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
• • 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
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56983—Viruses
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/81—Packaged device or kit

Definitions

• the invention relates to a method for the detection of influenza A / B viruses, a corresponding test kit and the use of saliva as sample material for the detection of influenza A / B viruses.
• Influenza A and / or Influenza B viruses are responsible for the real virus flu, which affects several hundred million people worldwide every year.
• the influenza A and B viruses primarily affect the nose, mouth and throat and initially cause general respiratory symptoms in those affected.
• influenza infection Due to the high medical importance of influenza infection (flu), almost every country in the world now has a nationally organized flu monitoring system. Resident doctors take suspicious patients' nose and / or throat swabs and send them to the respective national reference center. Influenza A / B viruses are then usually detected by elution of the swab and subsequent cultivation of the patient samples on mammalian cells, for example MDCK cells (Madin-Darby Canine Kidney cells).
• MDCK cells Medin-Darby Canine Kidney cells
• the cultivation in these special laboratories can take up to 14 days and is therefore not of immediate relevance as a diagnosis for the individual patient. Rather, the task of the national reference centers is to type the cultivated viruses and subtype, and report the results to the World Health Organization (WHO). It is then up to the vaccine manufacturers to adapt next year's flu vaccines to the latest circulating strains of nirus based on the WHO's annual recommendation.
• WHO World Health Organization
• influenza A viruses The reason for the high genetic and associated immunological variability, especially in the case of influenza A viruses, is that, in addition to the usual genetic drift (point mutation), in rare cases there is also a genetic shift ("reassortment", ie re-sorting of the virus genes) This is due to the fact that the genome of the influenza viruses, in contrast to other viruses, is segmented and that influenza A is both human and animal pathogenic.
• the immunodominant antigens on the surface of the virus are hemagglutinin (H) and neuraminidase (N).
• H hemagglutinin
• N neuraminidase
• 15 subtypes of hemagglutinin (Hl - H15) and 9 subtypes of neuraminidase (Nl -N9) are currently known.
• a host e.g. a pig
• the virus genome can be re-sorted and thus become a new Influenza A virus virus.
• Subtype come, which then completely undermines its immune system when it is transferred back to humans, the most recent example of which was the so-called bird virus flu in May 1997 in Hong Kong (type A / H5Nl / Hong Kong / 156/97), in which despite early detection and Intensive care treatment 6 of the 18 sick patients died.
• influenza A / B viruses influenza A and / or influenza B viruses
• the quality of the sampling therefore has a direct influence on the positivity rate of the rapid tests, whose Indian sensitivity is usually around 70%, i. H. in 70% of all samples assessed as positive by the reference method (cell culture), the immunological rapid test is also positive.
• the diagnostics manufacturers therefore indicate in the package leaflets of their rapid influenza tests that the sampling should only be carried out by specially trained medical personnel to ensure that the sample material actually contains sufficient amounts of influenza A / B viruses.
• pharynx posterior pharynx, pharynx, tonsils
• the areas to be wiped out in the pharynx may well differ depending on the virus infection. This means, for example, that in order to detect a streptococcal infection, other areas of the throat must be removed than, for example, for the detection of influenza A / B viruses.
• the positivity rate of an influenza test is determined not only by the quality of the smear but also by the correct elution, ie transfer of the smear material into the liquid phase, which serves as the actual "sample material" for the downstream test.
• the object of the present invention is to eliminate the disadvantages of the prior art.
• the object on which the invention is based is to provide a method for the detection of an influenza A and / or influenza B virus infection which is simple to carry out by untrained personnel or ideally the patient himself.
• the object of the invention is to find a sample material which can be obtained easily and without problems by ideally untrained staff or ideally the patient himself, from which influenza A and / or influenza B viruses can be reliably detected.
• the invention relates to a method for detecting an infection with the influenza
• a and / or influenza B virus comprising the steps i) obtaining a saliva sample from the individual to be examined ii) preparing the saliva sample for the detection or a detection reaction; and iii) detection of the influenza A and / or influenza B virus in the saliva sample.
• the invention furthermore relates to a test kit for detecting an infection with the influenza A and / or influenza B virus comprising i) a device for collecting a saliva sample; and ii) reagents and aids for the detection of influenza A and / or influenza B viruses.
