュラス インコーポレイテッド デジタルマイクロ流体デバイスおよびその使用方法
WO2019226919A1 (fr) 2018-05-23 2019-11-28 Miroculus Inc. Contrôle de l'évaporation dans la microfluidique numérique
CN109722491A (zh) * 2019-01-22 2019-05-07 南方医科大学 Hiv-1 rna定量检测的实时荧光定量pcr引物和探针及其应用
CN113543883A (zh) 2019-01-31 2021-10-22 米罗库鲁斯公司 非结垢组合物以及用于操控和处理包封的微滴的方法
WO2020176816A1 (fr) 2019-02-28 2020-09-03 Miroculus Inc. Dispositifs micro-fluidiques numériques et leurs procédés d'utilisation
CN119158636A (zh) 2019-04-08 2024-12-20 米罗库鲁斯公司 多盒式数字微流控装置和使用方法
WO2021016614A1 (fr) 2019-07-25 2021-01-28 Miroculus Inc. Dispositifs microfluidiques numériques et leurs procédés d'utilisation
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EP2809784A4 (fr) 2015-07-15
WO2013116039A1 (fr) 2013-08-08

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