CA2038839A1 - Method for detection of viral rna - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

There is disclosed a process for detecting an RNA virus in a sample, e.g. clinical, suspected of containing the virus. The process comprises treating the sample with heat for denaturing protein coating the RNA in the presence of a non-proteinaceous RNase enzyme inhibitor such as diethylpyrocarbonate (DEPC). RNA freed from the protein coat is 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 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

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The present invention relates to a simple procedure for treatment of samples containing or suspected of containing RNA
viruses, efficient recovery of the genomic RNA and detection of viral RNA by polymerase chain reaction or by other methods.
Polymerase chain reaction (PCR~ can amplify a single copy of DNA into detectable, often microgram quantities within hoursl.
Following reverse transcription of RNA, complementary DNA can also be amplified23. 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 viruses45, as well as for detection of the proviral DNA of retroYiruses6 (~NA
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 makes 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 tha 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 daproteinization 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 samples has been accomplished by the present inventors and is 2~3~3~

disclosed herein.
Currently availabl~ methods o~ deproteinization do not facilitate reproducible production of the PCR product: When the RNA
in a virus ~ample is not completely destroyed, the PCR is successful; when, on the other hand, the RNA is degraded, due to very little virus in the sample, the high content of RNases or extended exposure Qf the uncoa~ed RNA to the working temperature of RNases, the result of PCR is usually negative. 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 removing protein coating RNA of RNA virus in a liquid sample, which process comprises heating the sample with a non-proteinaceous RNase inhibitor, to denature the protein without substantially degrading the RNA.
Preferably, the non-proteinaceous RNasa inhibitor is diethylpyrocarbonate (DEPC) or vanadyl ribonucleoside (V-R). The concen~ration of DEPC is preferably in the range of from about .005%
to 5~ by volume of the sample, most preferably in the range of from about .01% to 1% by volume of the sample. The concentration of V-R is preferably in the range of from about 5mM to 50mM and mostpreferably in the range of from about lOmM to 25mM. Further preferably, the heating is to a temperature in the range of from about 90 C to about 100 C and for a duration of from about 3 to ~0 2 ~

minutes. 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. A highly specific heat labile proteinaceous RNase inhibitor such as RNasin~, which is harmless to the subsequent enzymatic reactions in RT and PCR, may be added when the temperature o~ the sample is lowered to just below the denaturation temperature of the inhibitor, which for RNasin i5 about 45 C, rather than at a lower temperature at which RNases may be more active. The overall result is an efficient deproteinization process with excellent protaction of the viral RNA, allowing efficient cDNA
synthesis by RT and PCR, as evidenced by the extreme sensitivity of the test.
In another embodiment, there is provided a process for deproteinizing RNA from RNA virus in a li~uid sample. The above described heating step is used followed by cooling the sample to a temperature below a denaturation temperature of a preselected proteinaceous RNase inhibitor, but above about 40 C. The preselected proteinaceous RNase inhibitor is 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. Pre~erably, the proteinaceous RNase inhibitor is RNasin~, RNade~ or Human Placental Ribonuclease Inhibitor (BRL, Gaithersburg, Md, U.S.A.) and is added to the sample when the sample temperature is b~low about 65 C and above about 40 C
and in a concentration in the range o~ from about 0.1 units to 20 units per ~1 of sample, most preferably about .5 to 4 units per ~1 2~3~
of sample. After addition of the proteinaceous RNase inhibitor, reverse transcription to form cDNA and then PCR to amplify the cDNA
may be performed.
In yet another embodiment, there is provided a process of treating a liquid clinical sample for detection of RNA virus, which process includes the above-described heating and cooling steps and then further treating the sampls, after addition of a preselected proteinaceous RNase inhibitor, for detection of RNA or a derivative thereof as an indication of the initial presence or absence of RNA
virus in the clinical sample. Praferably the sample is treated for detection of the RNA by a method including amplification of the amount of RNA or a derivative of the RNA. Further preferably such amplification is by PCRo PCR may be preceded by the step of treating the sample for reverse transcription of RNA and formation of cDNA.
The preferred application of the invention is to RNA
viruse~ such as BVDV and HID viruses, although the invention may be applied to a wide variety of RNA virusesO
In another embodiment of the invention wherein the process is used to detect BVDV in a clinical sample, a novel primer is used for the PCR steps, namely "BV06" having the following nucleotide sequence: (5' end)dCCAACCACCCTCCCGCT(3' end). The process preferably also includes, in the context of BVDV virus detection, use of the primer "BV05" having the following nucleotide sequence:
(5' end)dGCAGTCGTTCACCTCCA (3' end).
Description of Figures The figures attached illustrate preferred features of the inventive process.

2~3~3~

Figure 1 shows the results of an agar gel electrophoresis of EcoR I and Hind III digested plasmid pBV4p80 containing the nucleotide sequence 5644-79491 of BVDV genome.
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 polyacrylamide 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.

Detailed Description of the Invention The following materials and methods were used in the experiments supporting the present application:

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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 (~merican Type Culture Collection). Reading of the end point of titration of the noncytopathogenic strain was accomplished with the indirect fluorescent antibody me~hod, using bovine polyvalant BVDV antiserum and rabbit anti-bovine fluorescein labelled conjugate. The virus titters were calculated by the method of Viliet8. The titters of the NADL, CP and NCP BVDV viral stocks were 10563, 10436 and 1043 TCIDs~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 ~olume 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 nm and adjusted to 1 ug/~l with the above treated H20. The so isolated viral RNA was for use as a control template for PCR.
PlasmidpBV4p80encompassing nucleotide sequence 5644-79491 ~3~3~

of BVDV yenome was obtained from Dr. M.S. Collett (Molecular Vaccines Inc., Gaithersburg, MD. 20878, USA), and used to transform E. coll TB-1 by the calcium chloride procedurel1. The transformed E.Coli were grown in rich medium12, harvested and lysed in alkali solution followed by the equili~rium ultracentrifugation ~38,000 rpm, 40 hours, 70.1 Ti Rotor) as previously describedl2, to obtain purified plasmid DNA. After the digastion 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 TemperaturP, Bio-Rad, Richmond, CA, USA), Fig. ~. 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.
PCR Primer~ BV05 and BV06 were synthesized by the DNA
synthesis laboratory at the University of Calgary. BV05 contained the nucleotide sequence 5813-5829 of N~DL strain of BVDV, as previously established by Collett10. BV06 was complementary to the se~uence 5936-5952 (Table 1 and FigO 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 The following method was employed in demonstrating a preferred embodiment of the present invent.ion:

2 ~ 3 ~

Nine and a half microliters of a virus sample, 2.8 ~1 of 5 X RT buffer (BRL, Gaithersburg, MD, USA), 0.5 ~1 of 50 mM BV06 primer and 0.5 ~1 of 0.5% (34 ~M) DEPC in ethanol solution (or 1 ~1 of 20 mM vanadyl ribonucleo~ide) 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/~l 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 ~1 of 5 X
RT buffer, 2~1 of 2.5 mM of each of the deoxynucleotide triphosphate (dNTP) (SIGMA, St Louis, MO, US~) mixture and 100 u of Moloney Murine Leukaemia Virus Reverse Transcriptase (BRL, Gaithersburg, MD, USA) were added. The final reaction volume was 20 ~1. The reaction took place at 37C for 60 minutes.
Ten microliters of RT product were heated at 95 C for 5 minutes together with the equal volume of 10X PCR buffer (100 mM
Tris-HCl, pH 8.3, 500 mM XCl, 15mM MgC12 and 0.1% Gelatin), 74 ~1 of d H20 and 2 ~1 of 50 mM o~ 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 2 ~1 of 2.5 mM of dNTP mixture, 1 ~1 of 10% Triton X-100 (Schwarz/Mann Biotech, Cleveland, Ohio, USA) and 4 u of Taq DNA polymerase (BIO/CAN SCIENTIFIC, Mississauga, ontario, Canada) were added. To prevent evaporation, 100 ~1 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 PC~, rather than submitting it first to complementary DNA extraction.
Ten microliters of PCR product were electrophoresed in 3%
agarose (IBI, New Haven, CT, USA) gel, transfarred to nitrocellulose (Trans-Blot Trans~er ~edium, Bio-~ad, Richmond, CA, USA) and hybridized with ~2P-labelled probe (1-2 X 105 cpm/ml). The previously described procedure14, with the following modifications was used: 20X Sodium Chloride/Sodium Citrate (SSC) was used as transfer buffer and both nitrocellulose ~ilter and 3 mm paper were wetted in 6X SSC prior to use. After 17.5 hours of transfer, the nitrocellulose was subjected to prehybridiæation. Formamide ~SIGMA, St Louis, M0, 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 lX SSC-0~1% SD5 at 65 C ~or 30 minutes with moderate shaking for the last two washings. The filter was then exposed to a Kodak X-omat AR (XAR-5) Film (Eastman Kodak Company, Rochester, NY, USA) with an intensifying screen at -70 C for 21 hours.
In following the above method, 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 (Fig. 3). This translates into approximately 2 copies of the target sequence detected.
Under the inventive method, the RNA of the reference cytopathogenic strain of BVDV (NADL), as well as both NCP and CP

~3~

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). Addition of DEPC or V-R into the sample during dçproteinization played an important role in accomplishing PCR amplification of a complete BVDV
in a reproducible manner (Figs. 4, 4A).
Presence of the RT buffer did not have a negative effect on PCR reaction (Fig. 6). Actually, the amount of PCR product has been increased in the presence of the RT buffer.
The sensitivity o the inventive procedure for virus detection was documented in the experiment, where conventional virus isolation technique and PCR were per~ormed on the identical dilutions o~ the reference NAD~ strain of BVDV. Ten fold dilutions were divided into two parts, and virus isolation followed by fluorescent antibody test and PCR were perfo~med simultaneously.
The titter of the infactious virus was 10-563 while the PCR detected the BVDV specific sequences in the sample diluted 10-1 (Fig. 7).
PCR has been previously used for detection of DNA virus4l5, 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 o~ PCR amplification starting with a complete RNA virus, and without RNA extraction. The requirement ~3~3~

for RNA extraction neutralizes the greatest benefit of PCR, namely its sensitivity. We found that destruc~ion of the viral RNA during the deproteinization, i.2. by RNases, was limiting in the procedure, causing only intermittent success of PCR amplification. 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 always wants 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 non-proteinaceous RNase inhibitors was preferred for the detection of BVD virus (model RNA virus) reproducibly, with high sensitivity. Also praferred was the addition of the proteinaceous RNase inhibitor after the temperature was decreased below the protein-denaturing level, but not into the region of the strongest working temperature for RNases, i.e. 37 C. The proteinaceous inhibitor inhibits the RNA specifically, and it does not interfere with the action of reverse transcriptase and DNA
polymerase. Without these two procedural steps~ liberation of undamaged RNA was ineffiaient, and subject to great variation, making the whole process unsuitable for diagnostic use.
Several techniques are currently available for deproteinizationl6, but none was found suitable for the purpose of preparation of template RNA ~or RT and PCR amplificationO
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 thi~ treatment was unsuccessful in yielding the sufficient template for RT and PCR, as indicated by the 3 ~

failure to produce a speci~ic PCR product. Target viral RNA was rapidly degraded. RNA extraction, using guanidinium thiocyanate or phenol-chlorophorm could be used to liberate the target RNA, while inactivating RNases, but this is laborious, time consuming, and inevitably results in some loss of the RNA due to the incomplete recovery. The later procedure is suitable for extraction of large amounts of RNA, ~ut is unsatisfactory for very small quantities of RNA, as is the case in virus detection. In these instances, the maximum yield of RNA is desirable, facilitating the most sensitive detection.
Inspired by the work of other's with DNA virus17, who were 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 reverse transcriptase and DNA polymerase, we used the same treatment for bovine viral diarrhoea virus, an RNA virus. However, when such sample was subjected to RT and PCR, specific product was again 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 and V-R were employed, specific product was obtained reproducibly with high sensitivity (Figs. 4, 4A and 5, 5A).
This proves that the combination of heat deproteinization and addition of DEPC or V-R accomplished excellent protection of the target RNA. DEPC and V-R were previously employed in extraction of RNA from cellsl8 and RNA virusl9, but their use in heat deproteinization of an RNA virus, and ~ubsequent employment of this material in RT and PCR has not been reported. The relatively simple ~33~ 8 ~

chemical nature of these chemicals enables them to remain in operation at temp~ratures higher than proteinaceous inhibitors, and some amount may even outlast the deproteinizations (100C) and act in the cool-down period. This makes DEPC and V-R ideal for protection of virus RNA during its release ~rom envelope and capsid proteins against the action of fast acting ubiquitous RNases.
Another important problem encountered in an attempt to make the whole procedure simple, was the question of whether the product of reverse transcription could be directly us~d for the polymerase chain reaction without cDNA extraction. Residual ions from the RT
buffer could disrupt the PCR reaction. For this reason, the previous protocols used a cDNA extraction step prior to PCR
reaction. This concern was unfounded, since the presence of the RT
buffer actually increased the efficiency of PCR (Fig. 6), and the whole reaction (deproteinization, RT and PCR) would be thus achieved with one uninterrupted sequence of reaction steps.
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 objectiva for RNA
viruses. The currently available protocols for PCR ampli~ication of RNA, short of RNA extraction, failed to yield the specific product when whole virus particle~ 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 protocol was developed, allowing the sustenance of the viral RNA
throughout deproteinization. The reference cytopathogenic, as well as cytopathogenic and noncytopathogenic field isolates were ,t~

amplified.
The method provides the solution to the problem encountered universally with PCR amplification of RNA viruses, and can be applied in theory to PCR detection of all RNA viruses. Although the method of sample treatment is most use~ul in connection with PCR, the sample treatment provides the basis for impr~vement of sensitivity of other current (hybridization) or further (Q-Beta replicase and other) methods of detection of viral RNA.
The viruses from which RNA may be liberated by the procedure described, include but are not limited to: Food-and-mouth disease virus, vesicular exanthema virus, feline caliciviruses, equine encephalitides viruses, border disease virus, hog cholera virus, equine viral arteritis virus, newcastle disease virus, bovine respiratory syncytial virus, canine distemper virus, rinderpest virus, parainfluenza virusas, rabies virus, viral haemorrhagic septicaemia virus, infectious haematopoietic necrosis virus, spring viraemia of carp virus, red disease of pike virus, all members o~
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, measle virus, human T
cell leukaemia viruses (HTLVs), human immunodef~iciency viruses (HID), hepatitis A, C and D viruses.
The inventive procedure accomplishes deproteinization and 3 ~

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 pre~erves extremely well the target 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 all RNA viruses found in the serum, buffy coats, and as well as in suspensions made o~ solid tissues could be detected by the method.
It should be obvious to persons skilled in the art that the above-described embodiments are merely exemplary of the invention and that modifications and variations of such embodiments can be made without departing from the scope and spirit of the invention.

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Claims (22)

1. A process for removing protein coating RNA of RNA virus in a liquid sample, which process comprises heating the sample with a non-proteinaceous RNase inhibitor, to denature the protein without substantially degrading the RNA.

2. A process for deproteinizing RNA of RNA virus in a liquid sample, which process comprises:
heating the sample with a non-proteinaceous RNase inhibitor to denature protein coating the RNA without substantially degrading the RNA, cooling the sample to a temperature below a denaturation temperature of a preselected proteinaceous RNase inhibitor but above about 40 C:
and adding the preselected proteinaceous RNase inhibitor to the sample.

