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WO2004097040A1 - Procede de detection de virus d'un brin(+) d'arn infectieux, en particulier, d'enterovirus infectieux - Google Patents

Procede de detection de virus d'un brin(+) d'arn infectieux, en particulier, d'enterovirus infectieux Download PDF

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Publication number
WO2004097040A1
WO2004097040A1 PCT/EP2004/004239 EP2004004239W WO2004097040A1 WO 2004097040 A1 WO2004097040 A1 WO 2004097040A1 EP 2004004239 W EP2004004239 W EP 2004004239W WO 2004097040 A1 WO2004097040 A1 WO 2004097040A1
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rna
detection
oligonucleotide
pcr
strand rna
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Despina Tougianidou
Manfred Bayer
Thomas Maier
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4base Lab Advanced Molecular Analysis GmbH
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4base Lab Advanced Molecular Analysis GmbH
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    • 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
    • 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
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present invention relates to a method for the detection of infectious (+) strand RNA viruses, in particular infectious enteroviruses, in a sample.
  • the (+) strand RNA viruses include Picornaviridae, which are among the smallest RNA-containing viruses and represent one of the largest and most important family of human pathogenic viruses.
  • Picornaviridae includes the group of enteroviruses, which in turn are divided into polio, coxsackie A and B and echoviruses. These viruses are all human-pathogenic viruses that can lead to different symptoms of the disease. They are taken up by os and multiply first in the lymphatic tissue of the throat and later especially in the intestinal wall, where, however, they usually do not cause any symptoms. Reach through the bloodstream it becomes its "target organ" and organ manifestation occurs, but most enterovirus infections are asymptomatic. Manifest enterovirus infections are often uncharacteristic with weak clinical symptoms. The viruses are usually excreted in the stool over a longer period of time and can thus be isolated.
  • the diagnosis can only be confirmed by pathogen detection in the laboratory.
  • Prophylactic measures against enterovirus infections are not known, hygienic measures have a purely preventive effect, with one major disadvantage being the high resistance of the viruses to disinfectants.
  • enteroviruses for example in aqueous samples such as concentrates from waste water or drinking water, in order to be able to take preventive measures in good time if the pathogen is present in the samples.
  • Laboratory diagnostics for enteroviruses include classic virus isolation by growing the viruses in cell culture. Usually, permanent cell lines are inoculated with the samples to be tested. If viruses are present in this sample, the virus can multiply, which leads to a characteristic change in the inoculated cultures, which can already be recognized as rounding off cells under the light microscope in the form of a cytopathic effect.
  • a disadvantage of this conventional method is, in particular, the long period of time that must be waited to be able to detect infectious viruses.
  • RNA Since the viral genome of the enteroviruses is present as a plus-strand RNA, the RNA must first be converted into DNA by means of a reverse transcription, which is referred to as RT-PCR. After reverse transcription of the RNA into DNA, it can then be amplified via the polymerase chain reaction, that is to say reproduced. This is necessary because the amounts of RNA present are too small for direct detection and because DNA can be reproduced simply and quickly using PCR. Only the amplified DNA is then available in sufficient quantities for detection.
  • PCR poly erase chain reaction
  • infectious pathogens can be present in the disinfected samples, as well as those that were inactivated during disinfection and are therefore no longer infectious.
  • virus DNA or indirectly virus RNA
  • RT-PCR is only used to check whether virus DNA (or indirectly virus RNA) is generally present or not. If a positive proof is provided, this means that no statement can be made as to whether or not the amplified gene sequences originate from viruses that are still infectious and thus pose a danger to humans. This question is among others of great importance in the area of water virology, e.g. when examining drinking water, water in swimming pools or other bodies of water, waste water etc.
  • virus detection is also required when examining patient samples (e.g. urine) or food.
  • this object is achieved in that the detection of infectious (+) Strand RNA viruses, especially enteroviruses, are carried out in a sample with the following steps:
  • a) inoculating a cell culture with the sample b) incubating the inoculated cell culture for a period of 0.5 to 24 hours, preferably four to six hours, c) lysing the cell culture, d) isolating RNA from the cell culture lysate, and e) detection of the viral (-) - strand RNA.
  • the object is achieved by using oligonucleotides with the oligonucleotides listed in the enclosed sequence listing with the SEQ ID numbers. 1 to 5 for the detection of infectious enteroviruses in a sample.
  • the method according to the invention creates a method by means of which the combination of the cell culture method and the RT-PCR can be used to detect replicative RNA forms of, for example, enteroviruses.
  • These replicative forms of RNA can only be found in the cells during the replication (ie virus propagation) of the viral RNA.
  • the method according to the invention therefore, only infectious pathogens are identified, so that, for example, enteroviruses which are dangerous for humans can be clearly identified.
  • At least one cell culture is first inoculated with the samples to be tested, as a result of which viral material which may be present in the samples is highly amplified.
  • monkey kidney cells or human cell lines are used as the cell culture.
