WO1992016661A1

WO1992016661A1 - Method for detection of viral rna

Info

Publication number: WO1992016661A1
Application number: PCT/CA1992/000117
Authority: WO
Inventors: Arnost Cepica; Jiang Jian Liu
Original assignee: University Of Prince Edward Island
Priority date: 1991-03-22
Filing date: 1992-03-23
Publication date: 1992-10-01
Also published as: AU1563292A; CA2038839A1

Abstract

There is disclosed a process for detecting an RNA virus in a tissue cell or fluid sample, e.g. clinical, suspected of containing the virus. The process comprises heating the sample to a temperature sufficient for protein uncoating the RNA virus, in the presence of an RNase enzyme inhibitor active at said temperature, such as diethylpyrocarbonate (DEPC). Uncoated RNA is preferably further protected from destructive RNase activity upon cooling of the sample by addition of a proteinaceous RNase inhibitor, such as RNadeTM or RNasinTM, when the temperature of the sample is below the denaturation temperature for the inhibitor. RNA can then be detected, for example by reverse transcription (RT) to form cDNA and then applying the polymerase chain reaction (PCR) method to amplify the amount of cDNA to detectable levels. Detection of cDNA so amplified confirms the presence of RNA virus in the initial clinical sample.

Description

METHOD FOR DETECTION OF VIRAL RNA 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) .

Polymerase chain reaction (PCR) can amplify a single copy of DNA into detectable, often microgram quantities within hours¹. Following reverse transcription of RNA, complementary DNA can also be amplified^2,3. This technology can thus be used for virus detection, and this can be accomplished with ultimate sensitivity. PCR has been successfully used for detection of DNA viruses^4,5, as well as for detection of the proviral DNA of retroviruses⁶ (RNA viruses that are, during their replication transcribed into DNA) . PCR amplification of purified viral RNA of RNA viruses has also been achieved⁷, but not from complete virions in which the RNA is surrounded by one or more layers of protein. The necessity of 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. Reverse transcription and PCR of bovine viral diarrhoea virus (BVDV) RNA from complete viral particles in serum and other clinical sampler has been accomplished by the present inventors and is disclosed herein. Also disclosed is such procedure when applied to human immunodeficiency virus (HIV) , feline infectious peritonitis virus (FIPV) , feline enteric coronavirus (FEVC) and infectious pancreatic necrosis virus (IPNV) .

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) .

In our experience, use of heat alone for virus deproteinization led to variable results with regards to virus RNA detection by RT/PCR. When the RNA in a virus sample was not completely destroyed, the PCR was successful; when, on the other hand, the RNA was degraded, due to very little virus in the sample, the high content of RNAses or extended exposure of the uncoated RNA to the working temperature of RNAses, the result of PCR was insensitive, or no detection of virus. Since there has been no report in the literature of PCR applied to a sample with complete RNA virus i.e. without extraction, it may be that others have encountered the same difficulties. Summary of the Invention

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. Preferably, the RNase inhibitor is non-proteinaceouε, further prefereably it is diethylpyr carbonate (DEPC) or vanadyl ribonucleoside (V-R) . The 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.

Use of a preferred concentration range of DEPC or V-R in a .sample will not inhibit subsequent reverse transcription (RT) and PCR if such are to be performed.

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. Preferably 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.

In the said cool down, 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. Preferably, the proteinaceous RNase inhibitor is RNasin™, RNade™ 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 μl of sample, most preferably about .5 to 4 units per μl of sample. After addition of the proteinaceous RNase inhibitor, reverse transcription to form cDNA and then PCR to amplify the cDNA may be performed.

The preferred application of the invention is to 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

The figures attached illustrate preferred features of the inventive process. Figure 1 shows the results of an agar gel electrophoresis of EcoR I and Hind III digested plasmid pBV4p80 containing the nucleotide sequence 5644-7949¹⁰ 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 3.

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.

