NL1002781C1 - Isolation and / or amplification of hepatitis C virus (HCV) nucleic acids from samples suspected of containing HCV. - Google Patents

Description

Titles Isolation sn / or amplification_ £ BH_btPftt lfcl> -C ~ Tigttt- (flCY) _- BuelalMiurêB_ttiS_ «OMf g> _ ¥« ΓΥ «_Υ · ΠΒΡ · β containing those HCV.

Description

The invention relates to the field of purification and amplification of nucleic acids from starting materials containing nucleic acid, in particular from biological materials such as urine, faeces, sperm, saliva, whole blood, serum or other body fluids, fractions of such liquids such as leukocyte fractions (buffy coats [orange-yellow coatings]), cell cultures and the like.

Until recently, isolation and / or purification of 10 nucleic acids from complex mixtures as described above has been a labor-intensive, multi-step process. EP 0389063, which is to be considered incorporated herein, discloses a simple and rapid purification of nucleic acid material from a complex mixture. This method comprises treating the complex mixture, such as whole blood, with a chaotropic agent in the presence of a nucleic acid-binding solid phase silica material under conditions that allow binding of all the nucleic acid material to said solid phase, and separating said solid from the mixture. The reference shows that both single-stranded and double-stranded nucleic acids are bound to the solid phase when it is present in a mixture. The reference also discloses amplification (PCR) of a particular nucleic acid with a known sequence suspected to be present in a mixture.

1002781 -2-

Accordingly, 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 in advance or there may be many different targets that need analysis. In these cases, the rapid, but rather rough, process described above may not be sophisticated enough and further separations of the raw material may be desirable. Fractionation of mixtures of double-stranded (ds) and single-stranded (ss [single-stranded]) nucleic acids (NA [nucleic acids]) into single and double-stranded forms is often required, eg when separating labeled ss-NA probes from ds hybrids, in the separation of 15 in vitro transcripts from ds DNA reading strands and in the separation of genomic DNA from mRNA. In particular, it may also be necessary to identify the presence of an infectious agent such as HCV, in a sample containing other (ds) nucleic acids. Currently, the separation of different types of nucleic acids can be achieved by several methods are accomplished. Electrophoresis can be used to fractionate different forms of nucleic acids due to differences in size and shape. Centrifugation takes advantage of density differences and more recently, high-performance liquid chromatography (HPLC) technology has been used to separate and purify single and double stranded DNA and RNA molecules.

RNA purified from eukaryotic cells by the currently most widely used method (1) has been found to contain significant amounts of genomic DNA, an adaptation that reduces contamination of the ss-RNA fraction with genomic DNA has recently been described (2).

It is not possible to look at single stranded and / or double stranded material separately using the 1002781-3 method of EP 0389063, because the method does not distinguish between the two. Accordingly, there remains a need for a simple and rapid test to detect the presence of single stranded material (from an infectious agent) in a sample containing double stranded material. This is particularly the case in the hepatitis region where there are various causative agents and associated agents, which may be double-stranded or single-stranded, RNA or DNA. These agents may all be present in one sample together with the host's nucleic acid material, or may be present as the sole causative agent or in any combination.

With the 1989 discovery of hepatitis C virus (HCV) (5), the causative agent for most cases of viral non-A-non-B (NANB) post-transfusion hepatitis has been identified and has been intensively investigated ever since . As early as 1983, it was reported that a non-A non-hepatitis virus, which in hindsight was probably HCV, could interfere with the infection process of hepatitis B virus (HBV) in chimpanzees (6-8). Although the number of experimentally infected animals was very low in these studies, the results suggested that expression of HBV was suppressed in the dual infections. In recent years, there have been several reports regarding the effects of dual HBV and HCV infections in humans. The results of these studies do not always point in the same direction. These range from the suggestion of suppression of HCV replication via concomitant HBV infection (9), the co-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 latest research was done when tests for HCV were yet to be developed. In recent years, it has become apparent that the replication of the HDV-1002781-4 genome of HBV is independent, except that transmission of co-infecting HBV is important. (20-22). The prevalences of HBV and HCV infections are very high in Egypt. As far as we know, there are no data regarding the prevalence of HDV in the Egyptian population. We reasoned that if we were able to analyze sera from Egyptians with liver disease, we could find a fairly high proportion of bipartisan infections and possibly some tripartite infections, which would allow us to consider some aspects of 10th in investigate in vivo interactions. The only criterion we used to understand patients as suffering from liver disease was an alanine aminotransferase (ALT) serum value equal to or greater than 45 E./L. We tested 48 serums for the presence of viral HBV, HDV, HCV antigens and / or 15 antibodies by means of serological tests and the presence of HBV, HDV, HCV genomes by reverse transcriptase (R.T.) and polymerase chain reaction (PCR).

