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WO2008155742A2 - Sonde d'acide nucléique peptidique (pna), coffret et mode opératoire pour la détection spécifique d'helicobacter pylori et ses applications - Google Patents

Sonde d'acide nucléique peptidique (pna), coffret et mode opératoire pour la détection spécifique d'helicobacter pylori et ses applications Download PDF

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Publication number
WO2008155742A2
WO2008155742A2 PCT/IB2008/052455 IB2008052455W WO2008155742A2 WO 2008155742 A2 WO2008155742 A2 WO 2008155742A2 IB 2008052455 W IB2008052455 W IB 2008052455W WO 2008155742 A2 WO2008155742 A2 WO 2008155742A2
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WO
WIPO (PCT)
Prior art keywords
probe
detection
helicobacter pylori
pna
sample
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.)
Ceased
Application number
PCT/IB2008/052455
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English (en)
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WO2008155742A3 (fr
Inventor
Nuno Filipe Ribeiro Pinto De Oliveira Azevedo
Nuno Miguel DA ROCHA GUIMARÃES
Maria Do Céu FONTES HERDEIRO FIGUEIREDO
Charles William Keevil
Maria João LOPES DA COSTA VIEIRA
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.)
Universidade do Minho
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Universidade do Minho
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Filing date
Publication date
Application filed by Universidade do Minho filed Critical Universidade do Minho
Publication of WO2008155742A2 publication Critical patent/WO2008155742A2/fr
Publication of WO2008155742A3 publication Critical patent/WO2008155742A3/fr
Anticipated expiration legal-status Critical
Ceased 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