Detailed Description of the Invention A. BVDV Experiments

(a) Preparation of Materials

Three strains of bovine viral diarrhoea virus (BVDV) were used: the reference cytopathogenic strain of BVDV (NADL) , B6356 cytopathogenic (CP) and B6356 noncytopathogenic (NCP) strains were local isolates from a single individual. 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 Viliet⁸. 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 (TCID₅₀)/ml respectively.

B6356 CP BVDV was used to prepare viral RNA according to Brock⁹ 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 H₂0 (d H₂0 ) 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 H₂0. The final concentration of viral RNA was determined by a Spectrophotometer (Ultrospec II, LKB, Cambridge, England) at 260 nirt and adjusted to 1 ug/μl with the above treated H₂0. 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 5644-7949¹⁰ 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 procedure¹¹. The transformed E.Coli were grown in rich medium¹², harvested and lysed in alkali solution followed by the equilibrium ultracentrifugation (38,000 rpm, 40 hours, 70.1 Ti Rotor) as previously described¹², to obtain purified plasmid DNA. After the digestion by EcoR I and Hind III, the 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. In 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 gel¹³ and used as a probe after labelling with ³²P-dCTP (Amersham Canada Limited, Oakville, Ontario, Canada) by nick-translation system (BRL, Gaithersburg, MD, USA) according to manufacturer's instructions. When the PCR was performed with the plasmid DNA containing BVDV as a cDNA sequence template, 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. In each of Figs. 3 and 3A lane "M" is the same as in Fig. 1, lane 1 is dH₂0 (no DNA) , and lanes 2- 9 are from serial dilutions of pBV4p80 DNA, amplified by PCR using BV05 and BV06 primers. In 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) .

Table 1. Sequences of PCR and RT Primers and Their Locations Primer 5' Sequence 3• Location of NADL

BV05 dGCAGTCGTTCACCTCCA 5813-5829

BV06 dCCAACCACCCTCCCGCT 5936-5952

(b) Experimental Method

A preferred embodiment of the present invention is disclosed under this heading.

Nine and a half microliters of a virus sample, 2.8 μl of 5 X RT buffer (BRL, Gaithersburg, MD, USA) preferably without Mg^**, 2 μl of 25 μM BV06 primer and 0.5 μl of 0.5% (34 μM) DEPC in ethanol solution (or 1 μl of 20 mM vanadyl ribonucleoside (V-R) ) were heated in a 1.5 ml microfuge tube at 100^*C for 10 minutes. The tubes were cooled to 45'C and 20 units (U) of RNAde™( 40 U/μl supplied by BIO/CAN SCIENTIFIC, Mississauga, Ontario, Canada) were added. Following vortexing for 1 minute, the samples were cooled on ice and another 1.2 μl of 5 X RT buffer preferably without Mg^**, 2μl of 0.1 M dithiothreitol, 4μl of 2.5 mM of each of the deoxynucleotide triphosphate (dNTP) (SIGMA, St Louis, MO, USA) mixture and lμl (200 u) of Moloney Murine Leukaemia Virus Reverse Transcriptase (BRL, Gaithersburg, MD, USA) were added. The final reaction volume was 20 μl. The reaction took place at 37^*C for 60 minutes. 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 Mσ*^* concentration on RT/PCR - BVDV. Tube # Final concentr. Final concentr. of Mα** in RT <?f Mg in PCF