Accordingly, the present invention provides a method of separating causative agents of hepatitis from one another and of valuable material by means of. applying differential bonding properties of single stranded and double stranded materials to a solid support at different conditions. In particular, the invention provides a method of separating HCV RNA from a sample suspected of containing said RNA, contacting said sample with a first liquid comprising a chaotropic agent and a nucleic acid binding solid phase, wherein the first liquid has a composition such that the double stranded nucleic acid binds to the solid phase and does not comprise a substantial amount of single stranded nucleic acid, 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 demonstrated or further purified or amplified.

1002781 -5-

Suitable conditions for separating double-stranded nucleic acids from the single-stranded HCV material can be determined by those skilled in the art.

Conditions where double-stranded material binds to the solid material and single-stranded material will not vary, important parameters for obtaining such differential binding, however, are the concentration of the chaotropic agent, which should be roughly between 1 - 10 M, preferably between 10 3 -6 M and in particular around 5 M; the concentration of the chelating agent, which in the case of using EDTA should be equal to or higher than 10 mM and preferably not higher than 1 M; the pH of the aqueous solution in which the separation is carried out must be above 2 when a thiocyanate is used as a chaotropic agent and must be below 10 otherwise there is a risk that the ds material becomes ss. The temperature at which the process is carried out seems uncritical, however it is probably best to keep it between 4 ° C and 60 ° C. Of course, it is important in the method that the ds material 20 remains double stranded during the separation. Under the conditions disclosed above, this will usually be the case if the ds nucleic acid is at least 50 bp in length and with 40% GC base pairs. The person skilled in the art knows how this length can vary with lower or higher GC content. In Van Ness et al. (3) 25 and / or Thompson et al. (4) it is shown that the whole method is dependent on complex interactions between, inter alia, the above factors. By applying this disclosure and the references cited, the skilled artisan will be able to adapt the circumstances to his or her particular method.

Chaotropic agents are very important in the present invention. These are defined as any material that can alter the secondary, tertiary and / or quaternary structure of nuclear acids. These should not have an important effect 1002781 -6- on the primary structure of the nucleic acid. If nucleic acids associated with other molecules are present, such as proteins, these associations can also be changed by the same or different chaotropic means. Many chaotropic agents are suitable for use in the present invention, such as sodium iodide, potassium iodide, sodium (iso) thiocyanate, urea or guanidine salts, or combinations thereof. A preferred class of chaotropic agents of the invention are guanidine salts, of which guanidine thiocyanate is most preferred.

Due to a tendency to chance, we found that ss nucleic acids and corresponding HCV nucleic acid material did not bind to silica particles or diatomaceous earth in the presence of buffer Lil (see examples), while ds nucleic acid did bond. Experiments with different conditions showed that addition of Mg2 * or other positive (divalent) ions to the unbound fraction was of great importance. Best results were obtained with a divalent ion (Mg2 *) concentration approximately equal to the chelating agent concentration (EDTA).

The solid phase to be used is less critical. Importantly, these nucleic acids should bind reversibly.

Many such materials are known, some of which are silicon-based, such as aluminum silicate and the like, preferably silica. Silica is intended 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 in any form, it may even be the vessel containing the nucleic acid mixtures or part of such a vessel.

It can also be a filter or any other suitable structure. In addition to silicon-based materials, other materials are also suitable, such as nitrocellulose (filters), latex particles and other polymeric materials. A preferred form of the solid phase is a 1 0 0 21 -7 particle form, which allows for easy separation of bound and unbound material, e.g. by centrifugation. The solid phase particle size is not critical. Suitable mean particle sizes are in the 5 range from about 0.05 to 500 μπ. 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 applies to the preferred ranges whose average particle sizes are between 0.1 and 200 µ, preferably between 1 and 10 200 µm. The binding capacity of a given weight of the particles increases with decreasing size, however, the lower limit of the size is reached if particles cannot easily be redispersed after separation by, for example, centrifugation. This will be the case with starting material which is rich in nucleic acids and which contains many higher molecular weight nucleic acids. The particles in the nucleic acids can form aggregations in these cases. The person skilled in the art is able to select the correct particle size for the particular intended application. The formation of aggregations can be avoided in a number of applications by using fractionated silica or diatomaceous earth.

A further embodiment of the present invention is a method as disclosed above further 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, wherein the second liquid has a composition such that the resulting mixture of supernatant and second liquid allows binding of the single stranded HCV nucleic acid material to the second solid phase.

In this way, the double-stranded nucleic acid material in the first step is removed from the crude mixture and the single-stranded nucleic acid is purified in another single step from the remaining, still crude mixture.

1 0 0 2 7 8 1 -8-

Both the double stranded material and the single stranded material are reversibly bonded to the respective solid phases so that it can 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-stranded or single-stranded) nucleic acid material.