• the invention relates to the use of saliva as sample material for the detection of an infection with the influenza A and / or influenza B virus. It was unexpectedly found that a reliable detection of influenza A / B viruses is possible from saliva as sample material using established diagnostic methods.
• saliva is to be understood to mean that for the detection of influenza A / B viruses there is no accumulation of the influenza A / B viruses in the sample when the saliva sample is taken. Only the saliva spontaneously located in the oral cavity is collected and, for example, transferred to a saliva collection vessel (e.g. by spitting or the like). It is also possible to collect the saliva sample in the mouth with the aid of an absorbent material, for example a cotton fleece (swab), or one known to the person skilled in the art
• Saliva collection devices such as the so-called Salivettes from Sarstedt or the OraSure Specimen Collection Device from Epitope Inc. Beaverton, OR 97008, USA) are used to collect the saliva sample.
• saliva as sample material for the detection of influenza A / B viruses, in which the viruses have been concentrated (concentrated) during the sampling or in subsequent processing steps.
• sample material saliva according to the invention and the simple type of sample collection in contrast to the sample materials known up to now in influenza diagnostics, enable nasal / pharynx swab and liquid obtained by nasal irrigation to obtain standardized samples from untrained persons or the patient himself and thus contribute to a higher diagnostic Safety of influenza A / B virus tests.
• saliva as a sample material is more homogeneous than the other materials previously used, which also contributes to diagnostic reliability.
• a saliva sample is first obtained. For example, as already described above, this can be done by spitting into a vessel, sucking up a saliva sample using an absorbent material, for example a cotton swab or the like, in the mouth or using a conventional saliva extraction device.
• an absorbent material for example a cotton swab or the like
• the saliva sample is then prepared for the detection reaction:
• the viral nucleoprotein of influenza A / B viruses is released from the saliva sample by lysis reagents.
• lysis reagents are known to the person skilled in the art and can, for example, contain salts or wetting agents as active constituents for the lysis.
• Lysis reagent for the detection of influenza viruses preferably contains a wetting agent (for example Triton ® X 100, Tween ® 20 or beta-octylglycopyranoside has proven itself), a mild reducing agent (e.g.
• N-acetyl-L-cysteine or DTT dithiothreitol
• physiologically beneficial Cooking salt ie 0.9% by weight NaCl in 20-50mM phosphate buffer
• a preservative e.g. 0.09% by weight NaN3
• the immunological detection can, for example, detect the viral matrix protein or the viral polymerases. It is also possible to detect hemagglutinin or the neuraminidase of influenza A / B viruses, for which - since these virus components are on the virus surface - no lysis is required.
• RNA viral nucleic acid
• RT-PCR Polymerase chain reaction
• influenza A / B viruses in the saliva sample is carried out according to methods known per se.
• the product of the RT-PCR is preferably labeled using a labeled primer and the labeling of the primer (e.g. an enzyme or fluorescence label) depending on the nature of the label (label) detected.
• a labeled primer e.g. an enzyme or fluorescence label
• the invention also relates to a test kit for carrying out the method according to the invention.
• the test kit contains, as an important component, first a device for collecting a saliva sample, for example a vessel for spitting into it, one or more absorbent cotton balls, or a commercially available saliva collection device, e.g. B. a Salivette from Sarstedt or an OraSure Specimen Collection Device from Epitope.
• the test kit also contains all reagents and aids for the detection of influenza A / B viruses in the saliva sample.
• these are, for example, a lysis buffer for releasing the viral nucleoproteins, possibly labeled antibodies and, if appropriate, a reaction medium (test strip, microtiter plate, reaction tube (tube)) for carrying out the immunological detection.
• a reaction medium test strip, microtiter plate, reaction tube (tube)
• the corresponding reagents and auxiliaries are known to the person skilled in the art in a large number of embodiments. The same applies to the detection of nucleic acid.
• the kit contains, for example, the necessary PCR reagents and reaction vessels.
• the throat swabs were removed using sterile disposable cotton swabs from Copan Italia (1-25125 Brescia; Order No. 167CS01) in the manner familiar to the person skilled in the art.
• the two throat swabs taken from a patient were obtained by wiping the throat once with a swab with two cotton cushions arranged directly next to each other. This ensured that the two smears were largely comparable.
• saliva extraction methods / devices known to the person skilled in the art such as, for example, the Salivettes from Sarstedt or the OraSure Specimen Collection Device from Epitope Inc. Beaverton, OR 97008, USA
• the use of such saliva extraction methods / devices is also possible according to the invention.
• the first swab was transferred into a tube with 1.5 ml of influenza virus transport medium from Virotest (Rosenbergstr. 85, D-70193 Stuttgart, Cat.-No. 0500300) and placed on MDCK cells (Madine -Darby Canine Kidney cells).
• the samples were cultivated in accordance with the method described in the literature (microbiological diagnostics, Georg Thieme Verlag, Stuttgart, New York, 1992, publisher Friedrich Burkhardt, page 371). The results are shown in Table 1, where "+” symbolizes a positive finding and "-” symbolizes a negative finding.
• the second swab was opened immediately after taking the sample from the patient's throat using an immunological rapid antigen test Presence of influenza viruses (influenza A / B rapid test from Röche Diagnostics GmbH, Mannheim, Germany, cat. No. 2 158 663) checked.
• the rapid test used here essentially corresponds to the immunological rapid test described as example 2 in EP-A 0 926498. Reference is expressly made to this document.
• the intensity of the detection line after the chromatography was terminated was measured quantitatively using a remission photometric measuring device (ring illumination using 24 green LEDs with a wavelength of 555 nm and a CCD camera with lens).
• the intensity of the detection line signal was examined as a percentage of remission (% rem .; based on a "white" area of the test strip to which the remission was assigned 100%): remission values from 98.5% are recognized by the user as a negative signal; Remission values between 96% and 98.5% are recognized as a weakly positive signal; Remission values of less than 96% are recognized as a clearly positive signal.
• Table 1 where "+” means a positive signal, "(+)” means a weakly positive signal, and "-” means a negative signal.
• RNA ribonucleic acid
• the analysis process consisted of the three sub-steps of sample preparation (for the removal of inhibitors), amplification and detection of the nucleic acid, which are common in nucleic acid diagnostics (see below under 1.3. A to 1.3. L.c). The procedure was as follows:
• the one under order no. 1 858 882 from Röche Molecular Biochemicals uses the commercially available "High Pure Viral RNA Kit".
• the procedure was carried out in accordance with the standard protocol listed in the product description, first by using 200 ⁇ l saliva per Reaction vessel (filter tube) was bound to the glass fleece of the filter tube in the presence of binding buffer and after removing any inhibitors present, the viral nucleic acid was eluted in 200 ⁇ l elution buffer by two washing steps, taking care to ensure that the sample volume before (saliva) and after (Eluate) of the sample preparation was identical, so that no concentration of the influenza viruses took place.
• the batch volume per PCR reaction vessel was 50 ⁇ l. This contained 10 ul sample volume (eluate from sample preparation) and 10 ul bicine buffer (5 x RT-PCR buffer). The other components of the master mix were contained in the following final concentration: 2.5 mmolar manganese acetate, each 0.2 mmolar dATP (2'-deoxy-adenosine-5'-triphosphate), dCTP (2'-deoxy-cytidine-5 ' triphosphate), dGTP (2'-deoxy-guanosine-5'-triphosphate) and dUTP (2'-deoxy-uridine-5-triphosphate) as well as 0.05 mmolar dTTP (2'-deoxy-thymidine-5 ?
• the sequence of the primers was taken from the literature (James C. Donofrio et al., Detection of Influenza A and B in Respiratory Secretions with the PCR, 1992, PCR Method and Applications 1, pages 263-268).
• the primers are type-specific for influenza A and detect a 212 base pair long, highly conserved gene segment (positions 101 to 312) of the matrix gene.
• the reverse primer was labeled with biotin at the 5 'end for the subsequent detection of the amplification product in the microtiter plate.
• Type-specific primers for influenza B are also known in the literature (James C. Donofrio et al., Detection of Influenza A and B in Respiratory Secretions with the PCR, 1992, PCR Methods and Applications 1, pages 263-268).
• the master mix was amplified in a Perkin Elmer 9600 thermal cycler with the following temperature profile: 20 minutes room temperature after UNG addition + 45 minutes 60 ° C + 2 minutes 94 ° C + 10 cycles (30 seconds 94 ° C + 60 seconds 50 ° C) + 90 seconds 68 ° C) + 35 cycles (30 seconds 94 ° C + 60 seconds 60 ° C + 90 seconds 68 ° C) + 7 minutes 68 ° C. 1.3.1.C Hybridization and Detection
• PCR ELISA DIG Detection
• Röche Molecular Biochemicals Ca - No. 1 636 111 The detection of the nucleic acid amplified by RT-PCR was carried out using the PCR ELISA (DIG Detection) from Röche Molecular Biochemicals Ca - No. 1 636 111. With the exception of the pipetting volumes, all steps were carried out according to the package insert.
• the sequence of the hybridization probe was taken from the literature (James C. Donofrio et al., Detection of Influenza A and B in Respiratory Secretions with the PCR, 1992, PCR Method and Applications 1, pages 263-268).
• the probe is directed against position 177-205 of the matrix gene (segment 7) of influenza A.
• Corresponding probes for influenza B are also known in the literature (James C. Donofrio et al, Detection of Influenza A and B in Respiratory Secretions with the PCR, 1992, PCR Method and Applications 1, pages 263-268).