1. 0.0 mM 2. 1.5 mM 10% PCR buffer (Bio/Can) 3. 3.0 mM 1.5 mM 4. 6.0 mM

5. 3-Q-O fflM

6. 0.0 mM

7. 5% RT buffer (BRL) 1.5 Mm 8. 3.0 mM 3.0 mM 9. 6.0 mM

UL 10.0 mM

11, o.o mM 0.0 M 12. 1.5 mM 1.5 mM 13, 3.0 mM 3.0 mM 14, 6.0 mM 6.0 mM

Ten microliters of RT product were heated at 95^βC for 5 minutes together with an equal volume of 10X PCR buffer (100 mM Tris-HCl, pH 8.3, 500 mM KC1, 15mM MgCl₂, 0.1% Gelatin and 1% Triton X-lθg, (Schwarz/Mann Biotech, Cleveland, Ohio, USA)), 71 μl of dd H₂0 and 2 μl of 25 μM of each of BV05 and BV06. This was followed by brief centrifugation in a microcentrifuge (15,800 g) , and by 10 minutes incubation .at 37^*C. Then 4 μl of 2.5 mM of dNTP mixture, and 5 u of Taq DNA polymerase (BIO/CAN SCIENTIFIC, Mississauga, Ontario, Canada) were added. To prevent evaporation, 100 μl of mineral oil (SIGMA, St Louis, MO, USA) was overlaid on top of the reaction solution. Finally, 30 thermal cycles were performed. Each cycle included 1 minute at 95^*C, 2 minutes at 55°C and 3 minutes at 72^°C. The 72'C incubation in the last cycle was extended to 15 minutes.

Note in the above-described preferred embodiment, the use of 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) . For Southern blot analyses, DNA was transferred to nitrocellulose (Trans-Blot Transfer Medium, Bio-Rad, Richmond, CA, USA) and hybridized with 32P-labelled probe (1-2 X 10⁵ cpm/ l) . The previously described procedure¹⁴, with the following modifications was used: 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. After 17.5 hours of transfer, the nitrocellulose was subjected to prehybridization. Formamide (SIGMA, St Louis, MO, USA) was added to final concentration of 50% in both prehybridization and hybridization solution. 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.

Under the inventive method, the RNA of the reference cytopathogenic strain of BVDV (NADL) , as well as 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) . In each of Figs. 5 and 5A, lane 1 is the same as for lane "M" for Fig. 1; lane 2 is dH₂0; lane 3 is from NCP BVDV; lane 4 is from CP BVDV; lane 5 is from NAD2 BVDV; and lane 6 is from purified BVDV RNA. Addition of DEPC or V-R into the sample during deproteinization played an important role in accomplishing PCR amplification of a complete BVDV in a reproducible manner (Figs. 4, 4A) . In each of Figs. 4 and 4A, lane 1 is the same as lane "M" for Fig. 1; lane 2 is dH₂0; lane 3 is DEPC-treated NADL strain of BVDV; lane 4 is untreated (i.e. - li ¬

no DEPC used) NADL strain of BVDV; and lane 5 is from purified BVDV RNA.

Presence of the RT buffer did not have a negative effect on PCR reaction (Fig. 6) . In Fig. 6, lane 1 is the same as lane "M" for Fig. 1; lane 2 is from 0.1 μg of plasmid DNA (pBV4p80) without RT buffer; lane 3 is from 0.1 μg of plasmid DNA (pBV4p80) with RT buffer; and lane 4 is dH₂0. Actually, 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. Ten fold dilutions were divided into two parts, and virus isolation followed by fluorescent antibody test and PCR were performed simultaneously on equal volumes. The highest dilution at which the infectious virus was detected was 10^"5*63, while the PCR detected the BVDV specific sequences in the sample diluted 10^"10 (Fig. 7) . In Fig. 7, lane 1 is the same as lane ^NMⁿ for Fig. 1; lane 2 is from dH₂0; and lanes 3-10 respectively are from dilutions of 10^"10 to 10^"3 of NADL strain of BVDV. Similar sensitivity of detection of BVDV genome from cell culture fluid is documented in Fig. 10, where 10^"2 TCID₅₀ yielded detectable PCR product, which was enhanced to 10^~3 by Southern blot analysis (not shown) .

The inventive method and a prior art 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. After centrifugation at ll,000x g for 20 minutes at 4 c, 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. With the use of the inventive method the BVDV was detected in the 10^"2 TCID₅₀ dilution by agar gel electrophoresis (Fig. 10) and in the 10^"3 TCID₅₀ dilution by Southern blot (not shown) . In Fig. 10A, the lanes correspond to the lanes of Fig. 10, i.e. in terms of dilutions, ddH₂0 and "M.M.". (c) Discussion

PCR his been previously used for detection of DNA virus^4,15, and purified viral RNA⁷, 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. When 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. However, 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 10⁴X increase in sensitivity. When a sample containing BVDV was not subject to RNA extraction, but directly incuded in the reaction, and heated for liberation of the RNA from the protein coat, positive RT-PCR results were obtained only occasionally and from those samples that contained high concentrations of virus particles. We found that destruction of the viral RNA during the deproteinization by RNases was the limiting step in the above procedure, causing only intermittent success of PCR amplification. Thus some product could be obtained occasionally even without use of DEPC or V-R during deproteinization and presumably in these instances the template RNA was not digested completely. However, in diagnostic situations one requires the maximum amount of undamaged virus genomes preserved, so that the probability of virus detection is maximized. Therefore, the inhibition of RNases during deproteinization, by suitable RNase inhibitors, was necessary for the detection of BVDV (model RNA virus) reproducibly, with high sensitivity. Suitable RNase inhibitors are those that are active at the temperature used for uncoating viral RNA. Preferably, 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. Optionally, 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.

Several techniques are currently available for deproteinization¹⁶ (also referred to herein as "protein uncoating" or RNA virus) , but none was found suitable for the purpose of preparation of template RNA for RT and PCR amplification. 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.

Other's¹⁷, have been able to achieve uncoating of DNA for the purpose of PCR simply with the use of high temperature (boiling for 10 minutes) , avoiding addition of any chemical potentially harmful to the nucleic acid or reaction enzymes. When the same treatment was used for bovine viral diarrhoea virus, an RNA virus, and the sample was subjected to RT and PCR, specific product was obtained irreproducibly, even when a relatively high quantity of the virus was initially present in the sample (as determined by the infectivity assay) .

When DEPC or V-R was employed, as preferred non- proteinaceous inhibitors for the heat deproteinization step, specific product was obtained reproducibly with high sensitivity (Figs. 4, 4A, 5, 5A, 7, 10 and 10A) . This shows that the combination of heat deproteinization and addition of suitable RNase inhibitors such as DEPC or V-R, accomplished excellent protection of the target RNA. DEPC and V-R were previously employed in extraction of RNA from cells¹⁸ and RNA virus¹⁹, but their or another RNase inhibitor's use in heat deproteinization of an RNA virus sample, and subsequent employment of such sample in RT and PCR has not been reported or hinted at. 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.

Another problem encountered was the question of whether the product of reverse transcription could be directly used for the polymerase chain reaction without cDNA extraction. Residual ions from the RT buffer theoritically could disrupt the PCR reaction. For this reason, the previous known protocols used in preparing for PCR, used a cDNA extraction step prior to PCR reaction. This concern was determined to be unfounded in applying one aspect of the inventive method, since the presence of the RT buffer actually increased the efficiency of PCR (Fig. 6) , and the whole reaction (deproteinization, RT and PCR) , as one aspect of the invention, was thus achieved with one uninterrupted sequence of reaction steps applied to a sample. 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. The currently available protocols for PCR amplification of RNA, short of RNA extraction, failed to yield the specific product when whole virus particles from tissue culture virus were utilized as templates. When the conventional deproteinization protocols were used, 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. Although 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.

The viruses 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 enσephalitides 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, equine infectious anaemia virus, mammalian and avian reoviruses, rotaviruses and orbi iruses, infectious bursal disease, infectious pancreatic necrosis virus, mumps virus, dengue virus, Japanese encephalitis virus, rubella virus, human respiratory syncytial virus, measles virus, human T cell leukaemia viruses (HTLVs) , human immunodefficiency viruses (HID) , hepatitis A, C and D viruses.

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. Thus 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. We used the method successfully for amplification of bovine virus diarrhoea virus (BVDV) from cell culture medium, serum, as well as from buffy coats and lymph node tissue suspension. 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. B. HIV Experiments

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. 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.

