DE19832050A1 - Detection of Hepatitis C and B viral genomes in serum or plasma using specific oligonucleotide primers and probes - Google Patents

Abstract

A method to detect Hepatitis C (HCV) and/or Hepatitis B (HBV) viral genomes in a serum or plasma sample using specific primers and probes (I) - (VIII), is new. A method to detect Hepatitis C (HCV) and/or Hepatitis B (HBV) viral genomes in a serum or plasma pool sample using specific primers and probes (I) - (VIII), is new and comprises: (1) extraction of viral nucleic acid; (2) reverse transcription of RNA to DNA; and (3) specific amplification of the DNA through PCR and detection of an increased reporter signal; where for reverse transcription and a first amplification of HCV the following primer pair was used: 5'-GGTGCACGGTCTACGAGACCTC-3' (I) 5'-AACTACTGTCTTCACGCAGAA-3' (II) for a specific second amplification the following primer pair were used: 5'-CCATAGTGGTCTGCGGAACC-3' (III) 5'-CAGGCAGTACCACAAGGCCT-3' (IV) the probe used was: 5'-CGACCCAACACTACTCGGCTAGCAGTCT-3' (V) for the detection of HBV the primer pair used for amplification was: 5'-TATCGCCGGATGTGTCTGC-3' (VI) 5'-GGACAAACGGGCAACATACC-3' (VII) and the probe was: 5'-CATCCTGCTGCTATGCCTCATCTTCCT-3' (VIII) An Independent claim is also included for an oligonucleotide for specific amplification and/or detection of viral nucleotide sequences in a sample chosen from (III) - (VIII).

Description

Various products are often used in medicine, that come from human blood. The different Components of the human serum or plasma are used for the different therapeutic applications used. At These products can be cleaned, for example Act coagulation factors or certain Antibody preparations used for prophylaxis or therapy various diseases can be used.

In recent years it has been found that through Blood plasma or blood serum and products made from it Diseases that are caused by viruses are transmitted that are present in the blood donations. For more precise Examination of products derived from serum or plasma can on the one hand the antibodies against the viral Pathogens are detected. The disadvantage of this However, the detection method is that frequently the antibodies only after a certain period of infection from the body of the Blood donors are formed. With fresh infections therefore a considerable risk that the immunological detection  is negative, although the blood donation is contaminated with a virus is. This can lead to an unwanted infection of the recipient of the product.

To ensure that the blood donation is used to manufacture of pharmaceuticals are used, not with viruses are contaminated, the test can on the other hand by the specific amplification of regions of the nucleic acids of the viruses can be detected. This Detection method is called the so-called polymerase Chain reaction has been known for a few years.

Problematic when performing the polymerase Chain reaction (PCR), however, is the very specific detection of viruses. Especially with those viruses that have a high genetic If there is variability, mutations often occur and this leads to whole family trees of such viruses related viruses exist, but are in their Partially differentiate sequence from each other. On An example of such a highly variable virus is that Immunodeficiency virus (HIV). Even the viruses caused by the existing methods are demonstrated, have a relatively high variability.

When the individual strains of a virus differ in terms of their Differentiate from each other is the exact selection of the individual verification steps and in particular the selection of for the PCR used primers and probes of crucial importance Importance. If the primers are specific to the DNA / (RNA) bind some virus subtypes, there is a risk that the PCR precisely those strains cannot be detected that don't bind the primers. The test result is negative although the sample is contaminated with a virus.  

The present invention therefore relates to a method for the detection of hepatitis C and / or hepatitis B virus genomes in a serum or plasma sample, which comprises the following steps:

• a) extraction of viral nucleic acids,
• b) Reverse transcription of ribonucleic acid in deoxyribo nucleic acid and
• c) specific amplification of the deoxyribonucleic acid via the polymerase chain reaction and subsequent detection about the increase in the reporter signal,

where the primer pair for reverse transcription and 1st amplification of hepatitis C virus genomes:

and for the specific 2nd amplification the pair of primers:

used and for detection the probe

is used and that the pair of primers for the detection of the hepatitis B virus genome

and the probe

is used.