• 200 ⁇ l of the reaction mixture were transferred into a well of a microtiter plate coated with streptavidin and incubated on a shaker at 37 ° C. for 1 hour. After the hybridization product has bound to the streptavidin of the microtiter plate wall, the contents of the well are suctioned off and washed 3 times with 300 ⁇ l of washing solution in each case. Then 200 ⁇ l of anti-digoxigenin peroxidase substrate (anti-DIG-POD conjugate) were added to the well and the solution was incubated for 30 minutes at 37 ° C. on a shaker.
• anti-DIG-POD conjugate anti-digoxigenin peroxidase substrate
• the extinction values of the negative controls carried in the microtiter plate were usually 100-150 mE.
• the values of the positive control included in the kit were usually 1000 - 1500 mE.
• Absorbance values> 300 mE were defined as positive signals for patient samples, which corresponds to a signal greater than twice the blank / zero value. The results can be found in Table 1, where "+” symbolizes a positive finding and "-" a negative finding.
• influenza A / B rapid test from Röche Diagnostics Cat.-No. 2 158 663 used.
• the rapid test used here essentially corresponds to the immunological rapid test described as example 2 in EP-A 0 926498. Reference is expressly made to this document.
• the test is normally used to detect type-specific throat swab viral nucleoprotein using an immunological chromatography test strip.
• the test does not differentiate between influenza A and influenza B viruses.
• the special lysis / elution buffer was composed as follows:
• Triton® X-100 0.9 wt% NaCl, 2 mM KH 2 PO 4 , 10 mM Na 2 HPO 4 , 0.095 wt% NaN 3 , 10 mM EDTA, 1.5 wt% bovine serum albumin (RSA), 1.5 % By weight of Triton® X-100.
• the further test was carried out according to the steps described in the package insert. This initially included the addition of 2 drops of antibody solution 1 (contains biotinylated monoclonal antibodies against nucleoprotein A and nucleoprotein B), the addition of 2 drops of antibody solution 2 (contains digoxigenylated monoclonal antibodies against nucleoprotein A and nucleoprotein B) and the final chromatography of this reaction mixture using one test strips.
• the test strip contains a conjugate fleece which is reversibly impregnated with a gold conjugate which can be removed in sample liquid.
• the gold particles are adsorptively loaded with a monoclonal antibody against digoxigenin.
• a nitrocellulose membrane on which polystreptavidin as the detection line and a polyclonal antibody PAK ⁇ mouse Fc ⁇ > S-IgG as the control line are irreversibly impregnated.
• the sandwich complex of biotinylated antibody / nucleoprotein / digoxigenylated antibody formed in the presence of the analyte in the reaction vessel chromatographs on the test strip, binds it after dissolving the gold conjugate via the anti-digoxigenin antibody to the digoxigenin-labeled anti-influenza antibody of the sandwich complex and then becomes trapped on the polystreptavidin line via the biotin-labeled anti-influenza antibody of the sandwich complex on the nitrocellulose membrane. A red line is visible on the nitrocellulose membrane, which represents a positive test signal.
• the intensity of the detection line after the chromatography was terminated was measured quantitatively using a remission photometric measuring device (ring illumination using 24 green LEDs with a wavelength of 555 nm and a CCD camera with a lens).
• the intensity of the detection line signal was expressed as a percentage of remission (% rem .; based on a "white" area of the test strip, the the remission was assigned 100%) examined: remission values from 98.5% are recognized by the user as a negative signal; Remission values between 96% and 98.5% are recognized as a weakly positive signal; Remission values of less than 96% are recognized as a clearly positive signal.
• Table 1 where "+” is a positive signal, "(+)" is a weakly positive signal and "-” is a negative signal.
• influenza A does not circulate in every winter season and that even if influenza A / B viruses coexist in one winter season, influenza B is always present with a significantly lower prevalence.
• influenza B-positive saliva samples pooled saliva samples which were spiked with culture supernatant from influenza B viruses
• saliva is suitable as sample material for the detection of influenza A / B viruses.
• the rapid test results from saliva coincide with the associated PCR results from saliva and thus prove that influenza A / B viruses can be reliably detected from saliva as sample material even without prior enrichment during sample collection.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Immunology (AREA)
• Engineering & Computer Science (AREA)
• Molecular Biology (AREA)
• Virology (AREA)
• Organic Chemistry (AREA)
• Physics & Mathematics (AREA)
• Hematology (AREA)
• Microbiology (AREA)
• Zoology (AREA)
• Urology & Nephrology (AREA)
• Biotechnology (AREA)
• Wood Science & Technology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Biomedical Technology (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Pathology (AREA)
• General Physics & Mathematics (AREA)
• Biophysics (AREA)
• Medicinal Chemistry (AREA)
• Tropical Medicine & Parasitology (AREA)
• Food Science & Technology (AREA)
• Cell Biology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• Genetics & Genomics (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Apparatus Associated With Microorganisms And Enzymes (AREA)