In these experiments the following samples were used: Sample nos. : 1. Virus Al 2. Virus A7 3. Virus A9B

4. Virus A10

5. Virus A25

6. Patient Serum LB 7. Patient Serum MG

8. Patient Serum MAC

9. Patient Serum SF

10. Patient Serum MAZ 11. CSF MAC

12. Water (ddH₂0)

13. Culture Medium (CM)

14. Negative Serum (NS)

The same inventive protocol was used in treating the above samples as for the BVDV samples discussed herein. Primers SK68 and SK69, respectively having the sequences from 5' to 3' ends as follows: AGCAGCAGGAAGCACTATGG and CCAGACTGTGAGTTGCAACAG; (see C-Y Oue et. al., Science, v. 239, pp. 295-297, esp. 296, January, 1988) were employed.

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. (Lanes M to 14 of Figure 8 correspond respectively to lanes M to NS of 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.

With respect to the negative line for the serum "LB", we note that this antibody positive serum was found virus negative. Since there is no independent equally sensitive test available for detection of HIV, we can not say whether the serum was in fact virus negative, or whether further optimization of the method would lead to a positive result.

C. IPNV Experiments

The method of the present invention has also been successfully applied in experiments for the identification of infectious pancreatic necrosis virus (IPNV) in tissue samples, as an illustration of the effectiveness of the method for detecting virions of the family Birnaviridae. In these experi e.rts the following samples were used:

Different tissue culture media containing IPNV strains of serotype A.

IBDV ^• Negative controls*

H₂0 (dd H₂OJ ; M.M. (Molecular marker)⁸

* IBDV is a different virus in the Birnaviridae family; ddH₂0 is double distilled H₂0. ^a The marker DNA fragment is pBR322 digested by Hinf I.

The same inventive protocol was used in treating the above samples as for the BVDV samples discussed herein. 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.

The results of agar gel electrophoresis are shown in Figure 12 and a Southern blot of such gel is shown in Figure 12A. All strains if IPNV in the samples were detected.

The sensitivity of the inventive method is shown in

Figure 13, parts A and B. Lanes 3-12 correspond to lO^"1 - 10⁴ 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 ddH₂0 (negative control) respectively. Detection in lane 4 of the Southern blot was achieved.

D. 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 ddH₂0 negative control.

Although the experiments conducted used strains of BVDV, HIV, IPNV, FIPV and FECV, it is reasonably predicatble to a person skilled in the art based on the results shown herein, that the inventive method will be useful in detecting other RNA viruses both within and outside the virus families represented by the viruses tested. Thus, the specific viruses tested and disclosed herein are representative of RNA viruses generally, and of the applicability of the inventive method to testing for the presence of RNA virus in a sample. References

¹ Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erhlich, H.A. , and Arnheim, N. (1985) Enzymatical amplification of beta-globin genomical sequences and restriction site analysis for diagnosis of sickle cell anaemia. Science. 230, 1350-1354.

² Chang, H.L., Zaroukian, M.H. and Esselman, W.J. (1989) T200 alternate exon use in murine lymphoid cells determined by reverse transcription-polymerase chain reaction. The journal of immunology. 143, 315-321.

³ Kinoshita, T. , Shimoyama, M. , Tobinai, K. , Ito, M. , Ito S.I., Ikeda, S., Tajima, K. , Shimotohno, K. and Sugimura, T. (1989) Detection of mRNA for the taxl/rexl gene of human T-cell leukaemia virus type I in fresh peripheral blood mononuclear cells of adult T-cell leukaemia patients and viral carriers by using the polymerase chain reaction. Pro.Natl,Acad.Sci.USA. 86, 5620-5624.

⁴ Hsia, K. , Spector D.H. , Lawrie J. and Spector S.A. (1989) Enzymatic amplification of human cytomegalovirus sequences by polymerase chain reaction. Journal of Clinical Microbiology. 27, 1802-1809.