  • the cell lines AGMK African Green Monkey Kidney
  • BGMK Biralo Green Monkey Kidney
  • Other suitable cell lines are, for example, RD (rhabdomyosarcom cell line) or Caco (colon carcinoma cell line). All cell lines are available, for example, from the German Collection for Microorganisms and Cell Cultures (DSMZ) or from the American Type Culture Collection (ATCC).
  • the inoculated cell culture is incubated for a certain period of time, a period of four to six hours at 37 ° C. and 5% CO 2 preferably being selected.
  • the time period is chosen such that there is sufficient time for the replication of the RNA, but the process as a whole is accelerated compared to the prior art.
  • the cell culture is first lysed with subsequent RNA isolation. Both steps can be carried out using various methods and are sufficiently described in the prior art.
  • RNA isolation is carried out, for example, by cell disruption under denaturing conditions.
  • a chaotropic salt in particular guanidinium salts such as guanidinium isothiocyanate and guanidinium hydrochloride, can be used.
  • the method using guanidium isothiocyanate was described, for example, by Chomczynski and Sacchi, "Single-step method of RNA isolation by acid guanidium thiocyanate-phenols-chloroform extraction", Anal. Biochem.
  • RNA 162 156-159 ( 1987) using a mixture of guanidinium thiocyanate and phenol-chloroform, which is particularly useful when processing large numbers of samples or isolating RNA from small amounts of cells or tissues.
  • the chaotropic salts make infected cells
  • the nucleic acids and proteins contained in the cell are released, and the use of phenol precipitates proteins, whereby only RNA and DNA can be isolated, the DNA being able to be removed, for example, by subsequent DNAse digestion However, phenol is not mandatory.
  • (-) strand RNA The final detection of (-) strand RNA clearly identifies infectious enteroviruses. Since the enteroviruses are "plus-strand" RNA viruses, ie viruses whose nucleic acid acts directly as RNA, (-) strand RNA is only found after infectious viruses have infected a cell. The method according to the invention thus ensures that even infectious viruses are clearly detected. In addition, the detection of (-) strand RNA can also be carried out simultaneously with (+) strand RNA.
  • the sample is an aqueous sample.
  • This aqueous sample can be, for example, a wastewater or drinking water sample, in particular sample concentrates, furthermore samples or sample concentrates from ponds, lakes, swimming pools, etc., in which infectious enteroviruses are to be detected.
  • the method according to the invention can also be used, for example, to detect viruses “directly” in the patient, for example in stool or urine samples, or on and in foods which may be contaminated with the pathogens.
  • samples that have already been disinfected can also be examined, since the great resistance of the enteroviruses means that there is a great possibility that infectious viruses will be present in one sample despite the disinfection.
  • Such samples can therefore, for example, be samples of skin that has already been disinfected, and also of disinfected surfaces such as, for example, floors, walls, toilets, instruments, and also of textiles and of drinking water and of water in swimming pools.
  • samples from hospitals, swimming pools and sewage treatment plants for example, which have been treated with the commonly used chemical, physical or other disinfectants.
  • disinfectants include chemical substances such as chlorine and iodine and their Compounds, also ethyl and propyl alcohol, formaldehyde and glutaraldehyde, etc. Dry or moist heat, ozone, UV, cathode and X-rays, ultrasound, etc. are used as physical disinfectants.
  • first total RNA and viral RNA is isolated from it in step d).
  • any RNA that is contained in the cells is first isolated, that is to say both viral RNA and the cell's own RNA.
  • viral and in particular the (-) strand RNA can finally be isolated and detected from the total RNA if infectious enterovirus material was present in the sample to be tested.
  • the total RNA can be obtained, for example, using the method described above using chaotropic salts.
  • the RNA can also be, for example, by fast prep with Trizol® (Gibco, USA) or with CTAB (cetyltrimethylammonium bromide; see Cheung et al., “A method to isolate RNA from gram-positive bacteria and mycobacteria”, Anal. Biochem 222: 511-514).
  • Viral RNA is then isolated from the total RNA.
  • the viral RNA can be isolated, for example, by magnetic separation. This method is well known in the prior art and has the advantage that the viral RNA can be easily and quickly separated from the total RNA of the cell cultures. Molecules with specific biological affinities are attached to magnetic particles via functional groups. Due to their affinity, the corresponding substances that bind to the molecules bind to that with the magnetic ones Particle conjugated molecule and by applying a magnetic field this complex can be removed from the suspension. Such a system for magnetic separation is available, for example, from Dynal Biotech, Norway.
  • streptavidin examples of such molecules with specific biological affinities are streptavidin and biotin.
  • streptavidin is bound to magnetic particles.
  • Biotin with which, for example, oligonucleotides can be labeled, then binds to these complexes.
  • the complementary RNAs can then be "fished” or isolated via the oligonucleotides labeled with biotin.
  • the complementary RNAs bind to the biotinylated oligonucleotides and, via this, to the streptavidin complexes. After application of the magnetic field, the bound RNAs can be isolated from the mixture in question and be cleaned in further steps.
