EP2326939A1 - Procédé d'identification de virus individuels dans un échantillon - Google Patents

Procédé d'identification de virus individuels dans un échantillon

Info

Publication number
EP2326939A1
EP2326939A1 EP09776005A EP09776005A EP2326939A1 EP 2326939 A1 EP2326939 A1 EP 2326939A1 EP 09776005 A EP09776005 A EP 09776005A EP 09776005 A EP09776005 A EP 09776005A EP 2326939 A1 EP2326939 A1 EP 2326939A1
Authority
EP
European Patent Office
Prior art keywords
viruses
sample
virus
scanning
height profile
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Withdrawn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
EP09776005A
Other languages
German (de)
English (en)
Inventor
Jürgen Popp
Volker Deckert
Dieter Naumann
Robert MÖLLER
Dana Cialla
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.)
Friedrich Schiller Universtaet Jena FSU
Original Assignee
Friedrich Schiller Universtaet Jena FSU
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 Friedrich Schiller Universtaet Jena FSU filed Critical Friedrich Schiller Universtaet Jena FSU
Publication of EP2326939A1 publication Critical patent/EP2326939A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y35/00—Methods or apparatus for measurement or analysis of nanostructures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01Q—SCANNING-PROBE TECHNIQUES OR APPARATUS; APPLICATIONS OF SCANNING-PROBE TECHNIQUES, e.g. SCANNING PROBE MICROSCOPY [SPM]
    • G01Q30/00—Auxiliary means serving to assist or improve the scanning probe techniques or apparatus, e.g. display or data processing devices
    • G01Q30/02—Non-SPM analysing devices, e.g. SEM [Scanning Electron Microscope], spectrometer or optical microscope
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01Q—SCANNING-PROBE TECHNIQUES OR APPARATUS; APPLICATIONS OF SCANNING-PROBE TECHNIQUES, e.g. SCANNING PROBE MICROSCOPY [SPM]
    • G01Q60/00—Particular types of SPM [Scanning Probe Microscopy] or microscopes; Essential components thereof
    • G01Q60/24—AFM [Atomic Force Microscopy] or apparatus therefor, e.g. AFM probes
    • G01Q60/38—Probes, their manufacture, or their related instrumentation, e.g. holders
    • G01Q60/40—Conductive probes
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
    • H01J37/26—Electron or ion microscopes; Electron or ion diffraction tubes
    • H01J37/28—Electron or ion microscopes; Electron or ion diffraction tubes with scanning beams
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
    • G01N2001/4088—Concentrating samples by other techniques involving separation of suspended solids filtration
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J2237/00—Discharge tubes exposing object to beam, e.g. for analysis treatment, etching, imaging
    • H01J2237/245—Detection characterised by the variable being measured
    • H01J2237/24571—Measurements of non-electric or non-magnetic variables
    • H01J2237/24578—Spatial variables, e.g. position, distance
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J2237/00—Discharge tubes exposing object to beam, e.g. for analysis treatment, etching, imaging
    • H01J2237/25—Tubes for localised analysis using electron or ion beams
    • H01J2237/2505—Tubes for localised analysis using electron or ion beams characterised by their application
    • H01J2237/2583—Tubes for localised analysis using electron or ion beams characterised by their application using tunnel effects, e.g. STM, AFM
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J2237/00—Discharge tubes exposing object to beam, e.g. for analysis treatment, etching, imaging
    • H01J2237/26—Electron or ion microscopes
    • H01J2237/28—Scanning microscopes
    • H01J2237/2802—Transmission microscopes
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J2237/00—Discharge tubes exposing object to beam, e.g. for analysis treatment, etching, imaging
    • H01J2237/26—Electron or ion microscopes
    • H01J2237/28—Scanning microscopes
    • H01J2237/2813—Scanning microscopes characterised by the application
    • H01J2237/2814—Measurement of surface topography