Both types can be amplified, or both types can be converted to each other so that they can be amplified. In another embodiment, the present invention provides a method of amplifying single-stranded nucleic acid material, comprising the steps of hybridizing the single-stranded nucleic acid with primers [initiators] and extending the probes using an enzyme that adds nucleotides to the primer sequence using the hybridized single stranded material as the reading strand, wherein at least one primer comprises a random hybridizing sequence and an amplification base.

The amplification criteria are well known in the art. The length of suitable primers, suitable buffers, suitable melting temperatures for strand separation and suitable hybridization conditions can all be determined using standard manual textbooks.

Of course, the sequences used as an example can be varied without departing from the present invention. It is not very important which sequence is used as an amplification basic form, as long as it is suitable for hybridization and primer extension. Suitable limits depend on the conditions which can be varied by the person skilled in the art. Usually, primers are at least 10 bases long and not much longer than 100 bases.

The amplification embodiments of the invention are illustrated using PCR (polymerase chain reaction).

1002781 -9-

Other amplification methods are of course equally suitable.

The exemplary marker (or label) on the primers is DIG (digoxygenin). However, other markers are available and well known in the art.

The invention is now explained in more detail in the following detailed description.

Separation / Isolation 10 MATERIALS AND METHODS Source of nucleic acids

Serums. Seruro samples were obtained from patients who had hepatitis and visited a general hospital in Cairo, Egypt. For this study, only serum samples were selected with an alanine aminotransferase (ALT) - value equal to or above the upper limit of normal (45 E./L). Selection of the samples was random. Serums from 26 men, mean age: 41 years (range 22-55); and 22 females, mean age: 42 years (range 31-52).

20

Chemicals

Guanidine thiocyanate (GuSCN) was purchased from Fluka (Buchs, Switzerland).

EDTA (Titriplex) and MgC12.6H20) were purchased from Merck 25 (Darmstadt, Germany). TRIS was purchased 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 Instruments Co., Inc., Downers Grove, 111.).

30 1002781 -10-

Buffer composition

The lysis / bind buffer L6, wash buffer L2 and TE (10 mM

Tris.HCl, 1 mM EDTA; pH = 8.0) have been described (27). 0.2 M EDTA (pH 8.0) was made by dissolving 37.2 g of EDTA (Merck, Germany) and 4.4 g of 5 NaOH (Merck, Germany) in water in a total volume of 500 ml. Lysis / binding buffer Lil was made by dissolving 120 g of GuSCN in 100 ml of 0.2 M EDTA (pH = 8.0). Binding buffer L10 was prepared by dissolving 120 g of GuSCN in 100 ml of 0.35 M TRIS.HCl (pH 6.4); then 22 ml of 0.2 M EDTA (pH 8.0) and 9.1 g of Triton-X-100 were added and the solution was homogenized; finally, 11 g of solid MgCl2.6H2 O was added. The final concentration of MgCl2 in L10 is approximately 0.25 M. L10 is stable for at least 1 month when stored in the dark at ambient temperature.

Fractionation of ds-DNA and sa-DNA by means of prescription R.

The method is outlined in Figure 1. A 50 μΐ specimen (containing a mixture of NA types in TE) was added to a 900 μΐ Lil and 40 μΐ SC mixture in an Eppendorf tube and then homogenized by vortexing. After binding for 10 min at room temperature, the tube was centrifuged (2 min at about 10,000 X g), resulting in a silica / ds-NA precipitate ('first silica precipitate *) and a supernatant containing ss-NA .

To recover ss-NA forms (Regulation R-sup), 900 μΐ of the supernatant was added to a mixture of 400 μΐ L10 and 40 μΐ SC and ss-NA was bound during a 10 min incubation at room temperature . The tube was then centrifuged (15 sec at about 10,000 x g) and the supernatant discarded (by suction). The resulting precipitate was then washed twice with 1 ml of L2, twice with 1 ml of 70% ethanol (V / V) and once with 1 ml of acetone. The silica precipitate was dried (10 min. At 56 ° C with open lid in an Eppendorf heating block) and eluted in 50 µl TE buffer 1002781-11 (10 min at 56 ° C; closed lid). After centrifugation (2 min at about 10,000 x g), the supernatant contains the ss-NA fraction.

To recover ds-NA forms (Prescription R precipitate) from the first silica precipitate, the remaining supernatant was discarded and the silica precipitate was washed twice with Lil to remove unbound ss-NA. The resulting silica precipitate was then washed twice with L2, twice with 70% ethanol, once with acetone, dried and eluted as described above. After centrifugation (2 min at about 10,000 x g), the supernatant contains the ds-NA fraction.

In the complete process (which takes about one hour) for fractionation of NA by regulation R, only two Eppendorf tubes are used.