The hepatitis B virus infection becomes predominantly parenteral transmitted, especially through contact with contaminated Blood, blood derivatives and / or other body fluids. The Hepatitis B virus is the family of the Hepatnaviridae assigned. It is about 42 nm in size DNA virus consisting of a core of approximately 27 nm, surrounded by a glycoprotein-containing lipid bilayer, which is in the subtypes adw, ayw, adr and ayr occurs. The virus has one circular, partially double-stranded DNA and has a specific HBV DNA polymerase, which with the partial double-stranded circular DNA is associated.

The clinical picture is characterized by fever in the acute phase, Body aches, fatigue and jaundice. Clinically falling especially increased in most of the acute infections Transaminase levels. However, the disease can also run asymptomatic. HBV infection can also assume chronic course, which is in similar Late effects, as described for chronic hepatitis C, manifested.

The hepatitis C viruses are assigned to the Flaviviridae and have at least six different subgroups that can be differentiated into several subtypes. The Virus particles are very small (around 40-70 nm), making the electron microscopic representation is difficult. Even if in acute infections before seroconversion viral loads of Several million copies per ml are not uncommon the viral genome at certain stages of infection only in a relatively low number of copies when infected Patients are present, which complicates the diagnosis.  

The viral genome consists of a single-stranded RNA. The Transmission is parenteral, with one Main source of infection is the transmission of blood and Blood derivatives is.

The clinical picture is similar to that of hepatitis B and is in the acute phase characterized by fever, body aches, Fatigue and jaundice. Clinically, above all, a large part of the acute infections increased transaminase values on. However, the disease can also be largely run asymptomatic. The problem with the disease is that they have a chronic course at a high percentage takes. The late effects can possibly turn out to be Cirrhosis of the liver or primary hepatocellular carcinoma manifest.

The method according to the invention is preferred in the Manufacture of pharmaceuticals used from blood donations. Here, relatively large amounts of blood plasma fall (Serum or plasma pools) that indicate the presence of Virus genomes need to be tested. To the polymerase It is first of all to be able to carry out a chain reaction required the viral nucleic acids from the serum or Extract plasma sample. Of course, for the Specialist that extraordinary in these steps is worked clean. In any case, it must be avoided that, for example, about laboratory equipment or reagents Nucleic acids of the viral genomes to be detected were introduced as this would lead to false positive results. An advantage of the method according to the invention is that Reduce the risk of contamination during amplification and detection by the process in the closed system or in the use of uracil N-glycosylase.  

In the method according to the invention, preference is given to the Extraction of the viral nucleic acid from the serum or plasma sample enzymatically and chemically digested. In the first Extraction step, the viruses are lysed. To be favoured the lipid envelopes of the viruses by detergents, such as for example Triton X-100, open-minded. The lysis of According to the invention, protein envelopes are carried out enzymatically by digestion with a proteinase, preferably proteinase K, and secondly, chemically through guanidinium chloride. The the latter reagent has the advantage that RNAsen as a result be inhibited, the detection of RNA viruses (hepatitis C) can complicate. The extraction is preferred Nucleic acids have a relatively high sample volume (for example 2 ml instead of the usual 200 µl), which improves the detection limit of the method.

The nucleic acids released by the lysis are then in Presence of a chaotropic (ammonium sulfate, or thiocyanate Perchlorate) specific to the surface of a salt Bound silica matrix. This silica matrix is located preferably in prepared small columns. As soon as the Nucleic acid has been bound to the silica matrix, this becomes then washed to quantify contamination as much as possible cut off. Then the purified nucleic acids eluted from the silica matrix. This is more preferred Way through nuclease-free water. These eluates can then amplified with the various PCR methods and be detected.