  • the viral RNA can, however, also be isolated using other methods, for example by immobilizing the corresponding oligonucleotides on matrices which, for example, consists of cellulose. After addition of the cell lysate to the matrix, the (at least partially) complementary RNA sequences bind to the oligonucleotides bound to the matrix and can then be eluted.
  • viral RNA is isolated in step d).
  • At least one of the oligonucleotides with the nucleotide sequence SEQ-ID-No. 1 and 2 is used.
  • bio-U 5'-GCG GAA CCG ACT ACT TTG GGT GTC CGT GTT (SEQ ID No. 1)
  • bio-R 5'-AAC ACG GAC ACC CAA AGT AGT CGG TTC CGC (SEQ ID No. 2).
  • oligonucleotides which are each complementary to a specific RNA sequence, have proven particularly suitable in the inventors' own experiments.
  • the oligonucleotides bind to a specific region of the viral poliovirus RNA, more precisely at positions 531 to 560 of PV1 (database number ACC J02281, see, for example, in the “Entrez Nucleotides” database of the NCBI, National Center for Biotechnology Information) , wherein the oligonucleotide with SEQ ID No. 1 binds to the (-) strand and the oligonucleotide with SEQ ID No. 2 to the (+) strand.
  • step e) is carried out via RT-PCR of the viral RNA and detection of the amplification products.
  • This measure has the advantage that the (-) - strand RNA - if infectious enteroviruses were contained in the sample to be tested - can be detected in a particularly simple and quick manner.
  • the isolated viral RNA is first transcribed into DNA by reverse transcription. This DNA can be amplified in the next step, namely by the PCR, so that sufficient quantities of the relevant sequences are available. These amplified sequences can then be detected by further methods.
  • AMV (Avian Myoblastosis Virus) reverse transcriptase (for example available from Stratagene, USA), which is inactivated in the denaturation step of the PCR, can be used as the enzyme.
  • the enzyme Tth can also be used, for example, which works both as a DNA polymerase and - but only if a certain manganese concentration (1 to 4 mM) is present - as a reverse transcriptase.
  • the reverse transcriptase function of the enzyme can be inactivated via a low manganese concentration (ie below 1 mM) in the PCR steps.
  • the RT activity of the enzyme also completely ceases as soon as magnesium ions are present (see Myers et al., "Reverse Transcription and DNA Amplification by a Thermus thermophilus DNA Polymerase", Biochemistry 6; 30 (31): 7661-7666 (1991)
  • An advantage of this enzyme is its thermostability, according to which the reverse transcriptase can also take place at higher temperatures.
  • the amplified product can be detected, for example, by FRET (fluorescence resonance energy transfer). Because of its speed, this method is particularly suitable for the direct detection of the amplified sequences.
  • the PCR can be carried out quickly and on the other hand the results of the PCR can be quantified and analyzed simultaneously by recording the fluorescence during the amplification.
  • FRET fluorescence resonance energy transfer
  • Kits and devices for performing this method are available, for example, from Roche-Applied Science, Germany (see “LightCycler TM” and related products; Wittwer et al., “Continuous fluorescence monitoring of rapid cycle DNA amplification”, Biotechniques 22 : 130-138 (1997)).
  • the advantage is exploited here that the emitted light is used directly in the PCR device. measure and evaluate, so no unnecessary
  • the detection of the amplification products can also be carried out, for example, by gel electrophoresis with visualization of the amplified DNA by conventional methods, such as, for example, using ethidium bromide, or by Southern transfer of the amplified products to membranes and subsequent oligonucleotide hybridization.
  • Another method is the PCR-ELISA (enzyme-linked immunosorbent assay), in which the PCR is carried out with a labeled oligonucleotide (for example digoxigenin).
  • a labeled oligonucleotide for example digoxigenin
  • biotin-labeled probes are hybridized to the amplificates and the hybrids are immobilized on streptavidin-coated microtiter plates.
  • the bound hybrids are then detected with a suitable substrate (anti-digoxigenin-peroxidase conjugate and ABTS®, available from Röche Diagnostics, Mannheim, Germany).
  • a region in the 5 ′ non-coding region of the viral RNA is amplified for the RT-PCR.
  • This measure has the advantage that the amplification of a region in the 5 'non-coding region can be used to detect a region which is highly conserved in the genomes, in particular of the enteroviruses, so that the detection this area always several prototypes of enterovirals
  • certain areas can be amplified in the 5 'non-coding area of the poliovirus PV1 (ACC J02281), and in particular the area 159 to 478.
  • step e) for the detection of the viral RNA at least one of the oligonucleotides with the nucleotide sequence SEQ-ID-No. 3 to 5 is used.
  • oligonucleotides have the following sequences and names:
  • EV-FL2 5 '-GAG CTA SNT RRT AGT CCT CCG GCC C (fl) (SEQ ID No. 4)
  • EV-iLC2 5'-CYR YGT TAG GAT TAG CCG CAT T (-LC640) CA (SEQ ID No. 5)
  • the degenerate oligonucleotide with SEQ ID no. 3 (EVmod) carries a wobble base ("W") at position 3, which stands for adenosine or thymidine as standard.
  • EVmod (SEQ-ID No. 3) is that Primer for the reverse transcription of the (-) strand RNA as well as one of the two PCR primers.
  • the binding position of the primer in the sequence of the poliovirus is PVl (ACC J02281, see above) is 159-179.
  • the oligonucleotide with the SEQ ID no. 4 (EV-FL2) is also degenerate, the letters used following the conventional code:
  • the "S” at position 7 stands for cytosine or guanosine, and the "N” at position 8 for adenosine, cytosine, guanosine or thymidine.
  • This oligonucleotide hybridizes to positions 429 to 453 with reference to ACC J02281 (see above).
  • the "Y" used at positions 2 and 4 is by default for cytosine or thymidine, the "R” used at position 3 is adenosine or guanosine.
  • This oligonucleotide serves as a primer for the reverse transcription of the (+) strand RNA and hybridizes there at positions 455 to 478.
  • the oligonucleotide which one of the nucleotide sequences SEQ-ID-No. 1 to 5 from the attached sequence listing comprises at least one marker.
  • Labels that are suitable for this are, for example, biotin, digoxigenin, fluorescein, light cycler red 640, 6-carboxyfluorescein (FAM), 6-carboxytetramethylrhodamine (TAMRA), dabcyl.
  • FAM 6-carboxyfluorescein
  • TAMRA 6-carboxytetramethylrhodamine
  • the oligonucleotide with SEQ ID no. 5 be marked with the dye Light-Cycler-Red640, and thus as Probe (acceptor) act on the FRET during the PCR in the LightCycler.
  • the oligonucleotide with the SEQ ID no. In this connection, 4 can be labeled with fluorescein, for example, so that it serves as a probe (donor) in the FRET.
  • the dyes by which the oligonucleotides are labeled in this example case emit - as shown above - fluorescence after excitation.
  • the oligonucleotide EVmod (SEQ-ID No. 3) can be used to reverse-transcribe the (-) strand RNA into DNA.
  • This oligonucleotide simultaneously functions as one of the two PCR primers, so that when the other PCR primer EV-1LC2 (SEQ ID No. 5) is added, a sequence of the DNA resulting from the reverse transcription is amplified, the previously or simultaneously reverse transcriptase is inactivated.
  • a sequence is amplified which is highly conserved in the group of enteroviruses and therefore offers the possibility of detecting different enterovirus strains.
  • the oligonucleotide EV-FL2 (SEQ ID No. 4), which acts as a hybridization probe and which may be present in the PCR reaction mixture, binds to the amplified products in the immediate vicinity (position 429-453) of the one which functions as a primer Oligonucleotide EV-iLC2 (SEQ ID No. 5), which is incorporated into the amplified products in its function as a primer (position 455-478).
  • FRET enables direct detection of amplified sequences via fluorescence.
  • the method according to the invention is used to check the effect of disinfection measures and / or disinfectants. This has the The advantage that in particular samples that have already been disinfected can be tested quickly and reproducibly for the presence of infectious viruses.
  • disinfection measures or agents can be chemical or physical agents or measures, such as chlorination, iodination, ozonation, UV radiation, treatment with dry or moist heat and others.
  • the method according to the invention offers a reliable control, in particular for the control of, for example, waste water and drinking water and of hospital facilities, whether or not infectious viruses are still present after disinfection.
  • the invention further relates to an oligonucleotide which contains one of the nucleotide sequences SEQ-ID-No. 1 to SEQ ID no. 5 from the attached sequence listing, and the use of at least one of these oligonucleotides for the detection of infectious enteroviruses in a sample.
  • the invention relates in particular to the use of at least one of the oligonucleotides which contain one of the nucleotide sequences SEQ ID no. 1 to SEQ ID no. 5 of the attached sequence listing, in the method according to the invention described above.
  • oligonucleotides with the nucleotide sequence SEQ-ID-No. 1 or SEQ ID no. 2 from the attached sequence listing is used to isolate viral RNA, and in particular the oligonucleotide with the nucleotide sequence SEQ-ID-No. 1 for isolating enteroviral (-) strand RNA and the oligonucleotide with the nucleotide sequence SEQ ID no. 2 for isolating enteroviral (+) strand RNA.
  • the invention further relates to the use of the oligonucleotide with the nucleotide sequence SEQ ID no. 3 from the attached sequence listing as a primer for the reverse transcription and / or for the PCR of enteroviral (-) - strand RNA.
  • the invention further relates to the use of the oligonucleotide with the nucleotide sequence SEQ-ID-No. 4 from the attached sequence listing as a probe in the detection of RT-PCR products of viral (-) strand RNA and the oligonucleotide with the nucleotide sequence SEQ-ID-No. 5 from the attached sequence listing as a primer for the PCR and / or probe for the detection of RT-PCR products of viral (-) strand RNA.
  • the oligonucleotides can optionally be labeled.
  • Example 1 Cell culture a) Freezing and storing the cells
  • the cell line Buffalo Green Monkey Kidney Cells from Flow, Rockville, USA was used for the cell culture.