15 Fractionation of genomic DNA and as-MA

Due to capture of ss-NA in high molecular weight genomic DNA, R, as described above, gives only low yields of ss-NA. This can be prevented by first isolating total NA by means of Assay Y / D (27) which causes some breakage of the high molecular weight genomic DNA sufficient enough to prevent capture of the ss-NA. Correspondingly purified total NA can then be used as input for regulation R.

25 Cell Electrophoresis

In all experiments, NA was electrophoresed (8 to 10 V / cm) by neutral agarose plate gels with ethidium bromide (1 μg / ml) in the buffer system (40 mM TRIS, 20 mM sodium acetate, 2 mM EDTA, adjusted to pH 7.7 with acetic acid; ethidium bromide was added to a concentration of 1 µg / ml buffer), described by AAij and Borst (25).

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Hybridization DNA fragments were transferred to nitrocellulose filters by means of Southern method and hybridized with [alpha-32P] dCTP-labeled pHC624 prepared by

5 random brands (Boehringer, Germany). Hybridization conditions were as previously described (29).

RESULTS

Comparison of different GuSCN-containing lysis-10 buffers for binding different NA types to silica particles revealed that only double-stranded forms were bound when Lil (which is about 100 mM in EDTA) was used as the binding buffer; on the other hand, both double-stranded and single-stranded forms were bound in binding buffer L6 (which is about 20 mM in EDTA) (Table 1). These observations formed the basis for the development of a protocol (Regulation R) for the fractionation of single-stranded nucleic acids and double-stranded nucleic acids (Fig. 1).

When double-stranded nucleic acid in Lil is bound by silica particles, a short spin separates the silica / ds-NA precipitate 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 in terms of Mg2 *) restores the binding of single stranded nucleic acids to the silica particles. Double-stranded and single-stranded shapes can then be purified by washing and eluting the silica-NA complexes (regulation R). Double stranded nucleic acid is recovered from the first silica precipitate (protocol R-30 precipitate), while single stranded forms are recovered from the first supernatant (protocol R-sup).

To optimize regulation R, we performed reassembly experiments in which previously purified or 1002781-13 commercially available nucleic acids were mixed and then fractionated by means of prescription R.

Practionation of a mixture of double-stranded DNA and 5 single-stranded DMA.

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. up 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 broken, as previously noted (27). At the level of proof 15 by means of UV exposure, fractionation to 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

20 (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 proof by means of UV exposure was fractionation to de and ss forms complete.

25

Practionation of a mixture of double-stranded DNA and single-stranded RNA.

Figure 4 shows that ds DNA can also be efficiently separated from ss RNA.

30 Again, the recoveries for both groups are at least

80%. Similar results were obtained as E. coli rRNA

(23S and 16S) were used as ss RNA input (not shown).

1002781 -14-

In the experiments described above, fractionation of ds and ss NA forms (as judged by visual check of band intensities after ethidium bromide staining and UV exposure) was complete. To determine the performance of the fractionation method for a mixture of ds-DNA and ss-RNA to ss and ds forms, NA which was purified from such mixture by prescription R-sup, examined by Southern blotting and hybridization with a 3JP-labeled DNA probe, homologous to the ds DNA used as fractionation input. This experiment revealed that the ss-NA fraction contained less than 0.1% of the ds-DNA input (Figure 5).

Reference of male yellow of genomic DNA and ankalatrangial RNA.

When we investigated the separation of (genomic) high molecular weight ds-DNA from ss-RNA by means of direct fractionation using £ j. coli as input for regulation R, it was found that the ds-DNA fraction was heavily contaminated with rRNA (Fig. 6, lanes 6 and 7) and that ss-RNA recovery was very low (Fig. 6, lanes 8 and 9 ). This was probably due to capture of RNA in high molecular weight (genomic) ds DNA when silica / NA complexes were formed. On the other hand, no genomic DNA in the ss-RNA fraction was observed. Total nucleic acid, which was first isolated using standard Assay Y / D (28) and then used as input to Assay R, showed significantly higher recoveries of the ss RNA fraction (Fig. 6, lanes 2 and 5) .

HCV Nucleic Acid Purification Layer.

HCV RNA, HDV RNA and HBV DNA were purified from serum 30 by means of methods as described herein. Briefly, 100 μΐ serum was added to a 1.5 ml Eppendorf tube (Type 3810) containing 900 μΐ L6 lysis buffer (see herein) and 20 μΐ of a silica suspension. The tube was immediately vortexed. During a 10 min incubation at room temperature, the nucleic acid in the specimens was allowed to complex with the silica gel particles. The tubes were then vortexed and centrifuged at 10,000 rpm for 15 s.

