JPH10327898A - Preparation of sample for diagnostic test based on nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new method for preparing a sample in which a liquid biological material is pooled, enabling to enhance the sensitivity of the diagnostic test based on nucleic acid. SOLUTION: A precipitating agent (e.g. polyethylene glycol, dextral sulfate or ammonium sulfate) is added to selectively precipitate nucleic acid, thereby improving the sensitivity of the diagnostic test for detecting the nucleic acid of an infectious substance in a sample in which a liquid biological material is pooled. A preferable precipitating agent is polyethylene glycol(PEG) having a mol.wt. range of 1,000-10,000 and having a final concentration of 1-5%(w/v). A monovalent or divalent salt, preferably the monovalent salt, is further added to accelerate the precipitation of the uncleic acid, The monovalent salt is preferably NaCl or KCl. The amount of the salt to be added depends on the concentration of a salt already existing in the biological sample, and is commonly about 145 mM salt which is a physiological salt concentration. The final salt concentration of the mixture is preferably 200-450 mM, most preferably 270-325 mM.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel sample preparation method for pooled samples of liquid biological material that increases the sensitivity of nucleic acid-based diagnostic tests. The present invention also provides kits that are particularly suitable for performing this method.

[0002] In order to improve the safety of blood bank products, national authorities require that such products be analyzed by nucleic acid-based diagnostic tests before being administered to patients. This is due to the fact that antibodies to the infectious virus are often not detected for weeks or months after the actual infection has occurred. On the other hand, often amplification of nucleic acids of infectious matter (eg,
Nucleic acid-based diagnostic tests, including the polymerase chain reaction (PCR), can detect the presence of infectious matter very early after infection.

For example, the human immunodeficiency virus (HI)
V) and the detection of hepatitis C virus (HCV) rely on antibody screening by enzyme-linked immunosorbent assay (EIA). In recent years, the sensitivity of HIV and HCV antibody detection methods has improved, but infection and seroconversion (seroconve
(rsion) is detected. About one third of HCV seroconversion occurs within two weeks after infection (Lim et al., J. Haemat.
ol. 78 : 398), but anti-HCV antibodies
Appears only months later (Van der Poel et al., Vox Sang, 1).
992, 62 : 208, and Farci.
Et al. Eng. J. Med. , 1991, 325 : 9.
8). Current anti-HIV-1 antibody assays are positive on average on day 22 after infection (Busch et al.,
Transfusion, 1995, 35: 91) . Therefore, donor blood that is negative for anti-HCV and anti-HIV antibodies collected during the window period may not be detected by the blood safety screening method. The window period can be shortened by an assay that targets viral nucleic acids. In particular, the PCR assay has a window period of H
For IV-1 infection, 11 days (Busc
h) et al., Transfusion, 1995, 35 : 9.
1), and 59 days for HCV infection (Alter HJ, Blood,
1995, 85 : 1681).

[0004] HCV from blood screened for antibodies
And the risk of transmitting HIV is 4 for HIV-1.
93,000 times, and for HCV, 103,
It is estimated to be 1/1000 (Schreiber et al., N. Eng. J. Me.
d. , 1996, 334 : 1685). This low incidence has raised concerns about the cost-effectiveness of systematic screening of blood by PCR assays (AuBuhon et al., Tansfusion,
1997, 37:45 ). That is, there is a need for a simple and inexpensive nucleic acid amplification-based screening assay.

[0005] Unfortunately, nucleic acid amplification-based methods still utilize some steps that are not easy to automate so that the machine can handle large numbers of test samples simultaneously. Therefore, it has been proposed to pool samples and perform nucleic acid based diagnostic tests on the pooled samples. The pool sample determined to be positive is then, for example,
Each sample in the pool is analyzed further, either individually or by testing different pools of duplicate samples to find out which sample in the pool is positive. That is, positive sample C is easily determined to be positive because pools of samples AB, AC, and BC are analyzed and pools AC and BC should be positive. Of course, in the case of the latter pool test, safety is increased by individually testing the sample C and performing a confirmation test.

A major drawback of the pool method is that pooling reduces sensitivity.

[0007] The need to increase the sensitivity of nucleic acid-based diagnostic tests also exists in tracking the treatment of infected individuals. In order to assess the optimal time for changing or stopping drug treatment, it is necessary to determine when the viral nucleic acid has dropped below the level of detection. Shock Mel (Sch
Ockmel et al. have investigated this problem using an enhanced measurement format for the detection of HIV-1 RNA and have been able to increase sensitivity until 20 copies of RNA can be detected per ml of plasma. (J. of A
cquired Immunity Deficiency
Syndrome and Human Retro
Virology, 1997, 14 : 149-18.
3).

[0008] The problem of further increasing the sensitivity of nucleic acid-based diagnostic tests is solved by the present invention, the basis of which is the method as defined in the appended claims. More particularly, the present invention provides a method for increasing the sensitivity of a diagnostic test for the detection of nucleic acid of an infectious agent in a pooled sample of liquid biological material, the method comprising the steps of: Glycol, dextran sulfate or ammonium sulfate) to selectively precipitate nucleic acids. Suitable precipitants have a molecular weight range of 1,000 to 10,000 and a final concentration of 1 to 1.
5% (w / v) polyethylene glycol (PEG). More specifically, the present invention provides that the precipitant is polyethylene glycol having a molecular weight range of 7,000-9,000 and a final concentration of 2.0-4.0% (w / v) in the pooled sample; More preferably, the final concentration is 2.5 to 3.0%.
(w / v), most preferably a final concentration of about 2.8-2.9%
(w / v) PEG6000. In the method of the present invention, a monovalent or divalent salt, preferably a monovalent salt, is added to enhance the precipitation of the nucleic acid. Preferred monovalent salts are NaCl and KCl. The amount of salt added depends on the salt concentration already present in the biological sample, which is often about 145 mM salt, the physiological salt concentration. The final salt concentration in the mixture is preferably between 200 and 4
50 mM, most preferably 270-325 mM.

In the process according to the invention, the precipitation is preferably carried out at a temperature below 5 ° C., for example from 0 to 4 ° C.

[0010] The method of the present invention is suitable for performing co-precipitation of multiple types of viruses, eg, HCV and HIV (eg, co-infected samples). For control purposes,
For example, defined viruses prepared by recombinant DNA technology can be used as internal controls for quantification purposes.

[0011] The precipitation efficiency is further improved by the addition of carrier nucleic acids. An example of such a carrier nucleic acid is calf thymus D
NA or salmon sperm DNA. The carrier nucleic acid may also be a defined sequence that allows, for example, to regulate the effectiveness of the nucleic acid precipitation of the infectious agent. That is,
For example, a fixed amount of phage λ DNA may be added for this purpose. Method of the present invention (amplification step and detection step)
Another nucleic acid can be added as an internal control to adjust the effectiveness of all diagnostic assays, including. This other nucleic acid may be RNA, DNA, or a synthetic oligonucleotide. If the nucleic acid of the infectious object is RNA, the internal control nucleic acid is also preferably an RNA molecule. The nucleic acid of the internal control preferably has essentially the same length and base composition as the target sequence amplified in the nucleic acid of the infectious agent, and is used for amplification of the target sequence at an appropriate position in the flanking region. Contains a sequence complementary to the sequence of the primer. Thus, the target sequence of the nucleic acid of the infectious object as well as the internal control nucleic acid sequence
Amplified with the same primers, producing an amplification product (amplicon) of the same size. Next, the amplified material is probed using a probe specific for the central region of the internal control nucleic acid.
Tested for the presence of the amplified internal control nucleic acid. Thus, even in samples that do not contain nucleic acids from infectious matter,
The efficiency of the precipitation and / or amplification reaction can be tracked. This confirms that the negative sample is negative, ie, not caused by false precipitation and / or incomplete amplification reactions. That is, when the detection step indicates the presence of an appropriate amount of the internal control nucleic acid in the sample at the end of the operation, it is assured that all steps of the operation (ie, precipitation, amplification and detection) worked well. .

The precipitate obtained by adding the precipitant and, if necessary, the salt, is separated from the liquid, as described above, wherein a centrifugation step is preferably used.

[0013] The next step is the lysis of the infectious substance, which is performed by a number of methods known to those skilled in the art. Suitable methods include addition of chaotropic substances (eg, guanidine thiocyanate), detergents (eg, NP40 or Triton X100), heat treatment, or digestion with a protease such as proteinase K (preferably in the presence of detergents). ). RNA released during the lysis step
In order to stabilize the molecule, inhibitors of RNA degradation (eg, RNasin®, Promega (Prom
ega), Madison, Wis., USA) may be added to the lysis buffer. Dissolution is about 15-
Performed at a temperature of 45 ° C., preferably about 37 ° C., suitably the precipitate is resuspended in 0.5-5 ml, preferably about 2.5 ml of lysis buffer. Suitable lysis buffers include a chaotropic substance, such as guanidine thiocyanate, and preferably dithiothreitol or a similar substance in a suitable buffer. The most preferred lysis buffer is 60 mM
Tris / HCl (pH 7.5), 68% (w / v) guanidine thiocyanate, 3% (w / v) dithiothreitol.

Following the lysis step, an additional purification step is performed to remove PEG from the nucleic acid (possibly inhibiting PCR).
And further remove the protein. This purification is performed in a number of established ways, and the method chosen here is:
AMPLICOR® Test (F. Hoffma-La Roche)
nn-La Roche Ltd. ), A product of a PCR-based test system manufactured by Basel, Switzerland). This test is described in Anal. Biochem. 1987, 16
2 : Chomczinsky P. and Satch N (S) described in 156-159.
acci N. et al. ). However, this method has been modified to increase the purity of the final nucleic acid product used for amplification. That is, for example, the volume of the lysing reagent has been increased to 2.5 ml.

After lysis, the nucleic acids are precipitated. This is usually
Carried out by adding an equal volume of isopropanol (e.g., methanol, ethanol, propanol, and butanol, any other C ₁ -C ₆ alcohols such as pentanol, and liquid forms of hexanol [at room temperature] can also be used ). After a few minutes, preferably after about 2 minutes,
The nucleic acids are pelleted by centrifugation. The nucleic acid is then preferably reprecipitated with about 70% (w / v) aqueous ethanol (instead of ethanol, methanol, propanol and isopropanol, and liquid forms of butanol, pentanol and hexanol [at room temperature] Any other C ₁ -C ₆ alcohol can be used, such as

After centrifugation, the pellet is resuspended in a sample dilution buffer adapted to the amplification method. In a preferred method, the pellet obtained after centrifugation is directly resuspended in a solution suitable for performing RT-PCR or PCR. In the most preferred method, HIV, as exemplified in Example 1,
Solutions adapted to perform each subsequent RT-PCR of HCV and HBV are used. However, one skilled in the art can adapt the method described in Example 1 so that nucleic acids of other infectious agents can be measured individually or in combination.
Also, the volume of the lysis buffer may need to be adapted as a function of the protein content of the sample being tested.

The efficiency of the method of the present invention is supported by the results shown in the accompanying figures.

FIG. Limit of detection of HCV RNA in pooled plasma samples containing decreasing amounts of HCV RNA copies (data from two separate experiments). HCV R
A dilution of the NA positive sample was used and added to a 5 ml pool (from 10 to 0.12 copies / pool). HIV-1 RNA was also added to each pool tested (1,000 copies / pool for experiment 1 and 100 copies / m for experiment 2).
l).

FIG. HIV-1 RNA in pooled plasma samples containing decreasing amounts of HIV RNA copies
Detection limit. A dilution of the HIV RNA positive sample was used and added to the 5 ml pool (from 300 to 33 copies / pool). HCV RNA was also added to each pool tested (100 copies / ml).

The method of the present invention is used to increase the sensitivity of a diagnostic test for the detection of nucleic acids of an infectious agent in a pooled sample of liquid biological material. The term "liquid biological material" means any body fluid, such as, but not limited to, urine, secretions, blood (plasma or serum) and saliva. Pooled samples are usually obtained from different individuals (eg, humans). However, this is not required in that the sample used in the method of the invention may also be a large number of single samples obtained from one individual to increase the sensitivity of the diagnostic method. The term "infectious object nucleic acid" includes any nucleic acid (eg, DNA or RNA) of an organism that can infect a particular individual. Examples of such organisms are fungi, bacteria and viruses. The method of the present invention relates to nucleic acids of hepatitis virus that causes hepatitis (for example, hepatitis B virus [HBV] and C).
A nucleic acid from a hepatitis virus such as hepatitis virus [HCV] or a nucleic acid that causes an immunodeficiency such as AIDS (eg a nucleic acid from a human immunodeficiency virus such as HIV-1 or HIV-2) It is particularly useful for increasing the sensitivity of detection.

The method of the present invention is preferably used to detect infection by a plurality of infectious agents using one common purification step for the purification and concentration of the infectious agents.
In this way, the infectious matter can be concentrated and then detected with higher sensitivity using appropriate amplification / detection methods (eg, RT-PCR and / or PCR). The preferred method allows for the simultaneous detection of HCV RNA and HIV-1 RNA from a pool of plasma samples.