  • the cells were trypsinized, washed once by centrifugation at 2,000 rp, 4 ° C, and by resumption in MEME (Minimal Essential Medium with Earle's Salts) with 10% fetal calf serum. After a further centrifugation step, the cells were taken up in MEME with 20% fetal calf serum and 20% dimethyl sulfoxide.
  • the cell suspension prepared in this way was filled into 1.5 l-containing cryotubes and stored at -70 ° C. for a few days. Long-term storage took place in liquid nitrogen.
  • the Buffalo Green Monkey Kidney Cells were cultivated in 250 ml cell culture bottles and reacted every seven days. For this purpose, the medium was removed and the cell monolayer was washed with about 3 ml of 37 ° C. warm trypsin solution (0.2 g of trypsin in 1 l of phosphate-buffered saline (PBS)). The cells were then preincubated with 2 ml of trypsin solution at 37 ° C. for 2 min. The trypsin was then removed and replaced with fresh (1.5 ml). After a further incubation at 37 ° C.
  • trypsin solution 0.2 g of trypsin in 1 l of phosphate-buffered saline (PBS)
  • PBS phosphate-buffered saline
  • the monolayer detaches from the bottle and the trypsin was neutralized with 5 ml growth medium (MEME, 8% fetal calf serum, 1% non-essential amino acids).
  • the cells were separated by pipetting up and down several times.
  • the cell suspension was diluted in a ratio of 1: 6 to 1: 8 in growth medium (see above) and transferred to small cell culture bottles from 20 to 25 ml.
  • the cells were also sown in 6-well plates (3 ml per well) or in 24-well plates (0.6 ⁇ l per well) and incubated at 37 ° C. and 5% CO 2 .
  • the cell onolayer formed within the following three to four days.
  • the medium was replaced with maintenance medium as required (MEME, 2% fetal calf serum, 1% non-essential amino acids).
  • the medium was removed from the cell culture plates and the cells were washed with PBS and inoculated with 0.2 to 0.5 ml of the suspension or dilution to be tested.
  • the following virus types were tested: poliovirus Sabin 1, -2, Coxsackie A virus types 7 and 9, ECHO virus types 4, 15 and 20 and the polio virus vaccine (polio types 1 to 3).
  • the viruses were adsorbed onto the cells during a 30-minute incubation at 37 ° C. The supernatant was then drawn off and 2 to 3 ml of maintenance medium (see above) were added to the cells.
  • the inoculation of the cell cultures in the cell culture bottles was carried out as follows: The cell monolayer was inoculated as soon as it was tight (approx. 3 to 4 days after the cells were sown). For this purpose, the supernatant was drawn off, the cells were washed with PBS and the virus suspension was pipetted onto the cells. After an adsorption time of one hour, the suspension was drawn off, the cells were overlaid with maintenance medium and incubated at 37 ° C. with 5% CO 2 for the necessary incubation time (for example six hours). Before the subsequent RNA isolation, the supernatant was drawn off and the cells were washed three times with tempered PBS.
  • RNA isolation was carried out using various methods. Examples of RNA isolation below are a) with the High Pure RNA Tissue Kit from Röche and b) with magnetic beads from Dynal Biotech:
  • the cells were lysed in a strongly protein-denaturing buffer and in the presence of chaotropic agents (guanidinium isothiocyanate salts).
  • chaotropic agents guanidinium isothiocyanate salts.
  • the culture supernatant of the infected cells was drawn off and washed with IxPBS. After adding lysis buffer to the cells, they were lysed by repeated pipetting up and down.
  • the cell suspension was transferred to a reaction vessel and vortexed strongly for 20 seconds. After a 2-minute centrifugation at 13,000 g at room temperature, 0.5 times the sample volume was added to the supernatant and the mixture was pipetted onto a silica fleece. The nucleic acids released were bound to the silica fleece by this step, the run was discarded.
  • RNAse-free DNAse After adding RNAse-free DNAse to the silica fleece, this mixture was incubated for 15 min at room temperature. After addition of washing buffer (5 M guanidinium-HCl, 20 mM Tris-HCl, pH 6.6; add 20 ml of absolute ethanol to 33 ml of buffer before use), the mixture was centrifuged for 15 seconds at 8,000 g. After addition of washing buffer II (20 mM NaCl, 2 mM Tris-HCl, pH 7.5; add 40 ml absolute ethanol to 10 ml buffer before use), the mixture was then centrifuged for 15 seconds at 8,000 g. After a further washing step, the RNA was eluted with nuclease-free, sterile water. b) RNA isolation using agnetic beads (Dynal Biotech, Norway)
  • the culture supernatant of the infected cells was drawn off and the cells were washed twice with PBS. Depending on the monolayer area, 100 to 200 ⁇ l of lysis buffer (4.5 M guanidine-HCl, 100 mM sodium phosphate, pH 6.6) are added to the cells. The cells began to detach and were suspended by repeated pipetting up and down. The detached cells were transferred to a reaction vessel, incubated at 95 ° C. for 8 min and then placed on ice.
  • lysis buffer 4.5 M guanidine-HCl, 100 mM sodium phosphate, pH 6.6
  • RNA ⁇ strand from the lysate, 40 ⁇ l lysate with 120 ⁇ l 8x SSPE (20x SSPE: 0.2 M NaH 2 PO 4 , pH 7.4; 2.98 M NaCl; 0, 02 M EDTA) and 48 p ol biotin oligonucleotide and incubated for 15 min at 55 ° C.
  • the following biotin-labeled oligonucleotide was used to isolate the (-) strand RNA: bio-U: 5'-gCg gAA CCg ACT ACT TTg ggT gTC CgT gTT (SEQ ID No. 1).
  • bio-R 5'-AAC ACg gAC ACC CAA AgT AgT Cgg TTC CgC (SEQ ID No. 2).
  • the oligonucleotides were made to order by BioSpring, Frankfurt. They each have a biotin label at their 5 'ends and bind to the following position of the viral RNA strand (J02281, see above): 531 to 560. Then 16 ⁇ l magnetic beads (Dynabeads® Streptavidin, Dynal Biotech, Norway) were added, mixed and incubated at 55 ° C. for a further 10 min.
  • the biotin-labeled oligonucleotides to which the corresponding RNA strands were bound bound to the Dynabeads® Due to the affinity of streptavidin for biotin, the biotin-labeled oligonucleotides to which the corresponding RNA strands were bound bound to the Dynabeads®.
  • the bead complexes were separated on the magnet and the supernatant was removed.
  • the Bea ' ds were each resuspended and washed twice with 400 ⁇ l washing buffer (10 mM Tris Cl, pH 7.5; 1 mM EDTA; 2 M NaCl; 0.05% Tween 20) and each transferred to a new cup. This was followed by a washing step with 400 ⁇ l washing buffer and 400 ⁇ l lx SSPE. After a short centrifugation step, any residues still present were removed and the RNA was eluted twice with 10 ⁇ l at 80 ° C. for 2 min.
  • LC-RNA Master Kit Hybridization Probes (“LC-RNA Master Kit Hybridization Probes”, Röche, Mannheim, Germany) was carried out in a conventional cycler (for example PTC-2000 MJ-Research, Biozym, Germany) carried out.
  • the oligonucleotide with the nucleotide sequence SEQ-ID-No. 3 used, while for the reverse transcription of the (+) - strand RNA the oligonucleotide with the nucleotide sequence SEQ ID NO. 5 was used.
  • the sequences of the primers are shown below: EVmod: 5'-GAW CAA GCA CTT CTG TTT CCC (SEQ ID No. 3)
  • EV-iLC2 5 '-CYR YGT TAG GAT TAG CCG CAT T- (iLC640) CA (SEQ ID No. 5)
  • the degenerate oligonucleotide with SEQ ID no. 3 (EVmod) at position 3 is a wobble base ("W"), this wobble base being standard for adenosine or thymidine.
  • W wobble base
  • the oligonucleotide binds to positions 159 to 179 with reference to ACC J02281.
  • the oligonucleotide Like the biotin-labeled probes (SEQ ID No. 1 and 2), EVmod was manufactured to order by Bio Spring, Frankfurt, Germany.
  • the "Y" used at positions 2 and 4 is by default for cytosine or thymidine
  • the "R” used at position 3 is adenosine or guanosine.
  • This oligonucleotide is marked internally on the thymidine at position 22 with the dye Light Cycler Red 640.
  • This oligonucleotide acts as a primer in the reverse transcription of the (+) strand RNA and hybridizes with reference to ACC J02281 at positions 455 to 478.
  • the amplification was carried out using the primers EVmod (SEQ-ID No. 3) and EV-iLC2 (SEQ-ID No. 5 ) instead of detection was carried out via the probe EV-FL2 (SEQ-ID No. 4) (donor) and via the oligonucleotide EV-iLC2 (SEQ-ID No. 5) (acceptor), which also acts as a probe.
  • the sequence of the oligonucleotide EV-FL2 is shown below:
  • EV-FL2 5 '-GAG CTA SNT RRT AGT CCT CCG GCC C (fl) (SEQ ID No. 4)
  • This oligonucleotide is also degenerate, the letters used following the conventional code: the "S” at position 7 stands for cytosine or guanosine, and the "N” at position 8 for adenosine, cytosine, guanosine or thymidine.
  • This oligonucleotide is also labeled with fluorescein at its 3 'end. This oligonucleotide serves as a probe and hybridizes to positions 429 to 453 with reference to J02281.
  • oligonucleotides EV-FL2 and EV-iLC2 were synthesized by Tib Molbiol, Berlin, Germany.
  • the missing primer was added, the mixture was transferred to the capillaries and the PCR was run in the LightCycler TM.
  • the amplification product is detected online using specific hybridization probes that bind to the resulting amplificate during the PCR.
  • the LightCycler TM is a small-lumen fluorometer, which has an integrated thermal cycler, which combines a fast-cyclic PCR with a real-time fluorescence measurement. With this LightCycler TM it is possible to detect and analyze PCR products using sequence-specific hybridization probes without the need for further processing of the samples after the PCR.
  • the fluorescence control of the amplification using hybridization probes is based on the concept that a fluorescence signal is generated when a fluorescence resonance energy transfer (FRET) takes place between two adjacent fluorophores.
  • FRET fluorescence resonance energy transfer
  • the antisense primer was internally with the dye LightCycler TM -red 640 marked. This requires the addition of only one hybridization probe that binds adjacent to the primer on the other strand.
  • the viral (-) strand RNA could already be detected by strand-specific RT-PCR for three hours.
  • Figure 1 shows the detection of (-) - strand and (+) - strand RNA after incubation of Buffalo Green Monkey Kidney Cells with 40,000 plaque forming units of virus material by RT-PCR in the LightCycler TM. For this purpose, the cells were infected with 40,000 plaque-forming units of poliovirus material, for 3, 3.5 and 4 hours.
  • 1 is the curve of the (+) strand RNA after three hours
  • 2 is the curve of the (+) strand RNA after 3.5
  • 3 is the curve of the (+) strand RNA shown after four hours.
  • Curves 4, 5 and 6 each show the development of the (-) strand RNA after 3, 3.5 and 4 hours of incubation of the cells with the virus material.
  • Curve 7 shows the detection of (-) strand RNA without incubation on cells and curve 8 shows the (+) strand RNA without incubation on cells.
  • the signals of the (-) - strand RNA were strong enough only after four hours (see Figure 1: curve 6).
  • the RT-PCR of the (-) strand RNA with the upstream primer oligonucleotide with SEQ ID No. 3 was strand-specific, ie without previous Incubation of the viruses on the cells amplified the (+) strand RNA (curve 8) and not the (-) strand RNA (curve 7).
  • thermostable enzyme Tth With the help of the thermostable enzyme Tth the reverse transcription can take place at high temperatures (61 ° C), whereby unspecific reactions during the amplification of (-) - strand RNA could be avoided.
  • the RT activity of the enzyme Tth completely ceased as soon as Mg ions were present and the Mn concentration was less than 1 mM. This was the case here in the PCR reaction (see reaction approaches).
  • Figure 2a shows signals of the RT-PCR of the (+) strand RNA from poliovirus after inoculation on the cells after six hours: curve 1 shows the data with 100 plaque-forming units (hereinafter PFU) on poliovirus Material shown per reaction; with 2 for 20 PFU, with 3 for 4 PFU, with curve 4 for 0.8 PFU, with curve 5 for 40 PFU without cells and curve 6 represents the negative control in which no virus material was used.
  • PFU plaque-forming units
  • Figure 2b shows the signals of the RT-PCR of the (-) strand RNA from poliovirus after inoculation on cells after six hours with different dilutions: curves 7, 8, 9 and 10 give 100, 20, 4 and 0.8 PFU poliovirus material per reaction again, curve 11 shows the positive control, ie 40 PFU poliovirus material without cells tiv control, ie 40 PFU poliovirus material without cells and curve 12 the negative control without poliovirus material.
  • Figure 3 shows the incubation of cells with 40,000 PFU poliovirus material, with the inoculation stopped after twelve hours.
  • curve family 1 shows (+) strand RNA
  • family of curves 2 (-) strand RNA
  • curve 3 (+) strand RNA without incubation on cells
  • curve 4 ( -) strand RNA without incubation on cells
  • curve 5 represents the negative control.
  • the amplification signals of the (+) strand RNA decrease with increasing dilution of the starting virus material (see Figure 2a). This regularity cannot always be observed when the (-) - strand RNA is detected (see Fig. 2b).
  • test method described is suitable for general checking of the infectivity of viruses, for example when determining the activity of disinfectants.
  • the method according to the invention is particularly suitable in the field of environmental water virology, it being possible here to use the method according to the invention to differentiate between inactivated and non-inactivated and consequently potentially infectious viruses. This is particularly important for assessing the risk to exposed persons.
  • Various virus-inactivating environmental influences or disinfection measures used in drinking water treatment (such as chlorination, ozonation or UV radiation) eliminate the infectiousness.
  • the genetic material of the viruses can continue to be protected and intact by the viral protein envelope.
  • the viruses can then no longer be detected via the cell culture, but they can be detected by conventional amplification of their nucleic acid, so that non-infectious but intact viruses can also be detected using the methods known in the prior art.

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Abstract

L'invention concerne un procédé de détection de virus d'un brin(+) d'ARN infectieux, en particulier, d'entérovirus, dans un échantillon. A cet effet, une culture cellulaire est tout d'abord inoculée avec l'échantillon, puis la culture cellulaire est incubée pendant une période de 0,5 à 24 heures, de préférence de 4 à 6 heures. Après quoi, la culture cellulaire est lysée, l'ARN est isolé du lysat de ladite culture, et le brin(-) d'ARN viral est détecté.
PCT/EP2004/004239 2003-04-30 2004-04-22 Procede de detection de virus d'un brin(+) d'arn infectieux, en particulier, d'enterovirus infectieux Ceased WO2004097040A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8329668B2 (en) * 2005-09-08 2012-12-11 Avi Biopharma, Inc. Antisense antiviral compound and method for treating picornavirus infection
WO2021243333A3 (fr) * 2020-05-29 2022-03-03 Biohsv Holdings, Inc. Test d'acide nucléique de masse

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102007031137A1 (de) * 2007-06-13 2008-12-18 Attomol Gmbh Molekulare Diagnostika Verfahren und Sonden/Primärsystem zum "real time" Nachweis eines Nukleinsäuretargets

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0789081A2 (fr) * 1995-02-17 1997-08-13 Instituto De Salud Carlos Iii Procedes d'amplification de genomes et melanges d'oligonucleotides initiateurs pour la detection et l'identification d'agents infectieux apparentes
US5728519A (en) * 1990-11-06 1998-03-17 The United States Of America As Represented By The Department Of Health And Human Services Assay for virulent revertants of attenuated live vaccines and kits therefor
US6258570B1 (en) * 1998-04-17 2001-07-10 University Of Pittsburgh PCR assay for bacterial and viral meningitis

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5075212A (en) * 1989-03-27 1991-12-24 University Of Patents, Inc. Methods of detecting picornaviruses in biological fluids and tissues
FI95398C (fi) * 1989-06-24 1996-01-25 Boehringer Ingelheim Int Menetelmä ihmisen nuhaviruksen serotyypittämiseksi
US6168917B1 (en) * 1996-10-02 2001-01-02 The United States Of America As Represented By The Department Of Health And Human Services Detection and identification of non-polio enteroviruses
CN1451050A (zh) * 2000-07-06 2003-10-22 比奥·麦利尤股份有限公司 控制水相介质的微生物质量的方法及其试剂盒

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5728519A (en) * 1990-11-06 1998-03-17 The United States Of America As Represented By The Department Of Health And Human Services Assay for virulent revertants of attenuated live vaccines and kits therefor
EP0789081A2 (fr) * 1995-02-17 1997-08-13 Instituto De Salud Carlos Iii Procedes d'amplification de genomes et melanges d'oligonucleotides initiateurs pour la detection et l'identification d'agents infectieux apparentes
US6258570B1 (en) * 1998-04-17 2001-07-10 University Of Pittsburgh PCR assay for bacterial and viral meningitis

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
CROMEANS T L ET AL: "Rapid detection of infectious RNA viruses by the presence of replicative form RNAs in cell culture", ABSTRACTS OF THE GENERAL MEETING OF THE AMERICAN SOCIETY FOR MICROBIOLOGY, vol. 101, 2001, & 101ST GENERAL MEETING OF THE AMERICAN SOCIETY FOR MICROBIOLOGY; ORLANDO, FL, USA; MAY 20-24, 2001, pages 614, XP009036248, ISSN: 1060-2011 *
REYNOLDS K A ET AL: "ICC/PCR detection of enteroviruses and hepatitis A virus in environmental samples", CANADIAN JOURNAL OF MICROBIOLOGY 2001 CANADA, vol. 47, no. 2, 2001, pages 153 - 157, XP009036181, ISSN: 0008-4166 *
REYNOLDS K S ET AL: "RAPID PCR-BASED MONITORING OF INFECTIOUS ENTEROVIRUSES IN DRINKING WATER", WATER SCIENCE AND TECHNOLOGY, ELMSFORD, NY, US, vol. 35, no. 11/12, 1997, pages 423 - 427, XP001156479, ISSN: 0273-1223 *
REYNOLDS KELLY A ET AL: "Detection of infectious enteroviruses by an integrated cell culture-PCR procedure", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 62, no. 4, 1996, pages 1424 - 1427, XP002295938, ISSN: 0099-2240 *
TOUGIANIDOU DESPINA AND BOTZENHART KONRAD: "Molecular techniques for the detection of enteroviruses in water", OECD WORKSHOP MOLECULAR METHODS FOR SAFE DRINKING WATER, 1998, pages 1 - 4, XP002295936 *
WANG W-K ET AL: "Detection of dengue virus replication in peripheral blood mononuclear cells from dengue virus type 2-infected patients by a reverse transcription-real-time PCR assay", JOURNAL OF CLINICAL MICROBIOLOGY 01 DEC 2002 UNITED STATES, vol. 40, no. 12, 1 December 2002 (2002-12-01), pages 4472 - 4478, XP002295935, ISSN: 0095-1137 *
WATKINS-RIEDEL T ET AL: "Rapid diagnosis of enterovirus infections by real-time PCR on the LightCycler using the TaqMan format", DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE 2002 UNITED STATES, vol. 42, no. 2, 2002, pages 99 - 105, XP002295937, ISSN: 0732-8893 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8329668B2 (en) * 2005-09-08 2012-12-11 Avi Biopharma, Inc. Antisense antiviral compound and method for treating picornavirus infection
WO2021243333A3 (fr) * 2020-05-29 2022-03-03 Biohsv Holdings, Inc. Test d'acide nucléique de masse

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