Priority Applications (7)

Applications Claiming Priority (2)

Publications (2)

Family

ID=7645396

Family Applications (1)

Country Status (9)

Families Citing this family (39)

Family Cites Families (5)

• 2000
  • 2000-06-15 DE DE10028837A patent/DE10028837A1/de not_active Ceased
• 2001
  • 2001-06-15 AU AU2001278452A patent/AU2001278452A1/en not_active Abandoned
  • 2001-06-15 EP EP01956475.6A patent/EP1297187B2/de not_active Expired - Lifetime
  • 2001-06-15 ES ES01956475.6T patent/ES2298244T5/es not_active Expired - Lifetime
  • 2001-06-15 AT AT01956475T patent/ATE385259T1/de active
  • 2001-06-15 CA CA002412121A patent/CA2412121C/en not_active Expired - Fee Related
  • 2001-06-15 JP JP2002510708A patent/JP2004503248A/ja active Pending
  • 2001-06-15 US US10/311,523 patent/US6811971B2/en not_active Expired - Lifetime
  • 2001-06-15 DE DE50113548T patent/DE50113548D1/de not_active Expired - Lifetime
  • 2001-06-15 WO PCT/EP2001/006735 patent/WO2001096595A2/de not_active Ceased

Also Published As

Similar Documents

Legal Events