In the method according to the invention, the various known variants of the polymerase chain reaction used become. The polymerase chain reaction requires two usually synthetically produced Oligodeoxynucleotide primers, their sequences to the initial and Final sequences of the sister strands of the DNA to be amplified are complementary. First, the double-stranded DNA of  desired sequence denatured by heating to about 94 ° C, so that it separates into the individual strands. Then you give the nucleotides and cool to about 50 ° C. Here hybridize the primer nucleotides to the ends of the Single-stranded DNA and thereby prevent reunification (reannealing) of the original DNA single strands. In one The next step is to mix the four Deoxynucleotide 5'-triphosphates and a temperature-stable DNA Polymerase, whereby usually the Taq Serves polymerase. At about 72 ° C the polymerase now supplements primed the single strand of DNA between the two Ends to double strand. The three steps of heat denaturation, Primerannealing, polyinerization are repeated several times and this increases the number of DNA fragments detected increased exponentially. These now amplified fragments are subsequently verified. For proof are known various processes. For example, the amplified DNA are separated on an agarose gel and it a certain, amplified piece is then visible in the gel.

In the polymerase chain reaction, the DNA fragment that located between the two primers. This Piece is demonstrated by the fact that to the reaction batch a probe is added that is complementary to one Strand of the amplified fragment, with the probe on the amplified fragment located between the two primers is. This probe can, for example, be radiolabelled, however, this is because of the environmental problems involved no longer preferred. Within the scope of the present invention the so-called "nested PCR" when detecting HCV genomes preferably used.

In the context of the present invention, however, is the so-called TaqMan polymerase chain reaction preferred. In contrast to The conventional PCR approaches use a fluorogenic probe to use.  

In the TaqMan PCR, the probe is at the 5 'end with a Fluorescent dye (fluorescent derivative) and at the 3 'end with a quencher dye (rhodamine derivative) marked. Will the intact probe at a specific wavelength (488 nm) Fluorescence is excited, so the emitted photons are on transfer the quencher molecule. So it is a Photon transfer before and the corresponding reporter signal is thereby suppressed. According to the invention, the probes preferably marked with the dyes FAM and TAMRA.

If a specific gene amplification by the PCR Reaction takes place, the primers and also the hybridize Probe to the corresponding complementary structures of the Matrix strands. During the extension phase, the taq migrates Polymerase along the matrix strand and strikes it the probe and displaces it. This creates a Y-shaped one Structure showing the 5'-3 'exonuclease activity of the polymerase activated. Due to the exonuclease activity, the probe cut and the fragment with the reporter dye is released. Now that the photon transfer to the Quencher molecule is interrupted, the increase in Reporter signal visible. Through the individual cycles of the PCR the signal becomes stronger and the evaluation of the signal is done with devices specifically designed for this reaction to be provided.

According to the invention, it has now been found that by using the following primers or probes particularly advantageous Analysis results can be achieved:

The primer pair for reverse transcription and 1st amplification of hepatitis C virus genomes:

and for the specific 2nd amplification the pair of primers:

The probe has the sequence:

The primer pair is used to detect the hepatitis B virus genome

used.

The probe has the sequence:

By combining the individual steps according to the invention and the choice of specific primers or probes can be one easy handling of the detection procedure can be achieved. At the preferred TaqMan PCR analysis can detect the Amplification without an additional, additional detection method respectively. It is particularly advantageous in the present method but that this enables a very high sensitivity to be achieved. In the method according to the invention, sensitivity can be of <80 genomes / ml can be achieved for both virus classes. For the hepatitis B virus had a sensitivity of 10 genomes / ml for subtype AD and subtype AY (according to HBV- Eurohepstandard of the Department of Medical Microbiology, University Clinics Göttingen).  

For the hepatitis C virus, a detection limit of <80 genomes / ml for HCV subtype 1 can be achieved (according to Pelipsy run control, CLB Amsterdam).

With the method according to the invention, all have so far been possible examined reference samples of the hepatitis B subtypes AD and AY and the HCV subtypes 1-5 are detected 100%.

The method according to the invention is preferably so performed that either contamination with hepatitis c or is detected with hepatitis B. Proof of both Viruses in one approach ("multiplexing") has the disadvantage a lower sensitivity.