Definitions

  • the invention relates to a method for low-effort, fast and reliable identification of any single virus in a sample.
  • the proposed method aims at the detection of single viruses of any (solid, liquid or gaseous) sample.
  • viruses or bacteriophages are to be accurately identified without time- and material-consuming sample preparation.
  • the invention makes it possible to obtain reliable information about the nature and composition of the virus particles in a sample, which leads to an accurate and unambiguous identification of the particles.
  • This method can be used universally for all viruses, regardless of which cells infect the virus and the nature of the individual virus. Since the method can determine viruses regardless of their origin, there are also many other possible uses (eg the detection of tobacco mosquito viruses in plants, the detection of virus particles in the air or the detection of viruses and bacteriophages in biotechnological production).
  • virus contamination tests can be considered. This bacterial or cell cultures are infected with supposedly virus contaminated material. After a certain incubation period, the virus infection is detected by a visually perceptible change (lyric scheme) in the cell or bacterial culture.
  • this detection is time consuming and allows only a general virus test and no accurate determination of the type of virus.
  • a virus attack does not always result in the lysis of the affected host cells.
  • the selected experimental conditions may not be optimal for virus proliferation or even inhibit it, or the virus is in a so-called lysogenic cycle whereby the DNA of the virus or phage is incorporated into the DNA of the host cell rather than forming a lytic scheme comes, although a virus attack is present, which is then not detectable or determinable in this way.
  • lysogenic cycle whereby the DNA of the virus or phage is incorporated into the DNA of the host cell rather than forming a lytic scheme comes, although a virus attack is present, which is then not detectable or determinable in this way.
  • ELISA eg BSS Nielsen, N.
  • Ante mortem diagnosis of paratuberculosis A review of accuracies of ELISA 5 interferon- ⁇ assay and faecal culture techniques, Veterinary Microbiology 2008, 129, 217-235) or Antigori, S. Calattini, E. Longhi, G. Bestetti, R. Piolini, C. Magni, G. Orlando, M. Gramiccia, V. Acquaviva, A. Foschi, S. Corvasce, C. Colomba, L. Titone, C. Parravicini, A. Cascio, M.
  • Corbellino Clinical Use of Polymerase Chain Reaction Performed on Peripheral Blood and Bone Marrow Samples for the Diagnosis and Monitoring of Visceral Leishmaniasis in HIV-Infected and HIV-Uninfected Patients: A Single -Center, 8-Year Experience in Italy and Review of the Literature, Clinical Infectious Diseases 2007, 44, 1602-1610; J. Peccia, M. Hernandez: Incorporating Polymerase Chain Reaction-based Identification, Population Characterization, and Quantification of Microorganisms into aerosol science: A review, Atmospheric Environment 2006, 4 0, 3941-3961; LA Benvenuti, A. Roggerio, NV Zambiase, A. Fiorelli, M.
  • RNA or DNA the genetic material (RNA or DNA) of the virus is amplified by an enzymatic reaction and then analyzed. It is necessary to isolate the genome of the virus from the sample and possibly of cells, cell components and other factors which can inhibit the enzymatic reaction. This sample preparation is associated with a considerable amount of work and time.
  • the isolated genetic material must then be mixed with expensive reagents and specific primers.
  • the primers are short single-stranded pieces of DNA that ensure that only certain specific parts of the virus genome are duplicated, which can later be used for accurate identification of the virus. Thus, if the PCR approach contains the wrong primers, DNA amplification and virus detection will not occur. In order to specifically detect viruses of different families and species, therefore, specific primers must be developed. However, slight changes in the genetic make-up of the virus can lead to the primers no longer binding to the DNA, and thus likewise no DNA amplification taking place. Viruses that do not have a reliable primer system can not be detected by PCR.
  • Atomic Force Microscopy is also used as a further imaging method (YF Drygin, OA Bordunova, MO Gallyamov, IV Yaminsky: Atomic force microscopy examination of tobacco mosaic virus and virion RNA, FEBS Letters 1998, 425, (2), 217 -221).
  • the virus particles immobilized on a solid and extremely smooth sample carrier are scanned with a very fine tip.
  • the size and shape of the individual imaged particles can then be used to assign the viruses to a specific family.
  • a major disadvantage of all imaging methods is that no information about the composition of the imaged particles is obtained. Thus, an assignment and determination is made only on the shape and size of the particles, which can easily lead to confusion and thus to false evaluations, especially in spherical viruses. Information about the composition of virus particles is obtained in principle with spectroscopic methods, such as
  • SERS surface-enhanced Raman spectroscopy
  • the invention has for its object to identify individual viruses in any solid, liquid or gaseous sample quickly, clearly and reliably and with the least possible preparative and technological effort without immobilization is required with antibodies and without any indication or at least a suspicion of any existing viruses must be given.