Definitions

  • PNA PNA NUCLEIC ACID PROBE
  • This invention relates to a method for the detection of clinically relevant microorganisms, and as such a peptide nucleic acid (PNA) probe has been developed to detect Helicobacter pylori.
  • PNA peptide nucleic acid
  • Another aspect of the current invention is the application of the PNA probe and detection procedure to a kit for the detection, identification and/or quantification of H. pylori in biological samples which might be used by the pharmaceutical industry.
  • Gastric biopsy specimens obtained by upper endoscopy can be analyzed for the presence of the bacterium by culture or by other molecular methods.
  • RAPD Random Amplified of Polymorphic DNA
  • PCR Polymerase Chain Reaction
  • FISH fluorescence in situ hybridization
  • FISH Fluorescence In situ hybridization
  • FISH methods have been based traditionally on the use of conventional DNA oligonucleotide probes containing around 20 bases. More recently, peptide nucleic acid (PNA) probes have been developed and optimized for bacterial detection.
  • PNA peptide nucleic acid
  • PNA molecules are DNA mimics, where the negatively charged sugar-phosphate backbone is replaced by an achiral, neutral polyamide backbone formed by repetitive units of N - (2- aminoethyl) glycine.
  • This technique has been shown to be a rapid, sensitive and very specific method for microbial detection and could be an important alternative or complement for the culture methods because using the probes produces faster results and additional information such as the location of the bacteria in gastric samples. Since the non-invasive techniques could not be adapted for the detection of the bacteria in different environments, this method has also more comprehensive application.
  • PNA probes have been designed and brought to public, through patent documents, to detect microorganisms of clinical interest such as Mycoplasm, Acholeplasm, or Ureaplasm (US2006252081; WO2006122049) ; species of Staphylococcus other than Staphylococcus aureus (WO2005054516; EP1709198); Enterococcus faecalis and/or other species of Enterococcus (WO2005018423; US2005153307) ; Pseudomonas (US2004259132) Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Streptococcus agalactiae, fungi, and Acinetobacter (WO2006130872) .
  • Hpy769 85 % 89 % This patent aSpecificity was calculated as NHp/ (TN) xlOO, where NHp stands for the number of H. pylori strains and TN for the total number of bacterial strains detected by the probe.
  • bSensitivity was calculated as NHp/ (TNHp) xlOO, wherein NHp stands for the number of H. pylori strains detected by the probe and TNHp for the total number of H. pylori strains in the databases .
  • the procedure of the present invention does not involve the use of reagents or enzymes for the formation of pores in the cellular membranes before the hybridization step and consequently the PNA probe can be directly applied immediately after the preparation of the sample in the slide.
  • kits that facilitate the application of FISH procedure by the users There are already kits that use PNA probes for the electrophoretic separation of DNA samples (US2005053944, WO9712995, EP1477572). Summary of the Invention
  • the PNA probes have physical and chemical characteristics inherent to their structure that allow a more rapid and sensitive analysis than the DNA probes, namely when applied to FISH.
  • the sequence of the nucleotides is the functional part of the probe and is constructed in order to hybridize with a complementary rRNA sequence in the target cell.
  • a successful hybridization allow us, for instance by fluorescence microscopy, to check the presence/absence and concentration of the target microorganism.
  • the probe has to meet the specific requirements of each trial, particularly the length and the sequence, to form a stable complex with the target in the ideal hybridization conditions.
  • the hybridization procedure consists in three steps: fixation, hybridization and washing step.
  • hybridization solution usually takes between 30 to 120 minutes, the probe of this invention with the sequence 5' -GAGACTAAGCCCTCC-S' is mixed in a solution named hybridization solution.
  • DNA FISH will easily identify formamide concentration (or other denaturating agent) , saline concentration (i. e. ionic force), hybridization temperature, detergent concentration, pH and the probe concentration as the usually referred factors that control the specificity of the hybridization.
  • the more similar other sequences of the sample are from the target sequence the more important this step is.
  • the immersion of the sample in a solution of 5 mM Tris Base, 15 inM NaCl and 1% (V/V) Triton X (pH 10) at hybridization temperature for 30 minutes is the optimal condition to ensure the desired specificity.
  • the sample can be observed using fluorescence microscopy with the right filters for the detection of the fluorochrome bonded to the probe.
  • the microscope is unable to detect any signal emission.
  • the procedure can be applied in the diagnosis and quantification of H. pylori in different kinds of samples, including biopsies, blood, air, food, water and other environmental samples.
  • the PNA probes can also be used in real time PCR, a process wherein the product amount is continuously evaluated during the amplification process, allowing the quantification of the genetic material in the initial sample.
  • the PCR product is detected through the bonding of dyes to the DNA double chain. Since these dyes are not specific, they recognize any product formed during the procedure, which requires a purification of the DNA of interest before the amplification step. To solve this problem, DNA probes have been developed to recognize specific sequences and such as with the FISH, the PNA molecules revealed additional vantages.
  • Hybridization During this step, an hybridization solution containing the probe is in contact with the sample. During this time period, the probe goes through the cellular membrane and binds to complementary sequences of the 16S rRNA. The hybridization usually occurs between 30 and 120 minutes, at 59°C and can be performed to both suspension and adhered H. pylori. In the first case, a concentrated hybridization solution is added in a way that the final products concentration should be as follows. In the second case, the necessary volume of hybridization solution to completely cover the sample is added directly in the sample. During the hybridization period, it is essential that the hybridization solution does not evaporate. For such, a cover slip is added on top of the hybridization solution and the incubation environment is kept moist by the insertion of a humid paper around the slide.
  • Hybridization solution A solution of 10% (wt/vol) dextran sulfate, 10 inM NaCl, 30% (vol/vol) formamide, 0.1% (wt/vol) sodium pyrophosphate, 0.2% (wt/vol) polyvinylpyrrolidone, 0.2% (wt/vol) Ficol, 5 mM disodium EDTA, 0.1% (vol/vol) Triton X-100, 50 mM Tris-HCl (pH 7.5) and 200 nM PNA probe was prepared.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention porte sur le développement d'une sonde d'acide nucléique peptidique (PNA) pour la détection spécifique d'Helicobacter pylori. Ces sondes sont utilisées dans un procédé à base de techniques de biologie moléculaire, à savoir l'hybridation in situ par fluorescence (FISH), et peuvent être appliquées dans une diversité d'échantillons, tels que des biopsies, du sang, de l'air, un aliment, de l'eau et autres échantillons environnementaux. En raison de caractéristiques physiques et chimiques propres à leur structure, ces sondes sont capables de fournir une analyse plus rapide et sensible que les sondes à ADN. Un autre aspect de la présente invention est le développement d'un coffret, sur la base de la sonde PNA et du procédé FISCH, pour la détection et/ou la quantification de l'H.pylori dans des échantillons biologiques, cliniques et/ou environnementaux.
PCT/IB2008/052455 2007-06-21 2008-06-20 Sonde d'acide nucléique peptidique (pna), coffret et mode opératoire pour la détection spécifique d'helicobacter pylori et ses applications Ceased WO2008155742A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PT103767 2007-06-21
PT10376707A PT103767B (pt) 2007-06-21 2007-06-21 Sonda de ácido péptido nucleico (pna), estojo e método para detecção específica de helicobacter pylori e respectivas aplicações.