The detection step of the method of the present invention can be designed to address the nucleic acid of each infectious object individually, or can be designed to simultaneously detect the presence of nucleic acids of several infectious objects ( So-called multiplex RT
-PCR and / or PCR). In the latter case, preferably, the so-called reverse dot blot method (for example, edited by Innis et al., 1995 PCR Strategy)
gies, Academic Press (Academic)
Press Inc. ), San Diego, Calif., Middle, Chehab et al., European Patent EP-A-237362, and European Patent Publication E.
PA-529493) is used, wherein the amplified material, which is preferably labeled, is
It is added to a solid support (eg, a membrane, filter, or well of a microtiter plate) containing sequence-specific oligonucleotide (SSO) probes suitable for detecting the infectious substance of interest. In this method, unlabeled SSO probes are exposed to labeled amplified nucleic acids of a pooled sample under appropriate stringent hybridization conditions. Next, the nucleic acids of the unhybridized labeled sample are removed by washing under appropriate stringency conditions, and the filter is followed for the presence of label bound to the probe sequence. The amplified material is
Labeling is performed using a labeled nucleic acid precursor (dNTP) or using a labeled oligonucleotide primer.

[0023] Labeled nucleic acid precursors (dNTPs) are commercially available. Oligonucleotides used in connection with the present invention are prepared by incorporating a detectable label by spectroscopic, photochemical, biochemical, immunochemical, or chemical means based on methods known in the art. Yes, it can be labeled. Useful labels include ³² P, fluorescent dyes, electron-dense reagents, enzymes (commonly used in ELISAs), biotin (eg, enzyme substrates and avidin-enzyme conjugates that are chromogens). Or haptens and proteins for which antigenic determinants or monoclonal antibodies are available.

When many oligonucleotide probes are involved in the detection step, the probes are preferably aligned. In such an alignment, a plurality of oligonucleotide probes are immobilized on the surface of a so-called microchip, preferably made of siliconized glass. Next, in a detection step, the surface of the alignment is scanned for a positive signal emitted by the labeled target bound to the probe. Suitable labels are molecules capable of emitting a fluorescent signal under appropriate conditions. For further details on this microchip technology, see MJ Kozal, et al., Natur.
e Medicine, 1996, 2 (7): 735-
739; Lipshutz et al., Biot.
echniques, 1995, 19 (3): 442-
447 and Eggers M. and Ehrlich D., Hema
tol. Pathol. , 1995, 9 (1): 1-1
See five publications.

The method of the invention is particularly useful for applications in the following fields: testing of donated blood; testing of intermediates and final preparations of biological origin of drug products for use in humans; -Products for human consumption (eg yogurt)
Testing of intermediates and final preparations of biological origin of the drug product for veterinary use;-samples of humans with low potential for infectious matter, eg after antiviral treatment ( (Eg, cerebrospinal fluid or synovial fluid).

The method of the present invention provides a purified preparation of nucleic acids that concentrates nucleic acids from infectious matter in small volumes and allows for more sensitive detection of such nucleic acids with suitable detection methods. I do. A preferred method is described in Example 1. If desired, the method can be adapted by changing the concentration and type of PEG and adapting the concentration of the salt (eg, NaCl). In a preferred method, the PEG and salt concentration are chosen such that several infectious substances can be precipitated simultaneously. In the first step of the most preferred method,
PEG and salts are added at concentrations suitable for the precipitation of HIV, HCV and HBV. It was found that HIV and HBV could be precipitated in the absence of salt and PEG.
HCV, on the other hand, precipitates only in the presence of salts and does not precipitate in a simple centrifugation step as described by Shockmel et al. (Supra). The optimal concentrations of PEG and NaCl have been determined for the three infectious entities described above, and the optimal incubation times and centrifugation speeds used for these entities (see Example 1). These various factors will vary depending on the infectious matter being precipitated. It has been found that the first precipitation step allows removal of more than 95% of the serum proteins in the test sample.

The present invention also provides a kit for determining the presence of nucleic acid of an infectious agent in a pooled sample of liquid biological material, preferably a viral nucleic acid in a blood sample (plasma or serum). A kit comprising a solution of a precipitant as defined above and other reagents required to detect the presence of nucleic acid (eg, a sequence-specific oligonucleotide probe). The latter probe is preferably bound to a solid support. The kit further comprises a monovalent or divalent salt, preferably NaCl or KCl. The kit may further comprise reagents necessary to carry out the amplification reaction, for example, a polymerase (eg, a thermostable DNA polymerase such as Taq polymerase or Tth polymerase) and primers (preferably a primer pair).
including. The primer is preferably labeled.
The reagents are preferably filled in one or more container units, which contain all the necessary to carry out the method of the invention and possibly the amplification and detection of the nucleic acid of the infectious substance. Contains useful ingredients. The kit may also contain instructions for performing the method.