  • the height profile of a carrier surface to which the sample to be examined is bound is scanned by a probe, for example according to the AFM method known per se. From this elevation profile obtained by the surface scan, scan locations are selected (either simultaneously with or after the said scan) which, due to their surface structure (height profile size), suggest a virus. These scanning locations selected according to the height profile are each irradiated with monochromatic excitation light and examined spectrometrically with regard to the Raman scattered light occurring as a result of the light excitation at the scanning location. By comparing these examination results of the said Raman scattered light with reference values, in particular comparative values of an electronic database, conclusions are drawn in each case on the single virus present at the scanning location.
  • this method combines information on the shape and size of suspected virus particles with data from vibration spectroscopy in order to enable a clear and rapid identification of viruses and even single viruses for the first time. This is achieved by the coupling of an imaging technique (surface scanning) with Raman spectroscopy.
  • the virus structures found and to be defined are selected at the sampling locations of the sample surface selected by their detection by comparing their respective vibration spectroscopic data all existing as reference data (all detailed virus information) evaluated and thus reliably detected.
  • reference data all detailed virus information
  • the detection is given even for single viruses, so that no reliable detection and determination
  • Minimum concentration is required and also in this regard Pre-cultivation omitted.
  • a size sorting for example by filtration, advantageous to separate the large particles from the virus, thereby purify the sample and in this way the evaluation, ie the selection of scan locations from the height profile of the support surface , as well as to simplify the virus detection and determination.
  • a filter array with decreasing pore size is used, through which the sample is filtered. The viruses are then enriched on a filter with the appropriate pore size, separated from larger particles, such as dust or bacteria.
  • FIG. 1 shows a schematic structure for the height profile scanning of a sample bound on a carrier by means of an AFM method (Atomic Force Microscopy) known per se and for the investigation of laser-excited Raman scattered light.
  • AFM method Atomic Force Microscopy
  • the irradiation of the sample with laser light takes place with the aid of a microscope objective opposite to the probe from below.
  • the amplified Raman signal is collected with the same microscope objective over which the sample was irradiated, and then passed to an evaluation detector of the laser microscope.
  • the sample to be examined consisting of a carrier 1 with a likewise schematically represented virus 2, is determined by means of an AFM
  • Metal particles 4 has scanned in their height profile. At this
  • Raman scattered light of the sample evaluated.
  • Exemplary embodiment 1 is a diagrammatic representation of Exemplary embodiment 1:
  • TMV tobacco mosaic virus
  • FMD is a highly contagious and notifiable disease of cattle and pigs; however, goats, sheep and other Cloven hoofed animals are also affected. There are even described infections of elephants, hedgehogs, rats and humans.
  • aphthous fluid for example, organ homogenates, pharyngeal mucus samples (probang samples), secretions and cell culture supernatants can be used. These are converted into a suitable lysis buffer, which leads to the release of the viruses from the cells. Subsequently, the liquid thus obtained are again passed through the filter arrangement mentioned in the embodiment 1 and the candidate filter, as described, analyzed.
  • the flu is triggered in humans of the genus influenza virus A or B. Infection often takes place via a so-called droplet or smear infection. Droplet infection is the direct inhalation of expiration droplets (exhalation droplets) of infected persons.
  • viruses As bacteriophages are generally called the viruses
  • the sample to be examined (culture medium, bacterial culture or the like) is first transferred to a suitable lysis buffer in order to break up the structures of the bacterial cells and release the viruses. Subsequently, this solution is passed through the above-mentioned filter arrangement and the corresponding filters, as described, analyzed and evaluated.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Analytical Chemistry (AREA)
  • Radiology & Medical Imaging (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Engineering & Computer Science (AREA)
  • Nanotechnology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Le but de l'invention est d'identifier des virus individuels dans un échantillon quelconque de manière rapide, univoque et fiable tout en réduisant autant que possible les préparatifs et les moyens technologiques sans qu'une immobilisation avec des anticorps soit nécessaire et sans qu'une indication ou au moins un soupçon sur la présence éventuelle de virus doive être donné. Selon l'invention, le profil en hauteur de l'échantillon est balayé. Des points balayés pouvant contenir des virus sont sélectionnés dans ce profil, sont exposés à une lumière d'excitation monochromatique et sont analysés par spectroscopie en termes de lumière diffusée Raman générée.
EP09776005A 2008-09-11 2009-07-21 Procédé d'identification de virus individuels dans un échantillon Withdrawn EP2326939A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102008047240.9A DE102008047240B4 (de) 2008-09-11 2008-09-11 Verfahren zur Identifikation von Einzelviren in einer Probe
PCT/DE2009/001031 WO2010028614A1 (fr) 2008-09-11 2009-07-21 Procédé d'identification de virus individuels dans un échantillon