The following advantages in particular can be achieved by the method according to the invention:

• - Easy handling of the method
• - Reduced analysis time for simple (HBV) - as well as for "nested" (HCV) PCR approaches using the two-step process, d. H. Annealing of the primer and chain extension are done in one step.
• - Time saving by eliminating follow-up analyzes regarding detection (e.g. gel electrophoresis, EIA, blotting, etc.). The result can be called up here directly after the analysis the.
• - Increased specificity by hybridizing a specific one Probe to the target sequence.
• - The on-line recording of all cycles enables the creation len of kinetics. This allows the analysis to be detailed be appraised. Anomalies during the run will be noticed lig.  
• - Carrying out the entire analysis in a closed system minimizes the risk of contamination. This is just one high sample throughput extremely important.

The present invention is illustrated by the following Examples explained in more detail.

example 1 Extraction of nucleic acids (carried out via the modified test kit: high pure viral nucleic acid kit, Boehringer Mannheim, 1 858 874)

1.8 ml of the sample to be examined (plasma pool sample or Controls) with 1.8 ml working buffer (6 M guanidinium chloride, 10 mM urea, 10 mM Tris-HCl, 20% Triton X-100, 150 µg poly (A) carrier-RNA, pH 4.4, always freshly prepared) or 360 µl proteinase K solution (accordingly 7.5 mg in distilled water), mixed and mixed for 10 min at 72 ° C incubated. After incubation 900 ul isopropanol added and the mixture mixed well again.

Each 810 µl of the lysis batch is placed in a filter tube (column with Silicamatrix) pipetted, centrifuged for 1 min at 8000 xg, the Eluate discarded and centrifuged again at 14000 xg for 30 sec.

This process is repeated 6 times until the entire Lysis approach has run over the column.

Then the column is washed 3 times with 450 µl wash buffer (20 mM NaCl, 2 mM Tris-HCl, pH 7.5) at 8000 xg for 1 min each washed (eluates are discarded) and then without buffer Centrifuged at 13000 xg for 30 sec.

The bound nucleic acids are finally with 50 ul nuclease-free aqua dest. (70 ° C) eluted at 8000 xg.  

The extracted DNA / RNA samples can be directly in the Reaction approaches for the amplification of HBV or HCV genomes pipetted or until further use at -80 ° C be stored.

Example 2 Amplification of HBV genomes

The concentration of the individual components in the reaction mixture were adapted in a series of experiments to the described primers and optimized the reaction conditions as follows.

The individual components were pipetted onto ice, the details corresponding to the respective final concentrations in the reaction mixture:
MgCl ₂ 8 mM; 1 × reaction buffer (10 mM Tris-HCl, 50 mM KCl, pH 8.3);
Nucleotide mixture (250 µM dATP, dGTP, dCTP, dUTP); Primer SEQ ID Nos. 6 and 7 each 0.4 µM;
Probe Seq. ID No. 8 0.4 µM. Uracil N-glycosylase in a concentration of 0.5 U or Taq polymerase in a concentration of 4 U is added to this approach. 5 µl of the nucleic acid preparation are then pipetted in and the reaction mixture is diluted to a final volume of 50 µl with distilled water. replenished.

The temperature profiles of the amplification are as follows:
Pre-incubation: 2 min at 50 ° C and 10 min at 94 ° C
Amplification: 1 min at 59 ° C and 15 sec at 94 ° C
The two-step PCR runs with a total of 50 cycles.

The following control scheme was chosen to check the quality of the amplification:
1 positive control: 100 ge / ml
2 inhibition controls: 100 ge / ml (the dilutions are made with plasma pool samples)
5 negative controls: corresponding to HBV negative human plasma

The test results can be evaluated using a Endpoint determination (fluorescence spectrometry, Excitation wavelength 488 nm and emission 518 nm). Here, the reaction on an external thermal cycler carried out. Alternatively, the kinetics of the Amplification (real time) can be recorded. Parallel studies showed that between the two Analysis modes no differences in results are noticeable.

Example 3 Amplification of HCV genomes

This method represents a "nested PCR", the reverse Transcription and 1st amplification using rTth- Polymerase can be carried out in one step.

The concentrations of the individual components in the reaction mixture were adapted in a series of experiments to the described primers and optimized the reaction conditions as follows.