5 (24-hole Hettich-KG centrifuge), the supernatant was discarded and the silica-gel-nucleic acid complexes were washed twice with 1 ml of buffer L2 (see herein), twice with 1 ml of 70% (V / V) ethanol and once with 1 ml acetone and dried at 56 ° C (10 min.); the nucleic acids were then eluted at 56 'C (10 min.) in 10 or 50 l of 10 mM Tris.HCl, 0.1 mM EDTA, pH 7.5 (TE) for HCV detection or in 25 μl; TE for HDV and HBV detection (see below).

The preparation of size fractionated silica, lysis buffer L6 and wash buffer L2 is described in detail in Boom et al. (27). Briefly, Lysis buffer L6 was made by dissolving 120 g of GuSCN 15 in 100 ml of 0.1 M Tris.HCl (pH 6.4) and then 22 ml of 0.2 M EDTA (pH 8.0) and 2.6 g of Triton-X-100 added. Wash buffer L2 was made by dissolving 120 g of GuSCN in 100 ml of Tris.HCl (pH 6.4).

20 Amplifications MATERIALS AND METHODS Chemicals and enzymes.

EDTA, KCl, MgCl2.6H20, NaCl and tri-sodium citrate dihydrate were purchased from Merck (Darmstadt, Germany). TRIS and BSA 25 were purchased from Boehringer (Mannheim Germany). Triton-X-100 was purchased from Packard (Packard Instruments Co., Ine., Downers, 111., U.S.A.). Sodium dodecyl sulfate (SDS) was purchased from Serva (Heidelberg, Germany).

The dNTPs and dextran sulfate were purchased from Pharmacia 30 (Uppsala, Sweden).

The chemicals used in regulation R are described herein.

1002781 -16-

Reverse transcriptase SuperScript II was purchased from Life Technologies (Gaithersburg, Maryland, U.S.A.). DNA polymerase Sequenase 2 was purchased from Amersham (United Kingdom). Ampli-Taq DNA polymerase was purchased from Perkin 5 Elmer (Norwalk, U.S.A.). RNA-ase-H was purchased from Boehringer (Mannheim, Germany). Salmon sperm DNA was purchased from Sigma (St. Louis, U.S.A.).

Composition of buffers and solutions.

The preparation of buffers used in regulation R are described herein, except that the lysis buffer and wash buffers (L10, Lil and L2) used in regulation R for the isolation of nucleic acids were filtered through a diatom-stacked column (27 ) to remove any endogenous nucleic acids in the lysis buffer and wash buffers.

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

The 10X 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).

Primer 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-read string [anti sense] -PCR primer for HCV was RB-6B: 5 'ACT.CGC.MG. CAC.CCT.ATC.AGG 3 * (nt. 292-312) and the reading strand 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.- 30 GM.AGG.CCT.TGT.GGT.ACT.G 3' (nt. 264291) was labeled at the 5-end with digoxyginine and used as a probe in hybridization experiments to determine the specificity of PCR products. The HCV oligos had specificity for the 5 'non-1002781-17 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 E22, the primer which was also used for reverse transcription, and E22: 5 'CGG.CTC.GGC.MC.ATT.CCG.AG 31 (nt 718-737). The plasmid pG4Z (D3) was a bounty from John Taylor (Fox Chase Cancer Center, Philadelphia) and contains 3 complete cDNA copies of the HDV genome in sequence, cloned into the Eco-RI site of 10 pGem4Z (Promega ). Via digestion of pG4Z (D3) with Bal-II. isolation of the 4.4 kb fragment. from an agarose gel, ligation and transfection to E. coli C 600, plasmid pG4Z (D1) containing a single cDNA copy of the HDV genome was obtained. pG4Z (D1) was labeled with digoxygenin-dUTP by means of random 15 primer DNA preparation according to the instructions of the manufacturer (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. 'l'l.TGC. 3 '(nt. 1939-1960, nt. Numbering according to (24)), and RAL: 5 "GCA.AGC. '1-1-1' .CTA.ACA.ACA.GTA.GTT.TCC. GG 31 (nt.

2332-2352); the underlined parts of the primers represent added restriction enzyme linkers [linkers]. The PCP10 plasmid contains two full copies of the HBV genome 25 in sequence, inserted at the Eco-R1 site of pBR 322 and was a generous gift from R. Heytink (Eramus University, Rotterdam). pHBt-III was derived from PCP10 by means of Eco-RI digestion. isolation of the linear form of HBV-Eco-RI from an agarose gel and ligating thereof into the Eco-RI-cleaved high copy number plasmid pHC 624 (28). pHBtIII was labeled with digoxygenin-dUTP by means of

random primer DNA preparation according to the manufacturer's instructions (Boehringer Mannheim GmbH) and used as a probe in hybridization experiments to determine the specificity of PCR products.

1002781-18-R.T.-PCR from HCV.

Reverse transcription was performed in a 25-1 reaction volume containing 20 E. RNA ase reimner (Promega Biotec, Madison, 5 Wis.) 67 mM Tris.HCl, pH 8.0, 17 mM ammonium sulfate, 1 mM

3-mercaptoethanol, 6 μΜ EDTA, pH 8.0, and 0.2 mg / ml bovine garden albumin [Bovine Serum Albumin] (BSA) (Boehringer), 6 mM MgCl2, 25 ng primer HCV-6, 0.6 μΐ 25 mM (for each) of deoxynucleoside triphosphates, 11.5 μΐ of the 50 μΐ eluate from the nucleic acid purification (see above) and 200 E.

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 means of Incubation at 95 ° C for 5 min (29). The PCR 15 was performed in a volume of 50 μΐ containing 2.5 E. Taq polymerase (Perkin Elmer Cetus), 50 mM Tris.HCl, pH 8.3, 20 mM KCl, 1.2 mM

MgCl2 and 1 mg / ml BSA), 12.5 μΐ of the R.T. reaction mixture, 200 μΜ of each deoxynucleoside triphosphate and 100 ng of each of primers RB-6A and RB-6B. Samples were taken at 95 ° C for 5 min

Denatured and subjected to 35 rounds of temperature cycling in a DNA thermal cycler (DNA temperature cycling device) (type 480; Perkin Elmer Cetus). A cycle consisted of 1 min. Denaturation at 95 ° C, 1 min. renaturation at 55 ° C and extension for 2 min at 72 ° C. After the recycle program 25, the samples were incubated for 10 min at 72 ° C. All samples analyzed for HCV-RNA by the RT-PCR were duplicate run on different days.

R.T.-PCR from HDV and PCR from HBV.

HDV RNA and HBV DNA were purified together by the method described above. However, HBV DNA in TE at 56 ° C, in contrast to HDV RNA, is poorly eluted from the silica particles, presumably due to co-purification of the protein covalently linked to the HBV DNA genome. 7 μΐ of the eluate (25 μΐ) was taken from the tube for HDV-R.T.-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 at 56 ° C for 10 min. After heating at 95 ° C for 10 min to inactivate the proteinase, and centrifuging (1 min at 12,000 x g), 20 μl of the eluate was used for HBV-PCR.

Naturation of the primer with reading strand HDV RNA was performed in a mixture containing 7 μl eluate, 1 ng anti-reading strand 10 primer E21, 50 mM Tris.HCl, pH 8.3, 40 mM KCl, 6 mM MgCl 2, 10 mM dithiothreitol and 150 μΜ (of each) deoxynucleoside triphosphates (dNTPs). After heating to 80 ° C for 1 min and cooling to room temperature, 12 E. AMV reverse transcriptase (Boehringer Mannheim GmbH) was added and R.T. 15 performed for 1 hour at 42 ° C in a total volume of 10 μΐ. Reverse transcriptase was denatured by 5 min. Incubation at 95 ° C (27). PCR was performed in a 100 μΐ reaction mixture containing 200 ng primer E21,200 ng primer E22, 50 mM

Tris.HCl, pH 8.3, 20 mM KCl, 1 mM MgCl 2, 0.1 mg BSA, 1.5 E. Taq polymerase, 200 μl of each deoxynucleoside triphosphate and 10 μl of the R.T. reaction. After 5 min incubation at 95 ° C, the sample was subjected to 35 cycles of amplification. The same cycle program as for HCV was used.

The HBV-PCR was performed in a reaction mixture of 100 μm containing 200 ng primer LBL, 200 ng primer RAL, 50 mM Tris.HCl, pH 8.3, 20 mM KC1, 1 mM MgC12, 0.1 mg BSA, 1 .5 E. Taq polymerase, contained 200 μΜ of each deoxynucleoside triphosphate and 20 μΐ of the eluate. After 5 min incubation at 95 ° C, the sample was subjected to 35 cycles of amplification. The same recycle-30 program as for HCV and HDV was used.

Agarose gel electrophoresis and Southern blotting

After completion of the amplification reactions, 10 μΐ of each reaction was analyzed by electrophoresis by horizontal 1002781 -20-

2% agarose plate gels in the buffer system described by AAy and Borst (25) and containing ethidium bromide (1 µg / ml). DNA

was transferred from the gel to nitrocellulose filter (Bio-Rad, U.S.A.), essentially as described by Southern 4. The transferred DNA was cross-linked to the filter by means of. incubation at 80 ° C for 2-3 hours.

Brands and hybridization.

The HBV and HDV containing plasmids were labeled with 10 digoxygenin-dUTP by means of random priming [initiate] (Boehringer Mannheim GmbH). Nitrocellulose flashers were pretreated as previously described (26). Hybridization and post-hybridization washes were as previously described. Detection of digoxygenin was done according to the instructions provided by the manufacturer of the kit (Boehringer Mannheim

GmbH).

Statistics

Statistical significance tests were done using the two-tailed Fisher exact test.

RNA fiber strand preparation

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

HCV reading strand RNA was transcribed from PMOZ.1 HCV in vitro. Briefly, 5 μg of plasmid DNA was linearized by means of Eco-RI and then incubated with 50 E. SP6 RNA polymerase for 2 hours at 37 ° C in the presence of 500 μΜ (of each) ribonucleoside triphosphate (GTP, ATP, UTP and CTP), 100 E.

1002781 -21- RNAsin, 10 inM 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 μΐ. After the transcription reaction, the DNA reading frame was broken down by means of two rounds of digestion with RNA-aseless DNA-ase 5 (Boehringer) for 30 min at 37 ° C, with 10 E. enzyme. After completion of the digestion, 2 rounds of extraction using phenol-chloroform-isopropyl alcohol followed by ethanol precipitation were done. The HCV RNA transcripts containing a poly (A) tail were further purified on an oligo-dT-10 cellulose column. The RNA concentrations were determined spectrophotometrically by UV-A.260. A partial volume was analyzed by agarose gel electrophoresis to verify its integrity. HDV RNA was prepared and, except for the oligo-dT cellulose selection, purified in a similar manner as described above for HCV-15 RNA. Hin-dlII linearized pG4B (D1) was the substrate for SP6 RNA polymerase.

Sensitivity of the (R.T.) PCR tests.

HCV and HDV RNAs prepared in vitro and the Eco-RI 20 linearized HBV plasmid pHBt-III were diluted in TE buffer to a concentration of approximately 10 copies / μ and stored at -20 ° C. Serial 10-fold dilutions in H 2 O of these parent solutions were made just prior to the R.T. PCR or PCR reactions. One hundred copies were routinely detected.

25

Demonstrate Southern hybridization and chemiluminescent

The electrophoresis of the PCR products, the transfer of the DNA to positively charged nylon membrane (Boehringer Mannheim GmbH) and hybridization with the probe were done as previously described. Detection of the hybridized digoxygenin-labeled probe was done with the DIG-Luminescence Detection Kit according to the instructions recommended by the kit supplier (Boehringer Mannheim GmbH).

1002781 -22-

VeraelUkand example «» ld

The methods of the invention were compared to commercial methods using antibodies.

5

Enzyme-linked immunosorbent assay [Enzyme-Linked Immunosorbent Assay] (ELISA) kits provided by Abott Laboratories (Chicago, Illinois) or Sorin Biomedica (Sallugia, Italy) were used to detect HBsAg, HBeAg, anti- HBe, IgG anti-HBc and IgM anti-HD. Antibodies to structural and nonstructural synthetic HCV peptides were detected with the enzyme immunoassay kit SP-NANBASE-C-96 provided by General Biologicals Corp. (Hsin Chu, Taiwan). The presence of anti-HCV antibodies was also determined using a recombinant immunoblot assay (RIBA-3, Chiron Corporation, Emeryville, CA). Tests were performed according to the manufacturer's directions. The results are presented in Tables 2-7. The serums were obtained from the previously identified group.

1002781 22a

Figure description:

Figure 1. Sketch of regulation R.

Recovery of ds-NA takes place from the first precipitate (R-precipitate), recovery of ss-NA takes place from the first supernatant (R-sup). Lil, L10, L6 and L2 are GuSCN 5 based buffers, SC is suspension of silica particles. See

Materials & Methods for Details.

Figure 2. Separation of da DNA and se-ONA

NA was purified Mn duplicate) by means of Prescription R, from a mixture of ds DNA (phage lambda, Hia dlII digest, 1 Hg) and ss DNA (phage M13 DNA, 500ng). Final elutions were in 50 μΐ TE and 25 μΐ were electrophoresed through a 1% agarose gel (with ethidium bromide) which was then photographed under UV exposure. Lane 1: 100% recovery marker for ds DNA fragments; lane 2: 100% recovery marker for ss DNA; lane 3: 100% recovery marker for mixture of ds-DNA / ss-DNA. Lanes 4 and 5: output of regulation R deposit; lanes 6 and 7: output of regulation R-sup.

Figure 3. Separation of da-RHA and axis RNA

NA was purified (in duplicate) by means of of R, from a mixture of ds-RNA (rotavirus-ds-RNA) and ss-RNA (phage-MS2-RNA, 800 ng). Final elutions were in 50 μΐ TE and 25 μΐ was electrophoresed through a 1% agarose gel (with ethidium bromide) which was then photographed under UV exposure. Lane 1: 100% - recovery marker for ds-RNA fragments; lane 2: 100% recovery marker for ss RNA; lane 3: 100% recovery marker for ds-RNA / ss-RNA mixture. Lanes 4 and 5: output of regulation R deposit; lanes 6 and 7: output for regulation R-sup.