Publications (1)

Publication Number Publication Date
EP2326939A1 true EP2326939A1 (fr) 2011-06-01

Family

ID=41170984

Family Applications (1)

Application Number Title Priority Date Filing Date
EP09776005A Withdrawn EP2326939A1 (fr) 2008-09-11 2009-07-21 Procédé d'identification de virus individuels dans un échantillon

Country Status (5)

Country Link
US (1) US20110165558A1 (fr)
EP (1) EP2326939A1 (fr)
JP (1) JP2012502282A (fr)
DE (1) DE102008047240B4 (fr)
WO (1) WO2010028614A1 (fr)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102010023099B3 (de) 2010-06-09 2011-11-17 Celltool Gmbh Verfahren und Vorrichtung zum Charakterisieren von biologischen Objekten
RU2491338C2 (ru) * 2011-06-30 2013-08-27 Федеральное государственное бюджетное учреждение "Научно-исследовательский Институт гриппа" Министерства здравоохранения и социального развития Российской Федерации (ФГБУ "НИИ гриппа" Минздравсоцразвития России) Применение моноклональных антител для идентификации ямагатской или викторианской эволюционных линий вируса гриппа типа в, штамм гибридомы 4н7 для получения моноклональных антител, предназначенных для определения вирусов гриппа в ямагатской ветви, штамм гибридомы в/4н1 для получения моноклональных антител, предназначенных для определения вирусов гриппа в викторианской ветви
US20130052636A1 (en) * 2011-08-22 2013-02-28 Spectral Platforms, Inc. Rapid detection of metabolic activity
WO2017041782A1 (fr) 2015-09-11 2017-03-16 Leibniz-Institut Für Photonische Technologien E.V. Dispositif pour l'analyse de sang individualisée d'un patient
DE102015115342A1 (de) * 2015-09-11 2017-03-16 Leibniz-Institut für Photonische Technologien e. V. Anordnung für die individualisierte Patientenblutanalyse
CN110940690A (zh) * 2018-09-21 2020-03-31 云南省农业科学院生物技术与种质资源研究所 一种黄瓜绿斑驳花叶病毒粒体的原位分离固定电子显微镜诊断方法
GB2580186B (en) 2018-12-24 2021-09-15 Cell Therapy Catapult Ltd Monitoring viral titre using Raman Spectroscopy

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH095237A (ja) * 1995-06-19 1997-01-10 Hitachi Ltd ラマンスペクトル測定装置及び測定方法
KR20070030263A (ko) * 2004-06-07 2007-03-15 그리펀 아날리틱스 엘엘씨 표면 강화 라만 분광법을 위한 기판 표면 제조 시스템과방법 및 이를 이용한 장치
US7738096B2 (en) * 2004-10-21 2010-06-15 University Of Georgia Research Foundation, Inc. Surface enhanced Raman spectroscopy (SERS) systems, substrates, fabrication thereof, and methods of use thereof
DE102006039492A1 (de) * 2006-08-21 2008-03-06 Nölting, Bengt, Dr. Ramanmikroskopie
WO2008028521A1 (fr) * 2006-09-07 2008-03-13 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Sonde, spectromètre de raman et procédé de fabrication de sonde

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2010028614A1 *

Also Published As

Publication number Publication date
WO2010028614A1 (fr) 2010-03-18
JP2012502282A (ja) 2012-01-26
US20110165558A1 (en) 2011-07-07
DE102008047240B4 (de) 2016-03-31
DE102008047240A1 (de) 2010-04-15

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