EP2326939A1

EP2326939A1 - Method for identifying individual viruses in a sample

Info

Publication number: EP2326939A1
Application number: EP09776005A
Authority: EP
Inventors: Jürgen Popp; Volker Deckert; Dieter Naumann; Robert MÖLLER; Dana Cialla
Original assignee: Friedrich Schiller Universtaet Jena FSU
Current assignee: Friedrich Schiller Universtaet Jena FSU
Priority date: 2008-09-11
Filing date: 2009-07-21
Publication date: 2011-06-01
Also published as: US20110165558A1; WO2010028614A1; DE102008047240A1; DE102008047240B4; JP2012502282A

Abstract

The problem of the invention consisted in identifying individual viruses in an arbitrary sample quickly, unambiguously and reliably, and with the least possible preparation-related and technology-related expenditure, without necessitating immobilization using antibodies and without requiring an indication or at least a suspicion of potentially present viruses. According to the invention, the height profile of the sample is scanned, from which scanning sites suspected of containing viruses are selected, exposed to monochromatic excitation light and analyzed spectroscopically with respect to the resulting Raman scattered light.

Description

Method for identifying single viruses in a sample

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. Here, 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).

As the oldest detection method for virus contamination, infection 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. However, this detection is time consuming and allows only a general virus test and no accurate determination of the type of virus. In addition, a virus attack does not always result in the lysis of the affected host cells. On the one hand, 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. Aufgrand these disadvantages today ^'often other molecular biological methods for the routine detection of viruses and bacteriophages are used. In particular, detection methods such as ELISA (eg BSS Nielsen, N. Toft: Ante mortem diagnosis of paratuberculosis: A review of accuracies of ELISA ₅ interferon-γ 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. de Lourdes Higuchi: Polymerase chain reaction in endomyocardial biopsies for monitoring reactivation of Chagas' disease in heart transplantation - A case report and review of the literature, Cardiovascular Pathology 2005, 14, 265-268) are used. A disadvantage of these methods, however, is that the detection of single viruses is very difficult or even impossible. Therefore, even for these detection methods often only an incubation of bacterial and cell cultures is used to increase the concentration of the virus. However, this cultivation is again associated with considerable time and effort. The actual detection of the viruses then takes place via the detection of their DNA or RNA (PCR) or via an immunological test (ELISA).

In PCR, 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. For the PCR, 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.

When immunological detection, such. B. by ELISA, the specific reaction between the virus particles and antibodies is used for the detection. However, viruses in low concentrations can only be detected after incubation and propagation. Again, specific biomolecules are required for detection. In the case of immunological detection, antibodies are used which are obtained from experimental animals or from cell cultures. As in the case of PCR, the correct biomolecules (PCR: primer, immunological detection: antibodies) must also be selected during immunological detection in order to detect a specific virus. However, if no suitable antibodies are present or the wrong ones are used, virus detection is not possible. Furthermore, the production of the antibodies is complicated and an additional immobilization step is necessary, in which either the viruses or the antibodies are bound to a solid substrate.

As a detection method for viruses and imaging methods are known. Thus, as an electron microscopic detection method in the

Diagnosis of Transmission Electron Microscopy (TEM) in Compound used with negative staining. With this method one can assign single viruses to the families, since the fine structure between different virus families differs sufficiently strongly (M. Gentile, HR Gelderblom: Rapid viral diadnosis: role of electron microscopy, The New Microbiologica 2005, 28, (1), 1- 12; PR Hazelton, HR Gelderblom: Electron microscopy for rapid diagnosis of infectious agents in emergent situations, Emerging Infectious Diseases 2003, 9, 294-303). An exact assignment beyond the virus family is therefore not possible by transmission electron microscopy. In addition, this method is associated with a considerable apparatus and preparative effort.

Atomic Force Microscopy (AFM) 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). In this imaging procedure, 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. However, it can be very easy to false evaluations (false-positive results) come, especially if the sample is contaminated with particles of another origin, but have a similar shape and size, and thus lead to confusion. If the surface of the sample tray is too rough or too many particles are present on the sample carrier, identification of the very small viras particles is also not possible.

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

Raman spectroscopy.

This vibrational spectroscopic examination allows the viruses to be assigned to a family or species on the basis of the spectra obtained. However, the Raman effect that underlies this method is only very weak, which makes it possible to study viruses with Raman spectroscopy only for bulk material (GJ Thomas Jr .: Raman spectroscopy and virus research, Applied Spectroscopy 1976, 30, (5), 483-94; TA Turano, KA Hartman, GJ Thomas Jr .: Studies of virus structure by laser-Raman spectroscopy, 3rd Turnip yellow mosaic virus, Journal of Physical Chemistry 1976, 80, (11), 1157-63). This means that the viruses must be present in a very high concentration and if possible with few impurities. Determination of single viruses is impossible with this method.