1002781 22b

Figure 4 .. Separation: Pre-DBA and ss-RNA

NA was purified (in duplicate) by means of prescription R from a mixture of. ds DNA (750 ng Hin-dlII digested phage lambda) and ss RNA (phage MS2 RNA, 800 ng). Final elutions were in 50 μΐ and 25 μΐ were electrophoresed through a 1% agarose gel (with 5 ethidium bromide) which was then photographed under UV exposure. Lane 1: 10 0% - recovery marker for ds DNA; lane 2: 100% recovery marker for ss RNA; lane 3: 100 * - recovery marker for ds-DNA / ss-RNA mixture. Lanes 4 and 5: output of regulation R deposit; lanes 6 and 7: output of 10 prescription R-sup.

Figure- 5. Separation of ds DNA. and ss RNA

NA was purified by means of R-sup, from a mixture of ds-DNA (1000 ng linearized pHC624, 2 kb.) and ss-RNA (phage-15 MS2-RNA, 800 ng). Final elution was in 50 μΐ TE and 25 μΐ or 10-fold serial dilutions of the ss-NA fraction was electrophoresed through a 1% agarose gel (with ethidium bromide) which was then photographed under UV exposure.

Panel A: Upper row: lane 1: Hin-dlII digested phage lambda-20 DNA; lane 2: 100% recovery marker for ds DNA and ss RNA and serial ten-fold dilutions thereof (lanes 3-6). Lower row: output of prescription R-sup (lane 2) and ten-fold serial dilutions (lanes 3-6).

Panel B: ds DNA was then transferred to a nitrocellulose filter and hybridized with a 32 P-labeled probe homologous to input ds DNA. ds DNA and ss RNA are indicated.

1002781 22c

Figure 6. separation of ganoaic DNA from sa-RNA

How to deal with capture of ss-RNA. E. coli bacteria were used directly as input for in-duplicate extractions by means of regulation R (lanes 6 and 7: R precipitation; lanes 8 and 9: R-sup). On the other hand, total NA was first purified by means of

5 regulation Y with the use of diatoms as an NA carrier (causing breakage of genomic DNA). The purified nucleic acids were then used as input for regulation R (lanes 2 and 3: R precipitate; lanes 4 and 5: R-sup). Final elutions were in 50 μΐ TE and 25 μΐ were electrophoresed through a 1% -10 agarose gel (with ethidium bromide) which was then photographed under UV exposure.

Marker lanes 1 and 10 (500 ng phage lambda DNA, with Hin-dlII

digested). 23S and 26S rRNA and ds DNA molecular weight markers (23 kb. And 2.0 kb.) Are indicated.

1002781 -23-

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10 1002781

Claims (13)

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, the first liquid has a composition such that double stranded nucleic acid binds to the solid phase and does not bind a substantial amount of single stranded nucleic acid, and separating the solid phase from the supernatant. The method of claim 1, further comprising treating the supernatant containing the single stranded nucleic acid material with a second fluid comprising a chaotropic agent and a second nucleic acid binding solid phase, the second fluid having a composition such that the resulting mixture of supernatant and second liquid allows binding of the single stranded nucleic acid material to the second solid phase. The method of claim 1 or 2, wherein the first liquid comprises at least 100 mM EDTA and which comprises a guanidine salt, preferably guanidine thiocyanate, as a chaotropic agent. The method of claim 1, 2 or 3, wherein a solid phase is based on silicon. The method of claim 4, wherein a solid phase is silica. 1002781 -28- A method according to claim 5, wherein the silica is in the form of particles having a size between 0.05 and 500, preferably between 0.1 and 200 µk. 7. A method according to any one of the preceding claims, wherein the solid phase is separated from the supernatant by means of spin. 8. A method of amplifying HCV nucleic acid material comprising the steps of hybridizing the single stranded nucleic acid with primers and extending the probes using an enzyme which adds nucleotides to the primer sequence using the hybridized single stranded material as a reading radar, wherein at least one primer comprises a random hybridizing sequence and a basic amplification form. The method of claim 8, wherein at least one primer comprising a random hybridizing sequence and an amplification sequence further comprises a label. 10. The method of claim 8 or 9, wherein at least one primer comprising a random hybridizing sequence and an amplification primer further comprises a direct sequence primer. 11. A method of isolating and amplifying HCV nucleic acid material originally present in a mixture of nucleic acids, comprising subjecting the mixture to a method according to any one of claims 1-6 followed by subjecting the isolated material to a method according to any of claims 8-10. 12. The method of claim 11, wherein the HCV nucleic acid material is converted to cDNA. 1002781 -29- The method according to any of the preceding claims, comprising a gel electrophoresis step. Ie. The method according to any of the preceding claims, which is followed by a sequencing step. 1002781