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WO1992016661A1 - Procede de detection d'arn viral - Google Patents

Procede de detection d'arn viral Download PDF

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Publication number
WO1992016661A1
WO1992016661A1 PCT/CA1992/000117 CA9200117W WO9216661A1 WO 1992016661 A1 WO1992016661 A1 WO 1992016661A1 CA 9200117 W CA9200117 W CA 9200117W WO 9216661 A1 WO9216661 A1 WO 9216661A1
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Prior art keywords
virus
sample
rna
process according
viruses
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PCT/CA1992/000117
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English (en)
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Arnost Cepica
Jiang Jian Liu
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University Of Prince Edward Island
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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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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

Definitions

  • the present invention relates to a simple procedure for treatment of water, tissue cell or fluid, or biological samples, for example clinical samples of blood, blood products, serum, body fluids, secretions, faeces, tissue homogenates and cell culture fluids, containing or suspected of containing RNA viruses, which will enable their direct inclusion, without RNA extraction, in a detection system (for eg. reverse transcription -polymerase chain reaction, or nucleic acid hybridization, Q-beta replicase, or ligase chain reaction) .
  • a detection system for eg. reverse transcription -polymerase chain reaction, or nucleic acid hybridization, Q-beta replicase, or ligase chain reaction.
  • PCR Polymerase chain reaction
  • RNA extraction from clinical samples containing RNA viruses has made the use of PCR for diagnostic virus detection laborious, but more importantly, the sensitivity of detection is compromised by the inevitable incomplete recovery of RNA by the extraction.
  • PCR has the capability to amplify even a single copy of a genome, and if RNA recovery would not be 100%, the most important benefit of PCR would be compromised.
  • No procedure for deproteinization of RNA viruses in a clinical sample, in a manner that would preserve RNA intact and for use in reverse transcription and subsequently in PCR, has been published. The present invention relates to such a procedure.
  • BVDV bovine viral diarrhoea virus
  • RNA viruses Currently available prior art methods of virus deproteinization were found by the present inventors to be not suitable for the sensitive detection of RNA viruses. Such methods are inadequate, in that they not only do not protect the uncoated RNA from the action of endogenous RNAses (heat) , but in addition they have a potential to introduce exogenous RNAses (detergents, proteases) .
  • the invention relates to a relatively simple method for detecting RNA virus in a sample, for example by using PCR, in which extraction of RNA from the sample can be avoided.
  • the invention provides a process for protein uncoating of RNA virus in a sample of water, tissue cells or fluid, or biological material without substantially degrading the RNA, which process comprises heating the sample to a temperature sufficient to uncoat RNA of the RNA virus of protein, in the presence of an RNase inhibitor which is active at the said temperature.
  • the RNase inhibitor is non-proteinaceou ⁇ , further prefereably it is diethylpyr carbonate (DEPC) or vanadyl ribonucleoside (V-R) .
  • DEPC diethylpyr carbonate
  • V-R vanadyl ribonucleoside
  • concentration of DEPC is preferably in the range of from about .00025% to .25% by volume of the sample to be heated, most preferably in the range of from about .0005% to .05% by volume of the sample to be heated.
  • the concentration of V-R is preferably in the range of from about .2mM to 8mM most preferably about 2mM in the solution to be heated. Further preferably, the heating is to a temperature in the range of from about 60 * C to about 100"C, most preferably 90 * C to 100"c Duration of heating depends on the temperature selected, i.e. more time at a lower temperature and vice versa. Heating for from 3 to 20 minutes is typical.
  • the invention also provides a process for treating a sample of tissue cells or fluid for detection of RNA virus, which process includes the above-described heating step and then further treating the sample or a derivative thereof, optionally after addition of a preselected proteinaceous RNase inhibitor during a cool down, for detection of RNA or a derivative thereof as an indication of the presence or absence of RNA virus in the tissue cells or fluid.
  • the sample is treated for detection of RNA after heating by firstly, reverse transcription of RNA to form cDNA, and then treatment of the sample to amplify the cDNA by polymerase chain reaction, so that cDNA may be detected, for example by gel electrophoresis and Southern blot methods.
  • the sample may be cooled to a temperature below a denaturation temperature of a preselected proteinaceous RNase inhibitor, but above about 40'c.