3.1 reverse transcription and first amplification

The individual components are pipetted onto ice, the details corresponding to the respective final concentrations in the reaction mixture:
Mn (OAc) ₂ 4 mM, 1 × reaction buffer (50 mM Bicine, 115 mM potassium acetate, 8% w / v glycerol pH 8.2); Nucleotide mixture (200 µM dATP, dGTP, dCTP, dTTP each); Primer SEQ ID Nos. 1 and 2 each 0.5 µM. RTth DNA polymerase in a concentration of 2.5 U is added to this approach. 10 µl of the heat-denatured nucleic acid preparation are then pipetted in and the reaction mixture is brought to a final volume of 50 µl with DEPC-Aqua dest. replenished.

The reverse transcription (cDNA synthesis) was carried out for 30 min 60 ° C. The mixture was then incubated at 94 ° C. for 1 min.

The first amplification is over 35 cycles (15 sec at 94 ° C and 50 seconds at 57 ° C). In conclusion, the Reaction batches incubated for 4 min at 74 ° C and until further Use tempered at 4 ° C.

3.2 Second amplification

The reaction mixture was pipetted into the following final concentrations (on ice):
MgCl ₂ 6 mM; 1 x reaction buffer (10mM Tris-HCl, 50mM KCl, pH 8.3);
Nucleotide mixture (300 µM dATP, dGTP, dCTP, dUTP each); Primer Seq. ID No. 3 and 4 each 0.24 µM;
Probe SEQ ID No. 5 0.48 µM. Uracil N-glycosylase in a concentration of 0.5 U and tag polymerase in a concentration of 2.5 U were added to this approach. Subsequently, 10 ul of the samples from the first amplification were pipetted in and the reaction mixture to a final volume of 50 ul with distilled water. replenished.

The amplification was carried out under the following reaction conditions:
Pre-incubation: 2 min at 50 ° C and 10 min at 94 ° C
Amplification: 1 min at 60 ° C and 15 sec at 94 ° C
The two-step PCR runs with a total of 40 cycles.

The pattern of the controls carried out is analogous to the HBV Amplification. The reactive controls each contain 180 ge / ml. In principle, both analysis modes (real time or end point determination) applicable.

The example shows that the method is contaminated with HCV Donate quickly and reliably, with relatively little Workload can be identified from a pool. Since the Measurement can be carried out directly after amplification, there are no subsequent steps for the detection of amplificates. Statements about the quality of the PCR analysis or the viral load can be used when using the real time mode (recording the Kinetics).

Claims (7)

1. A method for the detection of hepatitis C and / or hepatitis B virus genomes in a serum or plasma sample, in particular in a serum or plasma pool sample, comprising the following steps:

• a) extraction of viral nucleic acids,
• b) reverse transcription of ribonucleic acid in deoxyribo nucleic acid and
• c) specific amplification of the deoxyribonucleic acid via the polymerase chain reaction and subsequent detection via the increase in the reporter signal,

characterized in that for reverse transcription and 1st amplification of hepatitis C virus genomes the primer pair:
and for the specific 2nd amplification the pair of primers:
used and the probe
is used and that for the detection of the hepatitis B virus genome for amplification, the pair of primers
and the probe
is used. 2. The method according to claim 1, characterized in that at the extraction of the viral nucleic acid or the serum Plasma sample is broken down enzymatically and chemically. 3. The method according to claim 2, characterized in that the enzymatic digestion with a proteinase and chemical Digestion with a detergent and guanidinium chloride is carried out. 4. The method according to any one of claims 2 or 3, characterized characterized that after chemical digestion released nucleic acid in the presence of a chaotropic Salt specifically bound to the surface of a silica matrix becomes. 5. The method according to any one of claims 1 to 4, characterized characterized that the TaqMan Polymerase chain reaction is used. 6. The method according to any one of claims 1 to 5, characterized characterized in that the amplification in separate batches is carried out and that in the detection of hepatitis B- Virus genomes did not perform reverse transcription (b) becomes.   7. Oligonucleotides for the specific amplification and / or for the detection of viral nucleotide sequences in a sample, characterized in that the nucleotide sequence is selected from the group consisting of: