WO1997037040A2

WO1997037040A2 - Isolation and/or amplification of hepatitis c virus (hcv) nucleic acids from samples suspected to contain hcv

Info

Publication number: WO1997037040A2
Application number: PCT/NL1997/000167
Authority: WO
Inventors: Jaap Goudsmit; Marcellinus Gualbertus Hubertus Maria Beld; Cornelis Johannes Andreas Sol; Willem René BOOM
Original assignee: Akzo Nobel N.V.
Priority date: 1996-04-03
Filing date: 1997-04-03
Publication date: 1997-10-09
Also published as: WO1997037040A3; AU2180997A; NL1002781C1

Abstract

The invention relates to methods for easily separating single stranded (HCV) nucleic acid material from double stranded nucleic acid material in a sample containing both. By the right choice of at least one chaotropic agent, preferably a guanidine salt, at a selected concentration and other suitable conditions such as chelating agents, pH and the like it is possible to bind double stranded material to a solid phase such as silica particles, whereas single stranded material will not bind under those circumstances. By separating the silica particles from the sample the double stranded nucleic acid material is removed. It can easily be eluted from the silica particles. In a second step the single stranded HCV material may be bound to a solid phase by selecting a different set of conditions. The particles can again be separated from the sample and the single stranded material may now be eluted. For very efficient separations the process may be repeated. Following the separation of the two kinds of nucleic acid, either kind may be amplified. Methods of amplification are provided which do not need sequence data of the material to be amplified. In these methods a primer will be provided with an amplification motif and a random hybridization motif.

Description

ISOLATION AND/OR AMPLIFICATION OF HEPATITIS C VIRUS (HCV) NUCLEIC ACIDS FROM SAMPLES SUSPECTED TO CONTAIN HCV

The invention relates to the field of purification and amplification of nucleic acids from nucleic acid containing starting materials, especially from biological materials such as urine, faeces, sperm, saliva, whole blood, serum or other body fluids, fractions of such fluids such as

leucocyte fractions (buffy coats), cell cultures and the like.

Until recently isolation and/or purification of nucleic acids from complex mixtures as described above was a laborious, multi-step procedure. In EP 0389063, incorporated herein by reference, a simple and rapid purification of nucleic acid material from a complex mixture is disclosed. This procedure comprises treating the complex mixture, such as whole blood with a chaotropic agent in the presence of a nucleic acid binding silica solid phase material under conditions that allow for binding of all nucleic acid material to said solid phase and separating said solid phase from the mixture. The reference shows that both single stranded and double stranded nucleic acids are bound to the solid phase if present in a mixture. The reference also discloses amplification (PCR) of a certain nucleic acid with a known sequence, suspected to be present in a mixture.

Thus said reference teaches a simple and rapid

detection method for known nucleic acids suspected to be present in a sample.

In many cases the nature of the target nucleic acid (double stranded or single stranded) may not be known beforehand, or there may be many different targets necessary to be analyzed. In these cases the rapid but rather crude method described above may not be sophisticated enough and further separations of the crude material may be wanted. Fractionation of mixtures of double- (ds) and single- stranded (ss) nucleic acids (NA) into single- and double- stranded forms is frequently needed e.g. in the separation of labelled ss-NA probes from ds-hybrids, in the separation of in vitro transcripts from ds-DNA templates, and in the separation of genomic DNA from mRNA. Particularly it can also be needed in identifying the presence of an infectious agent such as HCV in a sample containing other (ds) nucleic acids. Currently, the separation of different kinds of nucleic acids can be accomplished by several techniques. Electrophoresis can be used to fractionate different forms of nucleic acids , because of differences in size and shape. Centrifugation takes advantage of differences in density, and more recently the technology of high-performence liquid chromatography (HPLC) has been applied to separate and purify single- and double-stranded DNA and RNA molecules.

RNA purified from eukaryotic cells by the currently most widely used procedure (1) appears to contain

significant amounts of genomic DNA, an adaptation which reduces genomic DNA contamination of the ss-RNA fraction has recently been described (2).

It is not possible to look at single stranded and/or double stranded material separately using the method of EP 0389063 because the method does not discriminate between the two. Thus there remains a need for a simple and rapid test for detecting the presence of single stranded material (from an infectious agent) in a sample containing double stranded material. This is particularly the case in the field of Hepatitis where there are different causative agents and agents associated therewith, which agents may be double stranded or single stranded, RNA or DNA. These agents may all be present in one sample together wist nucleic acid material of the host or they may be present as sole

causative agent or in any combination.

With the discovery of Hepatitis C Virus (HVC) in 1989 (5) the causal agent for most cases of non-A non-B (NANB) posttransfusion viral hepatitis was identified and has been studied since then intensively. As early as 1983 it was reported that a non-A non-B hepatitis virus, which in retrospect has probably been HCV, could interfere with the infection process of Hepatitis B Virus (HBV) in chimpanzees (6-8). Although the number of experimentally infected animals in these studies was very low, the results suggested that HBV expression was suppressed in the dual infections. During recent years there have been several reports about the effects of dual HBV and HCV infections in man. The results of these studies do not always point into the same direction. They vary from the suggestion of suppression of HCV replication through concurrent HBV infection (9), the coexistent replication of both viruses (10), to the

suppression and even usurpation of HBV, in terms of

expression and replication, by HCV (11-17). Hepatitis delta virus ( HDV) was initially described as a subviral agent that relies on HBV for propagation (18) and can effectively suppress HBV replication (19). The latter study was done when assays for HCV still had to be developed. In recent years it became evident that the replication of the HDV genome is independent of HBV but for transmission co- infecting HBV is important (20-22). The prevalences of HBV and HCV infections in Egypt are very high. To our knowledge there are no data on the prevalence of HDV in the Egyptian population. We argued that when we were able to analyze sera from Egyptians with a hepatic disease we could encounter a fairly high proportion of dual infections and possibly some triple infections, enabling us to study some aspects of the in vivo interactions. The only criteria we applied for patients as to suffer from a hepatic disease was an alanine aminotransferase (ALT) serum value equal to or higher than 45U/L. We tested 48 sera for the presence of HBV, HDV, HCV viral antigens and/or antibodies by serological tests and the presence of the HBV, HDV, HCV genomes by reverse transcriptase (RT) and Polymerase Chain Reaction (PCR).

The present invention therefore provides a method for separating causative agents of Hepatitis from each other and from host material by applying differential binding

properties of single stranded and double stranded materials to a solid phase under different conditions. In particular the invention provides a method for separating HCV RNA from a sample suspected to contain said RNA comprising contacting said sample with a first liquid comprising a chaotropic agent and a nucleic acid binding solid phase, whereby the first liquid has a composition such that double stranded nucleic acid binds to the solid phase and a substantial amount of single stranded nucleic acid does not and

separating the solid phase from the supernatant. In this embodiment the single stranded HCV nucleic acid material will be present in the supernatant where it can be directly detected or further purified or amplified.

Suitable circumstances to arrive at a separation of double stranded nucleic acids from the single stranded HCV material can be determined by the person skilled in the art.

Circumstances under which double stranded material binds to the solid material and single stranded material does not will vary, however important parameters to obtain such differential binding are the concentration of the chaotropic agent, which should roughly be between 1-10 M, preferably between 3-6 M and particularly about 5 M; the concentration of chelating agent, which in the case that EDTA is applied should be equal to or greater than 10 mM and preferably not higher than 1 M; the pH of the aqueous solution in which the separation is carried out should be above 2 when a thiocyanate is used as chaotropic agent and it should be below 10 because otherwise there is a risk that the ds material will become ss. The temperature at which the process is carried out seems to be non-critical, however, it is probably best to keep it between 4°C and 60°C. An

important aspect of the process is of course that the ds material remains double stranded during the separation.

Under the circumstances as disclosed above this will normally be the case if the ds nucleic acid is at least 50 bp long at 40% GC basepairs. The skilled artisan knows how this length may vary with lower or higher GC content. In Van Ness et al (3) and/or Thompson et al (4) it is shown that the whole process depends on intricate interactions between a.o. the factors mentioned above. Using this disclosure and the cited references the skilled artisan will be able to adjust the circumstances to his or her particular process.

Chaotropic agents are a very important feature of the present invention. They are defined as any substance that can alter the secondary, tertiary and/or quaternary

structure of nucleic acids. They should have no substantial effect on the primary structure of the nucleic acid. If nucleic acids are present associated with other molecules, such as proteins, these associations can also be altered by the same or different chaotropic agents. Many chaotropic agents are suitable for use in the present invention, such as sodium iodide, potassium iodide, sodium (iso)thiocyanate, urea or guanidinium salts, or combinations thereof. A preferred class of chaotropic agents according to the invention are guanidinium salts, of which guanidinium thiocyanate is most preferred.

By serendipity we found that ss -nucleic acids and thus HCV nucleic acid material did not bind to silica particles or diatomeous earth in the presence of buffer L11 (see examples), whereas ds nucleic acid did. Experiments with different circumstances showed that addition of Mg²⁺ or other positive (bivalent) ions to the unbound fraction was of great importance. The best results were obtained with a concentration of bivalent ion (Mg2⁺) about equal to the concentration of the chelating agent (EDTA).

The solid phase to be used is less critical. Important is that it should bind nucleic acids reversibly.

Many such materials are known, of which a number are silicium based, such as aluminium silicate and the like, preferably silica. Silica is meant to include SiO₂ crystals and other forms of silicon oxide, such as diatom skeletons, glass powder and/or particles and amorphous silicon oxide. The solid phase may be present in any form, it may even be the vessel which contains the nucleic acid mixtures or a part of such a vessel. It may also be a filter or any other suitable structure. Apart from silicium based materials other materials will also be suitable, such as

nitrocellulose (filters), latex particles and other

polymeric substances. A preferred form of the solid phase is a particulate form, which allows for easy separation of bound and free material, for instance by centrifugation. The particle size of the solid phase is not critical. Suitable average particle sizes range from about 0.05 to 500 μm.

Preferably the range is chosen such that at least 80, preferably 90 % of the particles have a size between the values just mentioned. The same holds true for the preferred ranges of which the average particle sizes are between 0.1 and 200 μm, preferably between 1 and 200 μm. The binding capacity of a given weight of the particles increases with decreasing size, however the lower limit of the size is when particles cannot easily be redispersed after separation through for instance centrifugation. This will be the case in starting material rich in nucleic acids containing many nucleic acids of a higher molecular weight. The particles and the nucleic acids may form aggregates in these cases. The person skilled in the art will be able to choose the right particle size for the particular application

envisioned. The formation of aggregates may be avoided by using fractionated silica or diatomaceous earth in a number of applications.

A further embodiment of the present invention is a method as disclosed above further comprising treating the supernatant containing the single stranded HCV nucleic acid material with a second liquid comprising a chaotropic agent and a second nucleic acid binding solid phase, whereby the second liquid has a compositon such that the resulting mixture of supernatant and second liquid allow for binding of the single stranded HCV nucleic acid material to the second solid phase.

This way the double stranded nucleic acid material is removed from the crude mixture in the first step and the single stranded nucleic acid is purified from the remaining still crude mixture in another single step. Both the double stranded material and the single stranded material are reversibly bound to the respective solid phases, so that they may be easily eluted from said solid phases to undergo further analysis or other treatments. A very useful further treatment is the amplification of the (double or single stranded) nucleic acid material.

Both types can be amplified, or both types may be converted into one another so that they can be amplified. The present invention provides in yet another embodiment a method for amplifying single stranded nucleic acid material comprising the steps of hybridizing the single stranded nucleic acid with primers and elongating the probes using an enzyme which adds nucleotides to the primer sequence using the hybridized single strand material as a template, whereby at least one primer comprises a random hybridizing sequence and an amplification motif.

The criteria for amplification are well known in the art. The length of suitable primers, suitable buffers, suitable melting temperatures for separating strands, suitable hybridization conditions can all be determined using standard handbooks in the field.

Of course the sequences which are exemplified can be varied without departing from the present invention. It is not so much important what sequence is used as an

amplification motif, as long as it is suitable for

hybridization and primer extension purposes. Suitable limits depend on the conditions which can be varied by the person skilled in the art. Usually primers will be at least 10 bases long and not much longer than 100 bases.

The amplification embodiments of the invention are exemplified using PCR (polymerase chain reaction). Other amplification methods are of course equally suitable.

The exemplified label (or tag) on the primers is DIG (digoxygenin). However other labels are available and well known in the art. The invention will now be explained in further detail in the following detailed description.

Separation / Isolation

MATERIALS AND METHODS

Source of nucleic acids.

Sera. Serum samples were obtained from patients

suffering from hepatitis and attending a general hospital in Cairo, Egypt. For this study only serum samples with an alanine aminotransferase (ALT) value equal or higher that the upper limit of normal (45 U/l) were chosen. Selection of the samples was at random. Sera were from 26 males, average age: 41 yr. (range 22-55) and 22 females, average age: 42 yr. (range 31-52).

Chemicals.

Guanidiniumthiocyanate (GuSCN) was obtained from Fluka (Buchs, Switzerland).

EDTA (Titriplex) and MgC12.6H2O were obtained from Merck (Darmstadt, Germany). TRIS was obtained from

Boehringer (Mannheim, Germany). The preparation of size- fractionated silica particles (silica coarse, SC) and diatom suspension has been described (11). Triton X-100 was from Packard (Packard Instrument Co., Inc., Downers Grove, I11).

Composition of buffers.

The lysis/binding buffer L6, washing buffer L2, and TE (10mM Tris.HCl, 1 mM EDTA; pH=8.0) have been described (27). 0.2M EDTA (pH 8.0) was made by dissolving 37.2 g EDTA

(Merck, Germany) and 4.4 g NaOH (Merck, Germany) in aqua in a total volume of 500 ml. Lysis/binding buffer L11 was made by dissolving 120 g of GuSCN in 100 ml 0.2M EDTA (pH=8.0). Binding buffer L10 was prepared by dissolving 120 g GuSCN in 100 ml 0.35M TRIS.HCl (pH 6.4); subsequently 22 ml 0.2M EDTA (pH 8.0) and 9.1 g Triton X-100 were added and the solution was homogenized; finally 11 g of solid MgCl₂-6H₂O was added. The final concentration of MgCl₂ in L10 is about 0.25M. L10 is stable for at least 1 month when stored at ambient temperature in the dark.

Fractionation of ds-NA and ss-NA by protocol R.

The procedure is outlined in Figure 1. A 50μl specimen (containing a mixture of NA-types in TE buffer) was added to a mixture of 900μl L11 and 40μl SC in an Eppendorf tube and was subsequently homogenized by vortexing. After 10 min. binding at room temperature, the tube was centrifuged (2 min. at approx. 10.000 x g) which resulted in a silica/ds-NA pellet ("initial silica pellet") and a supernatant

containing ss-NA.

To recover ss-NA forms (protocol R-sup), 900μl of the supernatant were added to a mixture of 400μl L10 and 40μl SC and ss-NA was bound during a 10 min. incubation at room temperature. The tube was subsequently centrifuged (15 sec. at approx. 10.000 x g), and the supernatant was discarded (by suction). The resulting pellet was subsequently washed twice with 1 ml of L2, twice with 1 ml ethanol 70% (vol/vol) and once with 1 ml acetone. The silica pellet was dried (10 min. at 56°C with open lid in an Eppendorf heating block) and eluted in 50μl TE buffer (10 min. at 56°C; closed lid). After centrifugation (2 min. at approx. 10.000 x g) the supernatant contains the ss-NA fraction.

To recover ds-NA forms (protocol R-pellet) from the initial silica-pellet, the remaining supernatant was

discarded, and the silica pellet was washed twice with L11 to remove unbound ss-NA. The resulting silica pellet was subsequently washed twice with L2, twice with ethanol 70%, once with acetone, dried and eluted as described above.

After centrifugation (2 min. at approx. 10.00.0 x g) the supernatant contains the ds-NA fraction.

In the complete procedure (which takes about one hour) for fractionation of NA by protocol R, only two Eppendorf tubes are used. Fractionation of genomic DNA and ss-NA.

Due to trapping of ss-NA into high-molecular-weight genomic DNA, protocol R as described above gives only low yields of ss-NA. This can be circumvented by first isolating total NA by protocol Y/D (27), which causes some shearing of the high-molecular-weight genomic DNA, sufficient enough to prevent trapping of the ss-NA. Total NA thus purified can subsequently be used as input for protocol R.

Gel electrophoresis.

In all experiments, NA was electrophoresed (8 to 10 V/cm) through neutral agarose slab gels containing

ethidiumbromide (1μg/ml) in the buffer system (40mM TRIS-20 mM sodium acetate-2mM EDTA adjusted to pH 7.7 with acetic acid; ethidium bromide was added to a concentration of 1μg/ml of buffer) described by Aaij and Borst (25).

Hybridization.

DNA fragments were transferred to nitrocellulose filters by the procedure of Southern and hybridized with [alpha-³²P]dCTP labelled pHC624 prepared by random labeling (Boehringer, Germany). Hybridization conditions were as described previously (29).

RESULTS

Comparison of different GuSCN-containing lysisbuffers with respect to the binding of different NA-types to silica particles revealed that only doublestranded forms were bound when using L11 (which is about 100 mM for EDTA) as binding buffer; on the other hand both double- and single-stranded forms were bound in binding buffer L6 (which is about 20 mM for EDTA) (Table 1). These observations formed the basis for the development of a protocol (Protocol R) for the

fractionation of single-stranded nucleic acids and double- stranded nucleic acids (Fig. 1)

Once double-stranded nucleic acid is bound by silica particles in L11, a brief centrifugation will separate the silica/ds-NA pellet from the supernatant containing the single-stranded forms. Addition of this supernatant to a mixture of silica particles and binding buffer L10 (which is about 250 mM for Mg²⁺) the binding of single-stranded nucleic acids to the silica particles is restored. Double-stranded and single-stranded forms can subsequently be purified by washing and eluting the silica-NA complexes (protocol R). Double-stranded nucleic acid is recovered from the initial silica-pellet (protocol R-pellet), whereas single-stranded forms are recovered from the initial supernatant (protocol R-sup).

For optimization of protocol R we performed

reconstruction experiments in which previously purified or commercially available, nucleic acids were mixed and

subsequently fractionated by protocol R. Fractionation of a mixture of double-stranded DNA and single-stranded DNA.

The fractionation of a ds-DNA/ss-DNA mixture, into double stranded- and single stranded forms is shown in

Figure 2. The recovery estimated from the band intensity of the ethidium bromide stained gel for ss-DNA was about 50%, the estimated recovery of ds-DNA in the range of 500 bp to 4, 6 kb was 80% -90% [similar recoveries were obtained for ds- DNA fragments in the range of 100-500 bp (not shown)], larger fragments were significantly sheared as noted before (27). At the level of detection by UV-illumination,

fractionation into ds- and ss-forms was complete.

Fractionation of a mixture of double-stranded RNA and single-stranded RNA.

Figure 3 shows the fractionation of a mixture of ds-RNA (human Rotavirus genome segments 1-11) and ss-RNA (phage MS2 RNA) into double stranded- and single stranded forms. The estimated recovery of ds-RNA and ss-RNA was at least 80%. At the level of detection by UV-illumination, fractionation into ds- and ss-forms was complete. Fractionation of a mixture of double-stranded DNA and single-stranded RNA.

In Figure 4 it is shown that ds-DNA can also

efficiently be separated from ss-RNA.

Again are the recoveries for both fractions at least 80%. Similar results were obtained when E.coli rRNA (23S and 16S) was used as ss-RNA input (not shown).

In the experiments described above, fractionation of ds- and ss-NA forms (as judged by visual inspection of band intensities after ethidiumbromide staining and UV

illumination) appeared to be complete. In order to establish the performance of the fractionation procedure for a mixture of ds-DNA and ss-RNA into ss- and ds-forms, NA purified by protocol R-sup from such a mixture was studied by Southern blotting and hybridization with a ³²P-labelled DNA probe, homologous to the ds-DNA used as input for fractionation. This experiment revealed that the ss-NA fraction contained less than 0,1% of the ds-DNA input (figure 5). Fractionation of a mixture of genomic DNA and single- stranded RNA.

When we investigated the separation of high-molecular- weight (genomic) dsDNA and ss-RNA by direct fractionation using E. coli as input for protocol R, it appeared that the ds-DNA fraction was heavily contaminated with rRNA (Fig. 6, lanes 6 and 7), and ss-RNA recovery was low (Fig. 6, lanes 8 and 9). This was likely due to trapping of RNA into high- molecular-weight (genomic) ds-DNA when silica/NA complexes were formed. On the other hand no genomic DNA was observed in the ss-RNA fraction. Total nucleic acid, which was first isolated using the standard protocol Y/D (28), and hereafter used as input material in protocol R showed significantly higher recoveries of the ss-RNA fraction (Fig. 6, lanes 2 and 5).

HCV nucleic acid purification.

HCV RNA, HDV RNA and HBV DNA were purified from

serum by methods as described herein. Briefly, 100 μl of serum was added to a 1.5 ml Eppendorf tube (Type

3810) containing 900 μl of L6 lysis buffer (see herein) and 20 μl of a silica suspension. The tube was

immediately vortexed. During an incubation of 10 min at room temperature, the nucleic acid in the specimens was allowed to complex with the silica particles. The tubes were subsequently vortexed again and centrifuged for 15 s at 10,000 rpm (24 - hole Hettich KG centrifuge), the supernatant was discarded and the silica-nucleic acid complexes were washed twice with 1 ml of buffer L2 (see herein), twice with 1 ml of 70% (vol/vol) ethanol and once with 1 ml of acetone and were dried at 56° C (10 min); the nucleic acids were then eluted at 56° C (10 min) in either 50 1 of 10 mM Tris.HCl 0.1 mM EDTA pH

7.5 (TE) for HCV detection or in 25 ul TE for HDV and

HBV detection (see below).

The preparation of size- fractionated silica, lysis buffer L6 and wash buffer L2 has been described in detail by Boom et al. (27). In short, lysis buffer L6 was made by dissolving 120 g of GuSCN in 100 ml of 0.1 M Tris. HCl (pH 6.4) and, subsequently, 22 ml of 0.2 M EDTA (pH 8.0) and 2.6 g of Triton X-100 were added.

Washing buffer L2 was made by dissolving 120 g of GuSCN in 100 ml of 0.1 M Tris.HCl (pH 6.4).

Amplifications MATERIALS AND METHODS

Chemicals and enzymes

EDTA, KCl, MgCl₂.6H₂O, NaCl and tri-Sodium citrate dihydrate were obtained from Merck (Darmstadt, Germany).TRIS and BSA were obtained from Boehringer (Mannheim, Germany). Triton X-100 was obtained from Packard (Packard Instruments Co., Inc., Downers, I11, USA). Sodium Dodecylsulfate (SDS) was obtained from Serva (Heidelberg, Germany).

The dNTP's and Dextran Sulphate were obtained from Pharmacia (Uppsala, Sweden).

The chemicals used in protocol R have been described herein.

Reverse transcriptase Superscript II was purchased from Life Technologies (Gaithersburg, Maryland, USA). DNA polymerase Sequenase 2 was obtained from Amersham (United

Kingdom). Ampli-Taq DNA polymerase was obtained from Perkin Elmer (Norwalk, USA). RNAse H was obtained from Boehringer (Mannheim, Germany). Salmon sperm DNA was obtained from Sigma (St. Louis, USA).

Composition of buffers and solutions.

The preparation of the buffers used in protocol R have been described herein, except that the lysis buffer and washing buffers (L10, L11, and L2) used in protocol R for the isolation of nucleic acids were filtered through a column packed with Diatoms (27) in order to remove any endogenous nucleic acids in the lysis buffer and washing buffers.

The 10 x reverse transcription buffer (CMB1) consists of 100 mM Tris.HCl (pH 8.5), 500 mM KCl and 1% Triton X-100.

The 10 x PCR buffer consists of 500 mM Tris.HCl (pH 8.3), 200 mM KCl and 1 mg/ml BSA.

The elution buffer Tris/EDTA (TE, pH 8.0) consists of 10 mM Tris.HCl (pH 8.0) and 1 mM EDTA (pH 8.0). Primers and probes

The primer used for reverse transcription of HCV RNA was HCV-6: 5'ACC.TCC 3' (nt 319-324, nt numbering according to (23). The anti sense PCR primer for HCV was RB-6B: 5' ACT .CGC.MG.CAC.CCT.ATC.AGG 3'(nt 292-312) and the sense PCR primer was RB-6A:5 ' GTG . AGG . AAC . TAC . TGT . CTT . CAC . G 3 '(nt 47- 68). The oligonucleotide RB-6P: 5 ' TTG.GGT.CGC.GM.AGG.CCT.TGT. GGT.ACT.G 3'(nt 264291) was labelled at the 5-end with digoxiginine and was used as a probe in hybridization experiments to determine the specificity of PCR products. The HCV oligos were specific for the 5' untranslated region of the HCV genome. The primer used for reverse transcription of HDV RNA was E21: 5 'CCT.CGA.GM.CM.GM.GM.GC 3' (nt

12571238, nt numbering according to (26). The primers used for HDV PCR were E21, the primer also used for reverse transcription, and E22: 5 ' CGG.CTG.GGC.MC.ATT.CCG.AG 3 (nt 718-737). The plasmid pG4Z(D3) was a generous gift of John Taylor (Fox Chase Cancer Center, Philadelphia) and it contains 3 complete cDNA copies of the HDV genome in tandem, cloned in the Eco RI site of pGem4Z (Promega). Through digestion of pG4Z(D3) with Bgl II , isolation of the 4.4 kb fragment from an agarose gel, ligation and transfection into E.coli C 600, plasmid pG4Z(D1) was obtained containing a single cDNA copy of the HDV genome. pG4Z(Dl) was labelled with digoxigenine-dUTP by random primer DNA synthesis according to the manufacturer's protocol (Boehringer

Mannheim GmbH) and was used as a probe in hybridization experiments to determine the specificity of the PCR product. The primers used for HBV PCR (core region) were LBL: 5

' GCG.GAT.CCG.TGG.AGT.TAC.TCT.CGT . '1'1'1'.TGC .3 ' (nt 1939- 1960, nt numbering according to (24) and RAL : 5GCA.AGC . '1-1- 1' . CTA.ACA.ACA.GTA.GTT.TCC. GG 3'(nt 2332-2352); the

underlined parts of the primers represent added restriction enzyme linkers. The plasmid PCP10 contains in tandem two complete copies of the HBV genome inserted at the Eco RI site of pBR 322 and was a generous gift of R. Heytink

(Erasmus University, Rotterdam). pHBt-III was derived from PCP10 by Eco RI digestion, isolation of the HBV Eco RI linear from an agarose gel and ligating it into the Eco RI cleaved high copy plasmid pHC 624 (28). pHBtlll was labelled with digoxigenine-dUTP by random primer DNA synthesis according to the manufacturer s protocol (Boehringer

Mannheim GmbH) and was used as a probe in hybridization experiments to determine the specificity of PCR products.

RT-PCR of HCV.

Reverse transcription was performed in a 25 I reaction volume containing 20 U of RNase inhibitor (Promega Biotec, Madison, Wis.), 67 mM Tris.HCl pH 8.8, 17 mM Ammonium

Sulfate, 1 mM 3 -mercapto-ethanol, 6 μM EDTA pH 8.0 and 0.2 mg/ml Bovine Serum Albumine (BSA) (Boehringer), 6 mM MgCl₂, 25 ng of primer HCV- 6, 0.6 μl of 25 mM (each)

deoxynucleoside Triphosphates, 11.5 μl of the 50 μl eluate from the nucleic acid purification (see above), and 200 U of superscript reverse transcriptase I (GIBCO-BRL,

Gaithersburg, U.S.A.). The mixture was incubated at room temperature for 5 min and then at 37°c for 60 min. Reverse transcriptase was denatured by incubation for 5 min at 95°C (29) The PCR was performed in a 50 μl volume containing, 2.5 U of Taq polymerase (Perkin Elmer Cetus), 50 mM Tris.HCl pH 8.3, 20 mM KCI, 1,2 mM MgCl₂ and 1 mg/ml BSA), 12.5 μl of the RT reaction mix, 200 uM of each deoxynucleoside

triphosphate and 100 ng each of primers RB-6A and RB-6B.

Samples were denaturated at 95°C for 5 min and subjected to 35 rounds of thermal cycling in a DNA thermal cycler (type 480; Perkin Elmer Cetus). A cycle consisted of denaturation for 1 min at 95°C, annealing for 1 min at 55°C, and

extension for 2 min at 72°C. After the cycling program the samples were incubated for 10 min at 72°C. All samples analyzed for HCV RNA by the RT-PCR were performed in

duplicate on different days.

RT-PCR of HDV and PCR of HBV.

HDV RNA and HBV DNA are co-purified by the method described above. However, contrary to HDV RNA, HBV DNA is poorly eluted from the silica particles in TE at 56°C presumably because of co-purification of the protein which is covalently bound to the HBV DNA genome. 7 μl of the eluate (25 μl) was taken from the tube for HDV RT-PCR. To the remaining eluate and the silica particles 18 μl TE containing 200 ng proteinase-K/ml was added and the tube was incubated for 10 min at 56°C. After heating at 95°C for 10 min to inactivate the proteinase and centrifugation (1 min at 12,000 x g), 20 μl of the elate was used for HBV PCR.

Annealing of the primer with template HDV RNA was performed in a mixture containing 7 μl eluate, 1 ng of antisense primer E21, 50 mM Tris.HCl pH 8,3, 40 mM KCI, 6 mM MgCI₂, 10 mM dithiothreitol, and 150 μM (each)

deoxynucleoside triphosphates (dNTPs). After heating to 80°C for 1 min and cooling to room temperature 12 U of AMV reverse transcriptase (Boehringer Mannheim GmbH) was added and RT was performed for 1 hr at 42°C in a total volume of 10 μl. Reverse transcriptase was denatured by incubation for 5 min at 95°C (27). PCR was performed in a reaction mixture of 100 μl containing 200 ng primer E21, 200 ng primer E22, 50 mM Tris.HCl pH 8.3, 20 mM KCI, 1 mM MgCI2, 0.1 mg BSA, 1.5 U Taq polymerase, 200 uM of each deoxynucleoside

triphosphate and 10 μl of the RT reaction. After incubation for 5 min at 95°C the sample was subjected to 35 cycles of amplification. The same cycling program was used as for HCV.

The HBV PCR was performed in a 100 μl reaction mixture containing 200 ng primer LBL, 200 ng primer RAL, 50 mM Tris.HCl pH 8.3, 20 mM KCI, 1 mM MgCI2, 0.1 mg BSA, 1.5 U Taq polymerase, 200 uM of each deoxynucleoside triphosphate and 20 μl of the eluate. After incubation for 5 min at 95°C the sample was subjected to 35 cycles of amplification. The same cycling program was used as for HCV and HDV.

Agarose gel electrophoresis and Southern blotting.

Upon completion of the amplification reactions, 10 ul of each reaction was analyzed by electrophoresis through horizontal 2 % agarose slab gels in the buffer system described by Aay and Borst (25) containing ethidium bromide (1 μg/ml). DNA was transferred from the gel onto

nitrocellulose (Bio-Rad, USA) filter essentially as

described by Southern 4 The transferred DNA was cross -linked to the filter by incubation for 2-3 hr at 80°C.

Labelling and hybridization.

The HBV and HDV containing plasmids were labelled with digoxigenine-dUTP by random priming (Boehringer Mannheim GmbH). Nitrocelluse filters were pretreated as described previously (26). Hybridization and post hybridization washings were as previously described. Detection of

digoxigenine was done according to the protocol provided by the manufacturer of the kit (Boehringer Mannheim GmbH).

Statistics.

Statistical significance tests were done using the two tailed Fischer exact test. RNA template production.

To construct an HCV-RNA transcription vector, HCV sequences from nt 47 to 1032 were cloned after RT PCR into the pSP 64 (poly A) vector (Promega, Madison, Wis). The presence of the right insert was confirmed by DNA sequence analysis. The construct was named pMOZ.1.HCV.

HCV template RNA was transcribed in vitro from

PMOZ.1.HCV. Briefly, 5 ug of plasmid DNA was linearized by Eco-RI and then incubated with 50 U of SP6 RNA polymerase for 2 hr. at 37°C in the presence of 500 uM (each)

ribonucloside Triphosphate (GTP, ATP, UTP, and CTP), 100 U of RNAsin, 10 mM dithiothreitol, 40 mM Tris-Hcl (PH 7.5), 6 mM Mgcl2, 2 mM spermidine, and 10 mM NaCl in a total reaction volume of 100 ul. After the transcription reaction, the DNA template was degraded by two rounds of digestion with RNase free DNase (Boehringer) for 30 min at 37°c with 10 U of enzyme. Upon completion of the digestion, 2 rounds of extraction using phenol-chlorophorm-isopropyl alcohol, followed by ethanol precipitation were done. The HCV RNA transcripts, which contained a poly (A) Tail, were further purified on an oligo dT cellulose column. The RNA

concentrations were determined spectrophotometrically by UV A260. An aliquot was analyzed by agarose gel electrophoresis to assess its integrity. HDV RNA was synthesized and except for the oligo-dT cellulose selection, purified in a similar way as described above for HCV RNA. pG4B (D1) linearized with Hind III was the substrate for SP6 RNA polymerase.

Sensitivity of the (RT)-PCR assays.

In vitro synthesized HCV and HDV RNAs and the Eco RI linearized HBV plasmid pHBt-III were diluted into TE buffer to a concentration of approximately 10⁶ copies/μl and stored at -20°C. Serial tenfold dilutions into H₂O of these stock solutions were made just prior to the RT-PCR or PCR

reactions. One hundred copies were routinely detected.

Southern hybridization and chemiluminescent

detection.

The electrophoresis of the PCR products, the transfer of the DNA onto positively charged nylon membrane

(Boehringer Mannheim GmbH) and hybridization with the probe were done as previously described. The detection of the hybridized digoxigenine labelled probe was done with the DIG Luminescence Detection Kit according to the protocols recommended by the supplier of the kit (Boehringer Mannheim GmbH).

Comparative example

The methods according to the invention were compared with commercial methods using antibodies.

Enzyme-Linked Immunosorbent Assay (ELISA) kits were, provided by Abbott Laboratories (Chicago, Illinois), or Sorin Biomedica (Sallugia, Italy) were used for the

detection of HBsAg,HBeAg, Anti-HBe, IgG anti-HBc and IgM anti-HD. Antibodies to HCV structural and nonstructural synthetic peptides were detected with the Enzyme Immunoassay kit SP-NANBASE C-96 provided by General Biologicals Corp. (Hsin Chu, Taiwan). The presence of antibodies to HCV was also determined using a recombinant immunoblot assay (RIBA- 3, Chiron Corporation, Emeryville, CA). Tests were performed according to the manufacturer's instructions. The results are presented in tables 2-7. The sera were obtained from the earlier identified group.

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Claims

1. A method for separating HCV RNA from a sample suspected to contain said RNA comprising contacting said sample with a first liquid comprising a chaotropic agent and a nucleic acid binding solid phase, whereby the first liquid has a composition such that double stranded nucleic acid binds to the solid phase and a substantial amount of single stranded nucleic acid does not and separating the solid phase from the supernatant.

2. A method according to claim 1 further comprising treating the supernatant containing the single stranded nucleic acid material with a second liquid comprising a chaotropic agent and a second nucleic acid binding solid phase, whereby the second liquid has a compositon such that the resulting mixture of supernatant and second liquid allow for binding of the single stranded nucleic acid material to the second solid phase.

3. A method according to claim 1 or 2, whereby the first liquid comprises at least 100 mM EDTA and comprising a guanidinium salt, preferably guanidinium thiocyanate as a chaotropic agent.

4. A method according to claim 1, 2 or 3, whereby a solid phase is silicium based.

5. A method according to claim 4 whereby a solid phase is silica.

6. A method according to claim 5 whereby the silica is in the form of particles having a size between 0.05 and 500, preferably 0.1 and 200 μm.

7. A method according to anyone of the aforegoing claims whereby the solid phase is separated from the supernatant by centrifugation.

8. A method for amplifying HCV nucleic acid material comprising the steps of hybridizing the single stranded nucleic acid with primers and elongating the probes using an enzyme which adds nucleotides to the primer sequence using the hybridized single strand material as a template, whereby at least one primer comprises a random hybridizing sequence and an amplification motif.

9. A method according to claim 8 whereby at least one primer comprising a random hybridization sequence and an amplification sequence further comprises a label.

10. A method according to claim 8 or 9, whereby at least one primer comprising a random hybridizing sequence and an amplification motif further comprises a direct sequencing motif.

11. A method for isolating and amplifying HCV nucleic acid material originally present in a mixture of nucleic acids comprising subjecting the mixture to a method according to anyone of claims 1 - 6 followed by subjection of the isolated material to a method according to anyone of claims 8-10.

12. A method according to claim 11 whereby the HCV nucleic acid material is converted into cDNA.

13. A method according to anyone of the aforegoing claims comprising a gel electrophoresis step.

14. A method according to anyone of the aforegoing claims followed by a sequencing step.