⁵ Olive, D.M., Simsek, M. and Al-Mufti S. (1989) Polymerase chain reaction assay for detection of human cytomegalovirus. Journal of Clinical Microbiology. 27, 1238-1242.

⁶ Kwok, S., Mack, D.H., Mullis, K.B., Poiesz, B. , Ehrlich, G., Blair, D. , Friedman-Kien, A. and Sninsky, J.J. (1987) Identification of human immunodefficiency virus sequences by using in vitro enzymatic amplification and oligomer cleavage detection. Journal of virology. 61, 1690-1694.

⁷ Carman, W.F., Williamson, C. , Cunliffe B.A. and Kidd, A.H. (1989) Reverse transcription and subsequent DNA amplification of rubella virus RNA. Journal of Virological Methods. 25, 21-30.

⁸ Viliet, J.H. and Roussel, P., (1985) Estimating viral concentrations: a reliable computation method programmed on a pocket calculator. Computer methods and programs in biomedicine. 21, 167-172.

⁹ Brock, K.V. , Brian, D.A. , Rouse, B.T. and Potgieter, L.N.D. (1988) Molecular cloning of complementary DNA from a pneumopathic strain of bovine viral diarrhoea virus and its diagnostic application. Can J Vet Res. 52, 451-457.

¹⁰ Collett, M.S., Larson, R. , Gold, C. , Strich, D. , Anderson D.K. and Purchic, A.F. (1988) Molecular Cloning and nucleotide sequence of the pestivirus bovine viral diarrhoea virus. Virology. 165, 191-199.

¹¹ Maniatis, T. , Fritsch, E.F. and Sambrook, J. (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, pp. 250-251.

¹² Maniatis, T. , Fritsch, E.F. and Sambrook, J. (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, pp. 88-94.

^{13 '} Maniatis, T. , Fritsch, E.F. and Sambrook, J. (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, p.170.

¹⁴ Maniatis, T. , Fritsch, E.F. and Sambrook, J. (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, pp. 383-389.

¹⁵ Olive, D.M. , Simsek, M. and Al-Mufti S. (1989) Polymerase chain reaction assay for detection of human cytomegalovirus. Journal of Clinical Microbiology. 27, 1238-1242.

¹⁶ Richards, F.M. and Wyckoff, H.W. (1971) Bovine pancreatic ribonuclease. In: Boyer, P.D. ed. The Enzymes. Vol 4. Academic Press. San Diego, pp. 647-806.

¹⁷ Berger, S.L. and Kimmel, A.R. (1987) Methods in enzymology, Volume 152. Guide to Molecular Cloning Techniques. Academic Press, Inc., San Diego, New York, Berkeley, Boston, pp. 44-48.

¹⁸ Demmler, G.J., Buffone G.J. , Schimbor CM. and May R.A. (1988) Detection of cytomegalovirus in urine from newborns by using polymerase chain reaction DNA amplification. The Journal of Infectious Diseases. 158, 1177-1184.

¹⁹ C.G. Rosen and I. Fedorcsak, Studies on the action of diethyl pyrocarbonate on proteins. Biochimica et biophysica acta, 130, (1966) 401-405.

Claims

WHAT IS CLAIMED II:

1. 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 the RNA, in the presence of a RNase inhibitor which is active at the said temperature.

2. A process of treating a sample of water, tissue cells or fluid or biological material for detection of a RNA virus, which process comprises: heating the sample to a temperature sufficient for protein uncoating of the RNA virus, in the presence of a RNase inhibitor which is active at the said temperature, without substantially degrading the RNA; and further treating the sample, or derivatives of the sample, for detection of the RNA, or a derivative of the RNA, as an indication of the presence of absence of the RNA virus in the tissue cells or fluid.

3. A process according to claim 2 wherein after the said heating, the sample is cooled to a temperature below a denaturation temperature of a preselected proteinaceous RNase inhibitor but above about 40°C, and the said proteinaceous RNase inhibitor is added to the sample in an amount sufficient substantially to inactivate RNase if present in the sample without degrading the RNA.

4. A process according to claim 1 or 2 wherein the RNase inhibitor is non-proteinaceous.

5. A process according to claim 4 wherein the non- proteinaceous RNase inhibitor is diethylpyrocarbonate.

6. A process according to claim 4 wherein the non- proteinaceous RNase inhibitor is vanadyl ribonucleoside. 7. A process according to claim 1, or 2, wherein the said heating is to a temperature in a range of from about 60'C to about 100°C.

^'8. A process according to claim 1, or 2, wherein the 5 said heating is to a temperature in the range of about 90°C to about 100"c.

9. A process according to claim 3 wherein the proteinaceous RNase inhibitor is selected from the group consisting of RNasin™, RNade™ and Human Placental 0 Ribonuclease Inhibitor and is added to the sample when the sample temperature is below about 55"C and above about 40^*C. *

10. A process according to claim 9, wherein the proteinaceous RNase inhibitor concentration is from about 5 .5 to 4 units per μl of sample.

11. A process according to claim 2, wherein after the said heating, the sample is treated for reverse transcription of the RNA to form cDNA, the sample is then treated for amplification of the cDNA by a polymerase chain 0 reaction, and the sample is then tested for the presence of cDNA.

12. The process according to claim 11, wherein the cDNA may be detected by gel electrophoresis.

13. A process according to claim 1, or 2, wherein the 5 RNA virus is a strain of foot-and-mouth disease virus, vesicular exanthema virus, feline caliciviruses, equine encephalitides viruses, border disease virus, hog cholera virus, equine viral arteritis virus, influenza A, B, C viruses, newcastle disease virus, bovine respiratory G 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, equine infectious anaemia virus, mammalian and avian reoviruses, rotaviruses and orbiviruses, infectious bursal disease, infectious pancreatic necrosis virus, mumps virus, dengue virus, Japanese encephalitis virus, rubella virus, human respiratory syncytial virus, measles virus, human T cell leukaemia viruses, human immunodefficiency viruses, hepatitis A, C and D viruses, bunyamwera viridae, coronaviridae (infectious bronchitis virus, human coronaviruses, murine coronaviruses, porcine coronaviruses, transmissible gastro-enteritis virus, haemagglutinating encephalomyelitis virus, feline infectious peritonitis virus, feline enteric coronaviruses, canine and bovine coronaviruses), filoviridae, flaviviridae, polio virus, or plant RNA viruses.

14. A process according to claim 1, or 2, wherein the RNA virus is a strain of bovine viral diarrhoea virus.

15. A process according to claim l, or 2, wherein the RNA virus is a strain of human immunodeficiency virus.

16. A process according to claim 1, or 2, wherein the RNA virus is a member of the family Coronaviridae.

17. _^ A process according to claim 16 wherein the RNA virus is a strain of feline infectious peritonitis virus.

18. A process according to claim 16 wherein the RNA virus is a strain of feline enteric coronavirus.

19. A process according to claim 1, or 2, wherein the RNA virus is a member of the family Birnaviridae.

20. A process according to claim 19, wherein the RNA virus is a strain of infectious pancreatic necrosis virus.

21. A process according to claim 5, wherein the diethylpyrocarbonate in the sample has a concentration in a range of from about .00025% to .25% by volume of the sample.

22. A process according to claim 5, wherein the diethylpyrocarbonate in the sample has a concentration in a range of from about .0005% to .05% by volume of the sample.

23. A process according to claim 6, wherein the vanadyl ribonucleoside in the sample has a concentration in a range of from about .2mM to 8mM of the sample to be heated.

24. A process according to claim 6, wherein the vanadyl ribonucleoside in the sample has a concentration of about 2mM in the sample to be heated.

25. A process according to claim 11, wherein buffer added to the sample for reverse transcription is substantially free of Mg^**.