Publications (2)

Publication Number Publication Date
WO2008155742A2 true WO2008155742A2 (fr) 2008-12-24
WO2008155742A3 WO2008155742A3 (fr) 2009-02-12

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2008/052455 Ceased WO2008155742A2 (fr) 2007-06-21 2008-06-20 Sonde d'acide nucléique peptidique (pna), coffret et mode opératoire pour la détection spécifique d'helicobacter pylori et ses applications

Country Status (2)

Country Link
PT (1) PT103767B (fr)
WO (1) WO2008155742A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011030319A1 (fr) 2009-09-11 2011-03-17 Universidade Do Minho Sondes d’acide nucléique peptidique, trousse et procédé pour la détection de helicobacter pylori et/ou de profil de résistance à la clarithromycine et applications
CN114705864A (zh) * 2022-03-11 2022-07-05 江苏海尔森生物科技有限公司 一种荧光偏振检测幽门螺旋杆菌的方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AZEVEDO N F ET AL: "ESTABLISHMENT OF A CONTINUOUS MODEL SYSTEM TO STUDY HELICOBACTER PYLORI SURVIVAL IN POTABLE WATER BIOFILMS" WATER SCIENCE AND TECHNOLOGY, ELMSFORD, NY, US, vol. 47, no. 5, 1 January 2003 (2003-01-01), pages 155-160, XP001179746 ISSN: 0273-1223 cited in the application *
GUIMARAES N ET AL: "Development and application of a novel peptide nucleic acid probe for the specific detection of Helicobacter pylori in gastric biopsy specimens" JOURNAL OF CLINICAL MICROBIOLOGY 200709 US, vol. 45, no. 9, September 2007 (2007-09), pages 3089-3094, XP002506952 ISSN: 0095-1137 *
MORENO Y ET AL: "Use of fluorescent in situ hybridization to evidence the presence of Helicobacter pylori in water." WATER RESEARCH, vol. 37, no. 9, May 2003 (2003-05), pages 2251-2256, XP002506951 ISSN: 0043-1354 *
STENDER HENRIK ET AL: "PNA for rapid microbiology" JOURNAL OF MICROBIOLOGICAL METHODS, ELSEVIER, AMSTERDAM, NL, vol. 48, no. 1, 1 January 2002 (2002-01-01), pages 1-17, XP002390590 ISSN: 0167-7012 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011030319A1 (fr) 2009-09-11 2011-03-17 Universidade Do Minho Sondes d’acide nucléique peptidique, trousse et procédé pour la détection de helicobacter pylori et/ou de profil de résistance à la clarithromycine et applications
CN102597267A (zh) * 2009-09-11 2012-07-18 米尼奥大学 用于检测幽门螺杆菌和/或克拉霉素抗性特征的肽核酸探针，试剂盒和方法及应用
JP2013504319A (ja) * 2009-09-11 2013-02-07 ウニベルシダージ ド ミーニョ ヘリコバクター・ピロリ及び／又はクラリスロマイシン抵抗性を検出するためのペプチド核酸プローブ、キット及び方法、及びその使用
CN114705864A (zh) * 2022-03-11 2022-07-05 江苏海尔森生物科技有限公司 一种荧光偏振检测幽门螺旋杆菌的方法

Also Published As

Publication number Publication date
WO2008155742A3 (fr) 2009-02-12
PT103767B (pt) 2009-09-16
PT103767A (pt) 2008-12-22

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