To detect lower virus concentrations, so-called surface-enhanced Raman spectroscopy (SERS) can be used. It uses metallic nanostructures and particles to increase the low sensitivity of Raman spectroscopy. However, this is not a procedure that is used in routine diagnostics, since the amplification of the Raman effect varies greatly with the nanostructures and nanoparticles used and thus reliable detection is not possible. Also, this method does not allow detection of single viruses (S. Shanmukh, L. Jones, J. Driskell, Y. Zhao, R. Dluhy, RA Tripp: Rapid and Sensitive Detection of Respiratory Virus Molecular Signatures Using a Silver Nanorod Array SERS Substrate , Nano Letters 2006, 6, (11), 2630-2636). In order to increase the spatial resolution of the Raman measurement, surface-enhanced Raman spectroscopy is combined with imaging techniques such as AFM and STM (scanning tunneling microscopy). This method is called tip-enhanced Raman spectroscopy (TERS) (RM Stöckle, YD Suh, V. Deckert, R. Zenobi: Nanoscale chemical analysis by tip-enhanced Raman spectroscopy, Chemical Physics Letters 2000, 318, 131-136). In recent years, an application has also been advanced for biological issues to analyze bacteria and RNA. When examining bacteria, a silver-coated AFM tip was placed on the bacterium. Due to the differences in size between a bacterium and the silver vaporized probe, only a very small portion of the bacterial surface can be sampled. Thus, these experiments have also shown no encouraging results. The detected TERS spectra are not reproducible among each other, which implies a mobility in the outer cell wall, so that this method must therefore be considered by those skilled in the art for the identification of bacteria as unsuitable (U. Neugebauer, P. Rösch, M. Schmitt, J. Popp, C. Julien, A. Rasmussen, C. Budich, V. Deckert: On the Way to Nanometer-Sized Information of the Bacterial Surface by Tip-Enhanced Raman Spectroscopy, Chem. Phys. Chem. 2006, 7, 1428-1430). Furthermore, TERS studies were performed on single-stranded polyC-RNA. This result should lead to a direct sequencing of isolated DNA or RNA. (E. Bailo, V. Deckert: Tip-Enhanced Raman Spectroscopy of Single RNA Strands: Towards a Novel Direct Sequencing Method, Angewandte Chemie International Ed., 2008, 47, 1658-1661).

The use of this method for the detection and identification of individual viruses has not become known in the art.

Considering all the methods currently known for viral diagnostics, it has to be stated that at present no method is known with which single viruses can be identified reliably and clearly, and which can be used sensitive to detection, quickly and effortlessly also in routine clinical control.

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.

According to the invention, 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.

For the first time ever, 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.

In this case, it is not absolutely necessary to have a priori information as to how many and which viruses are or could be present in the sample; instead, 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. As advantages over the classical methods described above, it should be mentioned that no expensive molecular biological reagents are needed, a clear and comparatively fast identification of viruses and even single viruses is possible and that the time-consuming and process-intensive precultivation and sample preparation can be dispensed with. Thus, the invention significantly exceeds the above-described methods for virus detection accuracy, reliability, detection speed and effort. In addition, even individual viruses, which, as described, not only by their form, but also according to the proposed additional spectroscopic analysis. In this way, in addition to size and structure, in each case exact information about the chemical composition of the investigated particles is obtained, which makes it possible for the first time to accurately and unambiguously determine these viruses. The method according to the invention does not use any molecular biological detection reactions, such as those used in the immunological or PCR detection of viruses, so that the elaborate sample preparation and preparation of these molecular biological methods is also eliminated. In addition, the often considerable costs of the primers, antibodies, enzymes and other reagents required for these detection reactions are saved.

By coupling an imaging method with a vibration spectroscopic method, the user, in contrast to the above-mentioned pure imaging techniques (AFM, TEM) for a high detection sensitivity very detailed information on the material composition of the sampled sample obtained, which also a very detailed structural comparison with reference data thus enabling extremely accurate and clear virus detection and identification. This is particularly interesting for viruses of the same or similar shape and size, whose unambiguous evaluation is only possible with the help of topographical information.

According to the invention, 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. As a sole sample preparation for the application of the invention, only 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. In this case, for example, 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.

The invention will be explained in more detail below with reference to exemplary embodiments illustrated in the drawing.