  • the preselected proteinaceous RNase inhibitor may then added to the sample.
  • RNase activity tends to be strongest at 37 * C, so it is preferable to add the RNase inhibitor when the sample temperature is as far above 37'c as possible, bearing in mind the addition must be at a temperature below the denaturation temperature of the proteinaceous RNase inhibitor.
  • the proteinaceous RNase inhibitor is RNasinTM, RNadeTM or Human Placental Ribonuclease Inhibitor (BRL, Gaithersburg, Md, U.S.A.) and when used, is - added to the sample when the sample temperature preferably is below about 55"c and above about 40°C and further preferably in a concentration in the range of from about 0.1 units to 20 units per ⁇ l of sample, most preferably about .5 to 4 units per ⁇ l of sample.
  • reverse transcription to form cDNA and then PCR to amplify the cDNA may be performed.
  • RNA viruses such as BVDV, HIV, FIPV, FECV and IPNV, although the invention may be applied to a wide variety of RNA viruses. Description of Figures
  • Figure 1 shows the results of an agar gel electrophoresis of EcoR I and Hind III digested plasmid pBV4p80 containing the nucleotide sequence 5644-7949 10 of BVDV genome.
  • the fragment "C” was used in making the Southern blots of Figures 3A, 4A, and 5A.
  • Figure 2 shows the location of the probe and primers on NADL genome.
  • Figure 3 shows the results of gel electrophoretic detection of serially diluted plasmid DNA containing BVDV sequence.
  • Figure 3A shows a Southern blot of the gel of
  • Figure 4 shows the results of an agarose gel electrophoresis of PCR products obtained from samples containing whole virus, with and without DEPC.
  • Figure 4A shows a Southern blot hybridization of the agarose gel shown in Figure 4.
  • Figure 5 shows the results of an agar gel electrophoresis of the PCR products obtained from complete - virions of various strains of BVDV.
  • Figure 5A shows a Southern blot hybridization of the gel of Figure 5.
  • Figure 6 shows the results of a polyacryla ide gel electrophoresis of the PCR products showing the effect of RT buffer on formation of PCR product.
  • Figure 7 shows the results of an agarose gel electrophoresis of the PCR products performed on serially diluted BVDV.
  • Figure 8 shows the results of an agar gel electrophoresis of the PCR products obtained from complete virions of various strains of HIV.
  • Figure 9 shows a Southern blot of hybridization of the gel of Figure 8.
  • Figure 10 shows the results of an agar gel electrophoresis of PCR products obtained from complete virions of NADL strain of BVDV using the inventive method.
  • Figure 10A shows the results of an agar gel electrophoresis of PCR products obtained after extracting RNA from virions of NADL strain of BVDV, for purposes of comparison with the results of using the inventive method as shown in Figure 10.
  • Figure 11 shows the comparative results of an agar gel electrophoresis of PCR products obtained from complete virions of BVDV strains using the inventive method, when Mg ** is removed from the RT solution.
  • Figure 12 shows the results of an agar gel electrophoresis of PCR products obtained from tissue culture media containing IPNV strains of serogroup A.
  • Figure 12A shows a Southern blot hybridization of the gel of Figure 12.
  • Figure 13 shows in part A, the results of an agar gel electrophoresis of PCR products obtained from complete virions of JASP strain of IPNV using the inventive method applied to various dilutions of such strain, and in part B, a Southern blot hybridization of the gel of part A.
  • Figure 14 shows the results of an agar gel electrophoresis of PCR products obtained from complete virions of FIP T ' 79 ⁇ 1146 and FECV 79"1683 strains using the inventive method, in order to demonstrate further how these different strains may be distinguished using PCR.
  • BVDV bovine viral diarrhoea virus
  • NADL reference cytopathogenic strain of BVDV
  • CP B6356 cytopathogenic
  • NCP B6356 noncytopathogenic
  • the viruses were grown and titrated in the cell line of bovine turbinate cells (American Type lture Collection) . Reading of the end point of titration cL the norf ytopathogenic strain was accomplished with the indirect fluorescent antibody method, using bovine polyvalent BVDV antiserum and rabbit anti-bovine fluorescein labelled conjugate. The virus titers were calculated by the method of Viliet 8 .
  • the titers of the NADL, CP and NCP BVDV viral stocks were 10 "5*63 , 10 ⁇ 4*36 and 10 " 4*3 tissue culture infectious doses 50 (TCID 50 )/ml respectively.
  • B6356 CP BVDV was used to prepare viral RNA according to Brock 9 with the following modification: Infected bovine turbinate cells were frozen and thawed once before the supernatant was collected for viral RNA isolation. A small portion of cells was kept to confirm presence of the virus by fluorescence antibody test. The RNA pellet was resuspended in deionised H 2 0 (d H 2 0 ) and precipitated twice with 0.2 M potassium acetate, pH 5.6 and 2.5 volume absolute ethanol at -20°C overnight.
  • the final precipitate was vacuum-dried in Speedvac System (Savant, Farmingdale, NY, USA) for 8 minutes and dissolved in diethyl pyrocarbonate (DEPC, from BDH, Poole, England) treated d H 2 0.
  • the final concentration of viral RNA was determined by a Spectrophotometer (Ultrospec II, LKB, Cambridge, England) at 260 nirt and adjusted to 1 ug/ ⁇ l with the above treated H 2 0.
  • the so isolated viral RNA was for use as a control template for PCR (See Figure 5, lane 6 and Figure 5A, lane 6) .
  • Plasmid pBV4p80 encompassing nucleotide sequence 5644-7949 10 of BVDV genome was obtained from Dr. M.S. Collett (Molecular Vaccines Inc., Gaithersburg, MD. 20878, USA) , and used to transform E. coli TB-1 by the calcium chloride procedure 11 .
  • the transformed E.Coli were grown in rich medium 12 , harvested and lysed in alkali solution followed by the equilibrium ultracentrifugation (38,000 rpm, 40 hours, 70.1 Ti Rotor) as previously described 12 , to obtain purified plasmid DNA.
  • plasmid DNA was divided into fragments A, B, C and D by the low-melting-temperature agarose electrophoresis (Low Gel Temperature, Bio-Rad, Richmond, CA, USA), Fig. 1.
  • lane "M” is for the marker DNA fragment (pBR322 digested by Hinf I) .
  • Lane 1 shows the fragments A, B, C and D from pBV4p80 containing BVDV sequence.
  • Fragment C was extracted from the gel 13 and used as a probe after labelling with 32 P-dCTP (Amersham Canada Limited, Oakville, Ontario, Canada) by nick-translation system (BRL, Gaithersburg, MD, USA) according to manufacturer's instructions.
  • the method was capable of detecting as little as 0.01 fg of the target DNA (Figs. 3 and 3A) . This translates into approximately 2 copies of the target sequence detected.
  • lane "M" is the same as in Fig.
  • lanes 2- 9 are from serial dilutions of pBV4p80 DNA, amplified by PCR using BV05 and BV06 primers.
  • lanes 2-9 respectively there are the following amounts of target DNA: 0.010 fg; 0.10 fg; 1.0 fg; 10 fg; 0.1 pg; 1 pg; 10 pg; and 0.1 ng.
  • PCR Primers BV05 and BV06 were synthesized by the DNA synthesis laboratory at the University of Calgary.
  • BV05 contained the nucleotide sequence 5813-5829 of NADL strain of BVDV, as previously established by Collett lc .
  • BV06 was complementary to the sequence 5936-5952 (Table l and Fig. 2) .
  • RNAdeTM ( 40 U/ ⁇ l supplied by BIO/CAN SCIENTIFIC, Mississauga, Ontario, Canada) were added.
  • the samples were cooled on ice and another 1.2 ⁇ l of 5 X RT buffer preferably without Mg ** , 2 ⁇ l of 0.1 M dithiothreitol, 4 ⁇ l of 2.5 mM of each of the deoxynucleotide triphosphate (dNTP) (SIGMA, St Louis, MO, USA) mixture and l ⁇ l (200 u) of Moloney Murine Leukaemia Virus Reverse Transcriptase (BRL, Gaithersburg, MD, USA) were added. The final reaction volume was 20 ⁇ l. The reaction took place at 37 * C for 60 minutes.
  • dNTP deoxynucleotide triphosphate
  • Fig. 11 The significance of removal of Mg ** from RT solution, excess of which was introduced by direct inclusion of, for example, serum tissue culture fluid, is demonstrated in Fig. 11.
  • the legend for Fig. 11 is as follows: Effect of M ⁇ * * concentration on RT/PCR - BVDV. Tube # Final concentr. Final concentr. of M ⁇ ** in RT ⁇ ?f Mg in PCF
  • RT product directly in PCR, rather than submitting it first to complementary DNA extraction.
  • 15 microliters of PCR product were electrophoresed in 3% agarose (IBI, New Haven, CT, USA) gel (at 100V for 2 hrs) .
  • DNA was transferred to nitrocellulose (Trans-Blot Transfer Medium, Bio-Rad, Richmond, CA, USA) and hybridized with 32P-labelled probe (1-2 X 10 5 cpm/ l) .
  • 2OX Sodium Chloride/Sodium Citrate (SSC) was used as transfer buffer and both nitrocellulose filter and 3 mm paper were wetted in 6X SSC prior to use.
  • nitrocellulose was subjected to prehybridization.
  • Formamide SIGMA, St Louis, MO, USA
  • the reactions were performed at 42 * C and the solution volumes were 20 ml for prehybridization and 10 ml for hybridization.
  • the washing was done in 2X SSC-0.5% SDS at room temperature for 15 minutes for the first two washings and in IX SSC-0.1% SDS at 65"c for 30 minutes with moderate shaking for the last two washings.
  • the filter was then exposed to a Kodak X- o at AR (XAR-5) Film (Eastman Kodak Company, Rochester, NY, USA) with an intensifying screen at -70 * C for 21 hours.
  • RNA of the reference cytopathogenic strain of BVDV (NADL)
  • both NCP and CP local isolates were amplified.
  • the electrophoresis showed a product 140 bases in length, corresponding to the length predicted from the position of the primers, and the specificity was further confirmed by Southern blot (see Figs. 4 and 5 and Figs. 4A and 5A) .
  • lane 1 is the same as for lane "M" for Fig.
  • lane 1 is the same as lane "M" for Fig. 1; lane 2 is dH 2 0; lane 3 is DEPC-treated NADL strain of BVDV; lane 4 is untreated (i.e. - li ⁇
  • lane 5 is from purified BVDV RNA.
  • lane 1 is the same as lane "M" for Fig. 1; lane 2 is from 0.1 ⁇ g of plasmid DNA (pBV4p80) without RT buffer; lane 3 is from 0.1 ⁇ g of plasmid DNA (pBV4p80) with RT buffer; and lane 4 is dH 2 0.
  • the amount of PCR product has been increased in the presence of the RT buffer.
  • the sensitivity of the inventive procedure for virus detection was documented in the experiment, where conventional virus isolation technique and PCR were performed on the identical dilutions of the reference NADL strain of BVDV.
  • lane 1 is the same as lane N M n for Fig. 1; lane 2 is from dH 2 0; and lanes 3-10 respectively are from dilutions of 10 "10 to 10 "3 of NADL strain of BVDV.
  • Fig. 10 Similar sensitivity of detection of BVDV genome from cell culture fluid is documented in Fig. 10, where 10 "2 TCID 50 yielded detectable PCR product, which was enhanced to 10 ⁇ 3 by Southern blot analysis (not shown) .
  • RNA extraction mehtod were compared in terms of their sensitivity of BVDV detection.
  • a modified extraction method of Chourczynski et al. (Analytical Biochemistry, 1987, 162, 156-159) was used. Briefly, two hundred microliters of serum was mixed in 2 volumes of the denaturing solution (4M guanidinium thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sarcosyl, 0.1M 2-mercaptoethanol) . Extraction was achieved by addition of 0.1 volume of 2 M sodium acetate (pH 5.2), 1 volume of water-saturated phenol, and 0.2 volume of chloroform-isoamyl alcohol (49:1).
  • the mixture was vigorously vortexed, chilled on ice for 15 minutes, and centrifuged to separate the phases at li,000x g for 20 minutes at 4 C.
  • the aqueous phase was removed, and the RNA was precipitated with a 0.3 volume of the denaturing solution and 1 volume of isopropanol, 15 -20 C for at least 1.5 hour.
  • the RNA pellet was again dissolved in the denaturing solution and precipitated with the isopropanol as above.
  • the final RNA pellet was resuspended in 0.1 ml of DEPC treated distilled water.
  • the BVDV was detected in the 10 "2 TCID 50 dilution by agar gel electrophoresis (Fig. 10) and in the 10 "3 TCID 50 dilution by Southern blot (not shown) .
  • the lanes correspond to the lanes of Fig. 10, i.e. in terms of dilutions, ddH 2 0 and "M.M.”.
  • PCR his been previously used for detection of DNA virus 4,15 , and purified viral RNA 7 , and from the theoretical attributes, as well as from the practical experience (Fig. 3) , the method has the potential to be the virus detection system of the ultimate sensitivity.
  • a single copy of the target genomic sequence can be amplified.
  • the BVDV sequence was detected from the plasmid DNA, approximately two copies of the genome were detected (Fig. 3) , confirming the theoretical predictions of extreme sensitivity.
  • Fig. 3 there has been no report of PCR amplification starting with a complete RNA virus and without RNA extraction.
  • the prior art method for RNA extraction neutralizes the greatest benefit of PCR, namely its sensitivity. This is demonstrated in Figs. 10 and 10A.
  • RT-PCR amplification was performed from 10 fold serial dilutions of tissue culture fluid containing BVDV, in parallel by the inventive method and with the use of RNA extraction.
  • the inventive method shows 10 4 X increase in sensitivity.
  • RNA was not digested completely.
  • RNase inhibitors are those that are active at the temperature used for uncoating viral RNA.
  • such inhibitors are non-proteinaceous, since thse are more likely to be heat stable, but it is conceivable that a porteinaceous, heat stable inhibitor might be bound.
  • Preferred non-proteinaceous inhibitors used in the exemplified inventive method are DEPC and V-R.
  • proteinaceous RNase inhibitor may be added after deproteinization and after the temperature of the sample is decreased to below the protein-denaturing level, but not into the region of the strongest working temperature for RNases, i.e. 37 * C, as a safety precaution to inactivate any RNases that might survive the heat treatment with the said suitable RNase inhibitor.
  • RNA virus also referred to herein as “protein uncoating” or RNA virus
  • Proteinase K has often been employed, since it has the theoretical capacity to degrade the virus protein coat, and at the same time to inactivate RNases, but even this treatment was unsuccessful in yielding the sufficient template for RT and PCR, as indicated by the failure to produce a specific PCR product.
  • Target viral RNA was rapidly degraded. The same happened when non-ionic detergents were used.
  • RNA extraction using guanidinium thiocyanate-phenol- chlorophorm was used to uncoat the target BVDV RNA, while inactivating RNases, but this laborious, time consuming method, resulted in some loss of RNA due to the incomplete recovery (See Figs. 10 and 10A) .
  • the latter procedure is suitable for extraction of large amounts of RNA, but is unsatisfactory for very small quantities of RNA, as may often be available in the case in virus detection in clinical samples. In these instances, the maximum yield of RNA is desirable, facilitating the most sensitive detection.
  • DEPC and V-R the relatively simple chemical nature of DEPC and V-R, the preferred chemicals, enables them to remain in operation at temperatures higher than typical proteinaceous inhibitors, and some amount may even outlast the deproteinizations and act in the cool-down period. This makes DEPC and V-R ideal for protection of virus RNA during its release from envelope and capsid proteins against the action of fast acting ubiquitous RNases.
  • BVDV is an enveloped, positive single stranded RNA virus, and serves here as a model virus, to demonstrate usefulness of the inventive procedure to achieve the above described objective for RNA viruses.
  • reverse transcription failed to take place, indicating degradation of the target RNA by the endogenous RNases.
  • the inventive protocol was developed, allowing the sustenance of the viral RNA throughout deproteinization.
  • the reference cytopathogenic, as well as cytopathogenic and noncytopathogenic field isolates of BVDV were then able to be amplified.
  • the method provides the solution to the problem encountered universally with PCR amplification of viral RNA for the purpose of sensitive detection, and can be applied in theory to PCR detection of all RNA viruses.
  • the method of sample treatment is most useful in connection with PCR, the sample treatment provides the basis for improvement of sensitivity of other current or future, non- amplifying or amplifying methods of detection of viral RNA, for example hybridization or Q-Beta replicase.
  • RNA from which RNA may be liberated by the procedure described include but are not limited to: Foot- and-mouth disease virus, vesicular exanthema virus, feline caliciviruses, equine en ⁇ ephalitides viruses, border disease virus, hog cholera virus, equine viral arteritis virus, newcastle disease virus, bovine respiratory syncytial virus, canine distemper virus, rinderpest virus, parainfluenza viruses, rabies virus, viral haemorrhagic septicaemia virus, infectious haematopoietic necrosis virus, spring viraemia of carp virus, red disease of pike virus, all members of retroviridae; avian leukaemia viruses, avian reticuloendotheliosis viruses, lympho- prolipherative disease of turkey virus, feline leukaemia virus, bovine leukosis virus, visna-maedi virus, caprine arthritis encephalitis virus,
  • the inventive procedure accomplishes deproteinization and reverse transcription of an RNA virus without extraction of the target nucleic acid.
  • the product of reverse transcription (cDNA) can be used in the PCR directly without extraction.
  • PCR of the genomic RNA can be accomplished in an uninterrupted reaction sequence.
  • the extreme sensitivity of BVDV detection achieved proves that the described method preserves extremely well the target viral RNA, and thus it supports in an excellent way the amplification power of the polymerase chain reaction.
  • BVDV bovine virus diarrhoea virus
  • Serum was particularly suitable as a sample of choice for BVDV, because of the absence of "contaminating nucleic acids" serving as a possible source of nonspecific templates for primers * Similarly it can reasonably be predicted that all RNA viruses found in the serum, buffy coats, and as well as in suspensions made of solid tissues could be detected by the method.
  • the method of the present invention has also been successfully applied in experiments for the identification of HIV in laboratory and clinical samples. Culture supernatants of 5 HIV isolates and five sera from seropositive patients were included. HIV was isolated from the patient's peripheral blood mononuclear cells (PBMCs) by cocultivation with normal umbilical cord PBMCs using standard procedures: Levy, J.A., and Shimabukuro, J. (1985) , "Recovery of AIDS-associated retroviruses from patients with AIDS or AIDS-related conditions and from clinically healthy individuals", J. Infect. Dis. 152:734- 738.
  • PBMCs peripheral blood mononuclear cells
  • HIV production was indicated by the detection of the viral core protein p24 in the culture supernatant fluid in a solid-phase immunoassay (Abbott Laboratories, North Chicago, 111.). HIV-seropositivity was ascertained using Biotech/Dupont HIV enzyme-linked immunoassay and Western Blot kits for HIV antibodies testing, as previously described (Schlect III, W.F., Lee, S.H.S., Cook, J., Rozee, K.R. , and Macintosh, N. (1989) , "Passive transfer of HIV antibody by Hepatitis B immune globulin", JAVMA 261:411- 413.
  • FIG. 8 The results of agar electrophoresis of the treated samples are shown in Figure 8.
  • a radiograph of a southern blot of the gel of Figure 8 is shown in Figure 9.
  • the probe used in making the southern blot analysis was prepared from the PCR product of sample no. 1 (Virus strain Al) : the band in lane 1 of the gel of Figure 8 was cut out, melted, eluted and labelled with P32 by the nick translation method. The size and specificity of the latter PCR product was confirmed by the presence of the Hae III restriction site.
  • IPNV pancreatic necrosis virus
  • IBDV is a different virus in the Birnaviridae family; ddH 2 0 is double distilled H 2 0. a The marker DNA fragment is pBR322 digested by Hinf I.
  • Novel primers used were as follows: 5'-GAAAGAGAGTTTCAACGTTA-3• and 5'-TTGTTTGTTAGAAAGGGCTT-3' ; these were synthesized at the DNA synthesis laboratory of the University of Calgary, and encompass an area of 93 nucleotides.
  • Lanes 3-12 correspond to lO "1 - 10 4 tissue culture infectious doses respectively of the JASP strain, and were subjected to the inventive method of detection using PCR. Lanes 1 and 2 are for the molecular marker (pBR322 digested by Hinf I) and ddH 2 0 (negative control) respectively. Detection in lane 4 of the Southern blot was achieved.
  • FIPV and FECV Experiments The method of the present invention was also applied successfully in experiments for the identification of feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV) . These are illustrative members of the family coronaviridae.
  • the results of RT-PCR applied to samples containing strains of FIPV and FECV are shown in Figure 14. This is a gel electrophoresis image showing FIPV detection at the 508 base pair marking and FECV detection at the 270 base pair marking.
  • the molecular marker in lane 1 was the DNA fragment resulting from Hinf I digestion of pBR322, and lane 2 is a ddH 2 0 negative control.

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Abstract

On décrit un procédé de détection d'un virus à ARN dans un échantillon de cellules tissulaires ou de fluide, par exemple un échantillon clinique suspecté de contenir le virus. Le procédé consiste à chauffer l'échantillon jusqu'à une température suffisante pour enlever l'enveloppe protéique du virus à ARN en présence d'un inhibiteur d'enzyme RNase actif à ladite température, tel que le diéthylpyrocarbonate (DEPC). L'ARN sans enveloppe est de préférence davantage protégé contre l'activité destructrice de la RNase lorsque l'échantillon est refroidi, au moyen de l'addition d'un inhibiteur protéique de RNase, tel que RNadeTM ou RNasinTM, lorsque la température de l'échantillon est inférieure à la température de dénaturation de l'inhibiteur. L'ARN peut alors être détecté, par exemple par transcription inverse (TI) pour former un ADNc, puis on applique le procédé de réaction en chaîne de polymérase (RCP) pour amplifier la quantité d'ADNc jusqu'à des niveaux détectables. La détection de l'ADNc ainsi amplifié confirme la présence du virus à ARN dans l'échantillon clinique initial.
PCT/CA1992/000117 1991-03-22 1992-03-23 Procede de detection d'arn viral WO1992016661A1 (fr)

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CA2,038,839 1991-03-22
CA 2038839 CA2038839A1 (fr) 1991-03-22 1991-03-22 Methode de detection de l'adn viral

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4433194A1 (de) * 1994-09-17 1996-03-21 Meier Ewert Herbert Univ Prof Technik zur molekularbiologischen Typisierung von Virusvarianten
WO1999047709A2 (fr) * 1998-03-18 1999-09-23 Chinook Acquisition Corporation Procede d'identification d'agents anticancereux
EP1409506A1 (fr) * 2001-07-23 2004-04-21 The Board of Trustees of The Leland Stanford Junior University Methodes et compositions pour l'inhibition induite par l'arni de l'expression genetique chez des mammiferes
US9018179B2 (en) 2001-07-23 2015-04-28 The Board Of Trustees Of The Leland Stanford Junior University Methods and compositions for RNAi mediated inhibition of gene expression in mammals

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4433194A1 (de) * 1994-09-17 1996-03-21 Meier Ewert Herbert Univ Prof Technik zur molekularbiologischen Typisierung von Virusvarianten
WO1999047709A2 (fr) * 1998-03-18 1999-09-23 Chinook Acquisition Corporation Procede d'identification d'agents anticancereux
WO1999047709A3 (fr) * 1998-03-18 2000-01-06 Genquest Inc Procede d'identification d'agents anticancereux
US6413717B1 (en) 1998-03-18 2002-07-02 Corixa Corporation Methods for indentifying anti-cancer agents
EP1409506A1 (fr) * 2001-07-23 2004-04-21 The Board of Trustees of The Leland Stanford Junior University Methodes et compositions pour l'inhibition induite par l'arni de l'expression genetique chez des mammiferes
EP1409506A4 (fr) * 2001-07-23 2004-11-17 Univ Leland Stanford Junior Methodes et compositions pour l'inhibition induite par l'arni de l'expression genetique chez des mammiferes
EP2280070A1 (fr) * 2001-07-23 2011-02-02 The Board Of Trustees Of The Leland Stanford Junior University Méthodes et compositions pour l'inhibition induite par l'ARNi de l'expression génétique chez des mammifères
US9018179B2 (en) 2001-07-23 2015-04-28 The Board Of Trustees Of The Leland Stanford Junior University Methods and compositions for RNAi mediated inhibition of gene expression in mammals
US10517887B2 (en) 2001-07-23 2019-12-31 The Board Of Trustees Of The Leland Stanford Junior University Methods and compositions for RNAi mediated inhibition of gene expression in mammals
US10590418B2 (en) 2001-07-23 2020-03-17 The Board Of Trustees Of The Leland Stanford Junior University Methods and compositions for RNAi mediated inhibition of gene expression in mammals

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