3. A process of treating a liquid clinical sample for detection of RNA virus, which process comprises:
heating the sample with a non-proteinaceous RNase inhibitor for denaturation of protein coating RNA of RNA virus without substantially degrading the RNA;
cooling the sample to a temperature below a denaturation temperature of a preselected proteinaceous RNase inhibitor but above about 40 C;
adding the preselected proteinaceous RNase inhibitor to the sample; and - Page 1 of Claims -further treating the sample for detection of RNA, or a derivative thereof, as an indication of the initial presence or absence of RNA virus in the clinical sample.

4. A process according to claim 1, 2, or 3, wherein the non-proteinaceous RNase inhibitor is diethylpyrocarbonate.

5. A process according to claim 1, 2, or 3, wherein the non-proteinaceous RNase inhibitor is vanadyl ribonucleoside.

6. A process according to claim 1, 2, or 3, wherein the said heating is to a temperature in a range of from about 90° C to 100° C
for a duration in a range of from about 3 to 20 minutes.

7. A process according to claim 2, or 3, wherein the proteinaceous RNase inhibitor is selected from the group consisting of RNasinTM, RNadeTM and Human Placental Ribonuclease Inhibitor and is added to the sample when the sample temperature is below about 65° C and above about 40° C and in a concentration in a range of from about .1 to 20 units per µl of sample.

8. A process according to claim 7, wherein the concentration range is from about .5 to 4 units per µl of sample.

9. A process according to claim 2, wherein after the proteinaceous RNase inhibitor is added to the sample, RNA of the sample is subjected to reverse transcription to form cDNA and the cDNA is then subjected to a polymerase chain reaction.

- Page 2 of Claims 2

10. A process according to claim 1, 2, or 3, wherein the RNA
virus is a strain of bovine viral diarrhoea virus.

11. A process according to claim 1, 2, or 3, wherein the RNA
virus is a strain of food-and-mouth disease virus, vesicular exanthema virus, feline caliciviruses, equine encephalitides 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 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, or hepatitis A, C and D viruses.

12. The process of claim 3, wherein the sample is treated for detection of RNA by a method including amplification of an amount of RNA or an amount of a derivative of RNA.

13. The process of claim 12, wherein the amplification is - Page 3 of Claims -achieved by means of a polymerase chain reaction.

14. The process of claim 13, wherein the polymerase chain reaction is preceded by a step of treating the sample for reverse transcription of RNA and formation of cDNA.

15. The process of claim 10, wherein the sample is treated for detection of the RNA using reverse transcription for formation of cDNA and then polymerase chain reaction for sufficient amplification of the cDNA for detection of the cDNA as a derivative of the RNA.

16. The process of claim 15, wherein primers BV05 and BV06 having the following nucleotide sequences are used in the polymerase chain reaction step:
BV05: (5' end) dGCAGTCGTTCACCTCCA (3' end) BV06: (5' end) dCCAACCACCCTCCCGCT (3' end).

17. The process of claim 4, wherein the diethylpyrocarbonate in the sample has a concentration in a range of from about .005% to 5% by volume of the sample.

18. A process according to claim 4, wherein the diethylpyrocarbonate in the sample has a concentration in a range of from about .01% to 1% by volume of the sample.

19. A process according to claim 5, wherein the vanadyl ribonucleoside in the sample has a concentration in a range of from about 5mM to 50mM.

- Page 4 of Claims -

20. A process according to claim 5, wherein the vanadyl ribonucleoside in the sample has a concentration in a range of from about 10mM to 25mM.

21. The process of claim 9 or 14, wherein buffer added to the sample for reverse transcription increases the amount of polymerase chain reaction product.

22. A primer nucleotide sequence as follows:
BV06: (5' end) dCCAACCACCCTCCCGCT (3' end) which is complementary to the nucleotide sequence 5936-5952 on NADL
strain of BVDV.

- Page 5 of Claims -