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. 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

Tip 3, which is at its end facing the test

Metal particles 4 has scanned in their height profile. At this

Height profile scanning of the sample is detected at the sampling location shown in Figure 1, the conspicuous and suspected virus structure of the bound on the carrier 1 virus 2. According to the invention, in such a recognition and selection of the scanning location (at the same time as said profile scanning or after complete scanning

Height profile sampling of the sample) at this specified sample location

Raman scattered light of the sample evaluated. For this purpose, a focused laser beam 5 via an objective 6 of a

For reasons of clarity not shown laser microscopes on the Tested. As a result of this light excitation of the sample by the laser beam 5 is produced scattered light, which is detected and evaluated via the lens 6 of the laser microscope. By comparison of the evaluation data of this scattered light with present reference values, in particular of a database likewise not shown in FIG. 1, the virus present at the sampling location of the sample is identified.

Exemplary embodiment 1:

Detection of tobacco mosaic virus (TMV) in a plant sample: The TMV causes the economically significant mosaic disease of the tobacco, but can also affect other plant families. The virus is particularly stable and can be easily transmitted, e.g. B. by direct contact between plants, by plant sap, in some plants by seeds and especially by agricultural practices in the handling of infected plants.

In order to avoid major economic damage, it is therefore necessary to identify the virus as quickly and accurately as possible. For this purpose, either plant juice or parts of plants (leaves, buds, fruits, stems, stems, roots or the like) are used. When plant parts are used, they are lysed in a first step mechanically or chemically with a suitable buffer to release the viruses from the cell structures. The recovered liquid is then passed over an array of filters of decreasing pore size (like the plant squeezed juice). Subsequently, only the filters on which the 15 nm to 400 nm large virus particles are collected, examined by means of the aforementioned device and method for viruses. The advantage here is that other phytopathogenic viruses can be identified with immediately. Embodiment 2:

Evidence of foot-and-mouth disease (FMD) 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.

For virus identification, for example, aphthous fluid, 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.

Embodiment 3

Detection of influenza viruses in air samples:

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.

Under contact infection or smear infection with the viruses, it is caused by highly infective expiration droplets that have fallen down on objects or body surfaces or, for example, by smeared nasal secretions.

To detect the viruses in an air sample, a previously defined volume of air is filtered via the filter arrangement already described above (see Example 1). Subsequently, the filters are again analyzed by the method described.

Embodiment 4

Detection of bacteriophages in a bacterial culture:

As bacteriophages are generally called the viruses

Use prokaryotes as host cells. Particularly interesting is the rapid identification of bacteriophages. These only attack bacteria and often result in the biotechnological production of active ingredients by bacteria to considerable damage. 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.

LIST OF REFERENCE NUMBERS

1 carrier

2 virus

3 AFM tip

4 metal particles

5 laser beam

6 Lens of a laser microscope

Claims

claims

A method of identifying single viruses in a sample in which the sample is preferably pretreated for size sorting, bound to a support surface, and by means of an imaging

Method is examined for viruses, characterized in that the height profile of the support surface is scanned with the bound sample by a probe that in addition to height profile scanning of the support surface at least selected and determined from the height profile of the support surface scanning each with monochromatic excitation light are irradiated and each the spectrum the Raman scattered light occurring as a result of the light excitation at the scanning location is detected in or on the probe, and that the Raman scattered light recorded at the scanning locations is compared with reference values in each case, the individual virus present at the scanning location being inferred from this comparison.

2. The method according to claim 1, characterized in that the Raman scattered light simultaneously with the Höhenprofilabtastung the

Carrier surface is detected for each scanning location of the support surface in or on the probe and that the synchronously detected with the Höhenprofilabtastung Raman scattered light is compared for each selected according to the height profile scanning locations for local virus identification with reference values.

3. The method according to claim 1, characterized in that first the height profile of the carrier surface is completely or partially scanned by the probe and that relevant scanning locations are determined by evaluating the scanned height profile, which are then irradiated with monochromatic excitation light and for which the resulting Raman Scattered light is detected in each case in or on the probe and compared to reference values for virus identification there.

4. The method according to claim I _5, characterized in that the Raman scattered light for virus identification is compared in each case with data of an electronic database.

5. The method according to claim 1, characterized in that liquid samples are pre-treated for size sorting by filtration.

6. The method according to claim 1, characterized in that solid samples for size sorting by homogenization (mechanical comminution) are pretreated.

7. The method according to claim 1, characterized in that gaseous samples for size sorting are pretreated directly by gas filtration.

8. The method according to claim 1, characterized in that gaseous samples for size sorting in order to dissolve the sample components are introduced into a liquid and these are then filtered.