Although the preferred embodiment involves RT-PCR amplification, amplification of the target sequence in the sample can be accomplished by any known method,
For example, ligase chain reaction (LCR), transcription amplification, and self-sustained sequence replication can be used, each of which allows the target sequence to be detected by nucleic acid hybridization to an SSO probe.
Give sufficient amplification. Tea Horn
T. ) And MRS Urdea (Urdea M.)
S. ), Nucl. Acids Res. , 1989,
17 (17): 6959-6967, and JA Running and M.M.
Urdea (Urdea M.), Biotechniq
ue, 1990, 8 (3): 276-279, it is also appropriate to detect the nucleic acid concentrated by the method of the present invention by the branched DNA signal amplification method.

Suitable amplification reactions are known in the art and are described in US Pat. No. 4,683,195; 4,683,2.
No. 02; and 4,965,188. Commercial vendors,
For example, Perkin Elmer (Norwalk, Connecticut) sells PCR reagents and publishes PCR protocols. A PCR summary is provided to facilitate understanding of the advantages of the present invention.

In each cycle of the PCR amplification, the double-stranded target sequence is denatured, primers are annealed to each strand of the denatured target, and the primer is a DNA polymerase (preferably a thermostable DNA polymerase, ie, heat- And is relatively stable and catalyzes the polymerization of nucleoside triphosphates to form a primer extension product complementary to one of the target sequence nucleic acid strands). The DNA polymerase starts synthesis at the 3 'end of the primer and proceeds in the direction of the 5' end of the template until synthesis stops. Purified thermostable DNA polymerase is commercially available from Perkin Elmer (Norwalk, Connecticut). Additional reagents present in a typical polymerase chain reaction mixture are oligonucleotide primers, nucleotide triphosphates, and a suitable buffer. Information on suitable amplification reaction mixtures and suitable temperature cycling conditions is known to those skilled in the art and is available from F. Hoffmann-La Roche.
Roche Ltd. ) Is described in the instructions for the AMPLICOR (R) test kit. The amplification process is typically repeated at least 25 times. The two primers anneal to opposite ends of the target nucleic acid sequence, and the extension product of each primer hybridizes to the other primer in a direction that is a complementary copy of the target sequence and when separated from its complement. Anneal so that you can soy. If each cycle is 100% efficient, the number of target sequences present will double.

[0031] Either DNA or RNA target sequences can be amplified by PCR. In the case of an RNA target (eg, amplification of HCV genomic nucleic acid), the first step consists of synthesizing a DNA copy (cDNA) of the target sequence. Reverse transcription is a separate step or, preferably, reverse transcription-polymerase chain reaction (RT-PCR) (RNA
(A modification of the polymerase chain reaction) to amplify RT-PCR amplification of RNA is known in the art and is described in U.S. Patent No. 5,322,77.
Nos. 0 and 5,310,652; Myers
s) and Gelfund, 1991, B
iochemistry 30 (31): 7661-7
666; U.S. Pat. No. 5,527,669; Young (Y
ung) et al., 1993, J. Am. Clin. Microb
iol. 31 (4): 882-886; and Young et al., 1995, J. Am. Clin. Micr
obiol. 33 (3): 654-657.

Because of the immense amplification potential of the PCR method, samples with high levels of DNA, positive control templates, or low levels of DNA contamination from previous amplifications can be used without the need to add template DNA. The product can be obtained. Laboratory equipment and methods to minimize cross-contamination
Kwok and Higuchi, 1
989, Nature, 339 : 237-238, and Ed. Innis et al., Eds., 1995 PCR P.
rotocols: A Guide to Metho
ds and Applications, Academic Press In
c. ), San Diego, Calif., In Kwok and Orrego. Enzymatic methods for reducing the problem of PCR contamination by nucleic acids amplified in previous reactions are described in US Pat.
18,149, 5,035,996, and Young et al., 1995 (supra).

[0033] The amplification reaction mixture is typically assembled at room temperature, a temperature well below that required to ensure the specificity of primer hybridization. At room temperature, the primers may nonspecifically bind to other only slightly complementary nucleic acid sequences and initiate the synthesis of undesired nucleic acid sequences, resulting in nonspecific amplification. These newly synthesized undesired sequences may compete with the target sequence of interest during the amplification reaction, greatly reducing the efficiency of amplification of the target sequence. Non-specific amplification is a "hot start" that prevents primer extension until the temperature is raised enough to achieve the required hybridization specificity
Can be reduced.

In one hot start method, one or more reagents from the reaction mixture are retained until the temperature is raised enough to obtain the required hybridization specificity. A hot start method using a thermally labile material such as a wax to separate or sequester the reactants is described in US Pat. No. 5,411,876, and Chou et al., 1992, Nucl. Acids R
es. 20 (7): 1717-1723. In another hot start method, reverse inactivation D, which does not catalyze primer extension until activated by high temperature incubation, prior to or as a first step in amplification.
NA polymerase is used. An example of such a reverse inactivated DNA polymerase is AmpliTaq.
GOLD (European Patent Application EP-A-717870; for a review, see Birch et al., 1996, Nature 381 :
445-446). Non-specific amplification also
As described in US Pat. No. 5,418,149, the extension product generated prior to the first high temperature step of amplification can be reduced by enzymatic degradation.

The amount of the nucleic acid of the infectious substance is increased to a detectable level by the PCR method. Methods for detecting amplified nucleic acids are known in the art. For example, the presence and amount of amplification products can be determined by gel electrophoresis using protocols known in the art (eg, Sambrook).
Et al., 1989, Molecular Cloning, Laboratory Manual (Molecular Cloning-AL).
laboratory Manual), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

In connection with the method of the present invention, the detection of the amplification product is preferably performed using an oligonucleotide probe. Preferably, a sequence-specific oligonucleotide (SSO) probe, ie, one or more oligonucleotide probes, wherein each probe is a "hybridizing region" that is completely complementary to the sequence to be detected.
Is used. Typically, such hybridizing regions will hybridize only to perfectly complementary target sequences under “sequence specific and stringent hybridization conditions”. When the stringency of the hybridization conditions is relaxed, non-matching sequences are also allowed. The degree of mismatch that can be tolerated can be adjusted by appropriately adjusting the hybridization conditions. Depending on the sequence to be analyzed, one or more sequence-specific oligonucleotides are used.

A sequence specific for a particular infectious agent, such as a particular isolate, is a sequence that is unique to the infectious agent or the isolate, ie, other infectious agents or such Sequences not shared by a particular isolate of the infectious agent. Probes containing subsequences complementary to sequences specific to the infectious agent are subjected to stringent conditions (eg, 2 × SSC, 0.1% SDS-PAGE at 70 ° C.).
Wash the solid support), will not hybridize to the corresponding portion of the genome of another infectious object. Suitable protocols for detecting hybrids formed between a probe and a target nucleic acid sequence are known in the art. An example of such a protocol is described in US Pat. No. 5,527,669.
No .; Young et al., 1993, supra; and Young et al., 1995, supra.

Another detection method used with PCR is
A so-called 5'-nuclease assay is described in U.S. Pat. No. 5,210,015 and Holland (Hol).
land) et al., Proc. Natl. Acad. Sc
i. USA, 1991, 88 : 7276-7280. In the 5'-nuclease assay, PCR
A labeled detection probe is involved in the amplification reaction mixture. The probe is modified to prevent the probe from acting as a primer for DNA synthesis. During the synthesis step (ie, primer extension), the probe that hybridizes to the target DNA is a DNA polymerase (r
Tth DNA polymerase) is degraded by the 5'-nuclease activity. The presence of the degraded probe indicates both that hybridization between the probe and the target DNA has occurred and that amplification has occurred. A method for detecting probe degradation is described in US Pat.
No. 0,015, EP-A-699768 and EP-A.
A-713921.

A further method for detecting the presence of certain amplified nucleic acids is described in Higuchi et al., 19
92, Bio / Technology 10 : 413-
417; Higuchi et al., 1993, Bi.
o / Technology 11 : 1026-1030;
And European Patent Publications EP-A-487,218 and EP-A-512,334.
In this method, an increase in the total amount of double-stranded DNA in the reaction mixture is tracked. Detection of double-stranded target DNA is performed by double-stranded D
Depends on the increase in ethidium bromide (EtBr) fluorescence when bound to NA and other bound labels. Amplification is double-stranded D
By increasing the amount of NA, an increase in fluorescence can be detected. Since non-specific amplification and primer-dimer generation also result in double-stranded DNA, this method should use appropriate primers that result in low non-specific amplification and primer-dimer generation.

However, because the amplified nucleic acids can also be used in cloning or sequencing methods (see, for example, US Pat. No. 4,683,195), the combination of the method of the invention and the amplification method does not necessarily require detection and measurement. It is not limited to law.

Further information on conventional methods of molecular biology and nucleic acid chemistry, which are known in the art, can be found in Sambrook et al., 1989, Molecular Cloning, Laboratory Manual (Molecular C).
loaning-A Laboratory Manua
1) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; Oligonucleotides
Synthesis) (M. J. Gate (M.
J. Gait), 1984); Nucleic Acid Hybridization
ation) (BD Ham)
es) and S.J. Higgins
ns), ed., 1984); and Methods in
Enzymology (Academic Press (Ac)
ademic Press Inc. ) Series. All patents, patent applications, and publications mentioned herein above and below are hereby incorporated by reference.

The PCR-based analysis of a pool of plasma donors uses a unique nucleic acid preparation to generate HCV RNA.
The following examples provide evidence of their applicability to the simultaneous detection of HIV-1 and HIV-1 RNA. This was done by including a preparation of virus particles that would increase the input of purified viral RNA in the PCR reaction. The input of RNA during the PCR reaction for each sample in the pool is comparable to 2 to 10 times more theoretical plasma volume using this pool method than is used for individual tests by commercial assays. The method of the invention allows detection of less than 3.3 HCV RNA copies per pool of 50 donated blood, corresponding to the detection of each positive plasma sample containing less than 33 copies / ml. This is the most sensitive measurement method currently available (Cobas Amplicor (AMPLI)
COR) (registered trademark) HCV). Therefore, the limit of HIV-1 RNA detection is 50
About 100 HIV-1 RNA per pool of donated blood
Copy.

Recent developments in enhanced assays for HIV-1 RNA detection have included a simple pre-purification step of virus particles using high speed centrifugation to increase the sensitivity of commercial assays by 10-30.
Can be doubled (G Shockmel (Sc
Hockmel G) et al. Acquir. Immu
n. Define. Syn. , 1997, 14 : 179).
As a result, in the course of the research leading to the present invention, the focus of the research was on the detection of HCV RNA. High speed centrifugation of HCV virus particles did not increase the recovery of virus particles in the pellet. The possibility of concentrating HCV virus particles by polyethylene glycol (PEG) precipitation was first investigated. 3% PEG, 145 mM Na
This was partially successful because approximately 50% of the total virus was recovered in the pellet using Cl and low speed centrifugation. The use of higher concentrations of PEG increased the recovery of virus in the pellet, but resuspension of the pellet in lysis reagent was very difficult. At higher frequencies, inhibition of the PCR reaction was observed. The use of large amounts of lysis reagent and complete removal of ethanol were important factors in eliminating inhibition of the PCR reaction. Using the PEG precipitation assay, no inhibition of the PCR reaction was observed in no more than 20 pools out of 50 donated blood tested for HIV RNA and HCV RNA. These results indicate that residual PEG does not inhibit the PCR reaction and that plasma with inhibitory activity is infrequent in the population of blood donors,
Alternatively, PEG precipitation indicates that the inhibitory activity was removed by eliminating the plasma components (Shockmel (S
chockmel) et al. ofAcquired I
mmune Deficiency Syndrome
and Human Retrovirology, 1
997, 14 : 179). In addition, testing pooled samples does not increase background levels. A clear cutoff between positive and negative results was always observed.

A major concern of PCR-based screening methods is the identification of plasma samples having viral nucleic acids in the absence of specific antibodies. These samples correspond mainly to pre-seroconversion samples from individuals with acute infection (De Saussure et al., Tra.
nsfusion, 1993, 33 : 164; MP Bush, Vox San.
g, 1994, 67 (extra number 3): 13; MP Bush (Busch MP) and GA Satin (S
atten GA), Am. J. Med. , 1997,
102 : 117). Samples of 8 individuals before seroconversion were H
CV RNA was positive, and no anti-HCV antibody was detected. When these samples are introduced into the pool, the P
The EG assay detected all of these, including three pooled samples with less than 5,000 copies / ml of RNA. This indicates that binding of specific antibodies to HCV virus particles is not a requirement for PEG precipitation of the virus particles. Unexpectedly low concentrations of HCV RNA were observed in some of the HCV pre-seroconversion samples. The results shown in the examples show the HC-based pool assay
This demonstrates that very sensitive assays are needed for the detection of V seroconversion. High levels of HIV-1 RNA have been reported throughout pre-seroconversion samples (M.
P. Bush (Busch MP) and GA Satin (Satten GA), Am. J. Med. , 1
997, 102 : 117; Kinrocchi de Roes (K
inloch-de Loes) et al. Engl.
J. Med. , 1995, 333 : 408; Baumberger et al., AIDS, 199.
3, 7 (Extra 2): 59) Therefore, the problem of HIV-1 RNA detection was different. Note that the relationship between the minimum detectable level of viral nucleic acid in the blood and the minimum infectious dose is a critical issue.

Another concern is the diversity of virus subtypes. In this sense, the genomic region that is the target of a PCR-based assay must be highly conserved among diverse virus strains. Selection of primers present in the highly conserved 5 'untranslated region of the HCV genome allows detection of the majority of HCV subtypes as shown in the examples below (Buck et al., Proc. Nat.
l. Acad. Sci. USA, 1992, 89:18.
7; Young et al. Clin. Micr
obiol. , 1993, 31 : 882). The detection of HIV subtypes is more problematic due to the reported high variability in the gag gene from which primers for HIV-1 amplification are obtained (Myers et al., Theoretical and Biophysical). Bio
logy and Biophysics), Los Alamos National Laboratory
alLaboratory), Los Alamos, New Mexico, USA, 1995). In addition, recombinant HIV virus strains with gene regions derived from multiple clades have recently been observed (Diaz et al., 19).
95, J.A. Virol. 69 : 3273). Recently, the risk of overlooking specific HIV-1 subtypes due to the realization of new primers that enable the detection of the majority of HIV-1 subtypes during the AMPLICOR® HIV Monitor Test Decreased (Michael
l) et al., IV Conference on Retrovirus and Opportunistic Infection (IV Conference on Retro).
viruses and Opportunistic
Infections), Washington DC,
(Abstract 279), 1997). Of note, however, is the constant worldwide assessment of the genetic evolution of HIV-1.

The following examples of the present invention are for illustrative purposes only and do not limit the scope of the present invention in any way. After reading the foregoing description and the following examples, it will be apparent to one skilled in the art that many embodiments of the invention are possible within the scope of the invention according to the guidelines of the examples.

Example 1 This example describes a suitable protocol for the detection of HIV, HBV, and HCV in pooled plasma. It should be understood that modifications of this protocol are possible, so long as the changes are within the scope of the appended claims.
Such modifications are within the scope of the claimed invention.

Protocol 1. 5000 with or without RNA
μl of plasma pool, 270 μl of 3M NaCl and 320 μl of 50% PEG 6000 (eg Fluka 81304) aqueous solution in a 15 ml sterile test tube (eg conical polypropylene tube such as Falcon tube # 2097); Becton Dickinson ( B
ecton Dickinson Co. 2. Mix and incubate on ice for 30 minutes; 3. Centrifuge at 5,000 rpm for 30 minutes at 4 ° C. (eg, Heraeus minfuge)
4. Discard the supernatant (eg, with a Pasteur pipette in a disposer); 5. 2.5 ml of lysis buffer (60 mM Tris / HCl (pH
7.5), 68% (w / v) guanidine thiocyanate, 3%
(w / v) dithiothreitol); 6. Incubate at 37 ° C. for 10 minutes (eg in a standard incubator or water bath); 7. Add 2.6 ml of isopropanol (methanol, ethanol, Propanol, and butanol,
Liquid form of pentanol and hexanol [at room temperature]
8. Incubate at room temperature for 2 minutes (about 15-25
9.) Centrifuge at 5,000 rpm for 15 minutes at room temperature (e.g., Heraeus minfuge (Heraeus min.
10. Discard the supernatant; 11. Add 1 ml of 70% (v / v) aqueous ethanol solution (instead of ethanol, liquid forms of methanol, propanol, isopropanol and butanol, pentanol and hexanol [ Other alcohols such as at room temperature can also be used); transfer to a 12.2 ml tube (eg, Salstedt (Sa)
rstted), No. 13. Centrifuge at 15,000 rpm for 5 minutes at room temperature (eg, Eppendorf 5415 centrifuge); 14. Discard supernatant; 15. 55 μl HIV sample diluent [10 mM Tris-HCl, 0.1 mM EDTA, 20 μg / ml polyrA
RNA, 0.05% (w / v) sodium azide] and resuspend the pellet; 16. Take 25 μl of the resuspended pellet and add 25 μl of sample diluent; 17. Add 50 μl of this mixture Take 25 μl of the resuspended pellet from step 15 and use 25 μl of 2 × HCV sample dilution (100 mM Bicine, 200 mM potassium acetate, 14 mM manganese (II) acetate, 0.05% (w / v) sodium azide)
19. Use 50 μl of this mixture for amplification and detection of HCV.

The above method may be used for HCV, HIV, and HBV
Combined with standard methods using the AMPLICOR (R) test kit for <RTI ID = 0.0> a </ RTI> allows specific detection of HCV, HIV, and HBV in pooled samples.

With respect to step 1, it should be noted that the sample input is important in that it can remove substances which may inhibit amplification only by precipitation. The addition of this suitable concentration of salt is critical for the precipitation of HCV. Recovery of HCV nucleic acid was less than 50% after 1 hour centrifugation at 25,000 xg in the absence of PEG and salts. Different concentrations of different types of PEG can also be used. However, it should be noted that changing the molecular weight of PEG and / or its concentration may also require changing the type and / or concentration of the salt added to aid precipitation. If the viruses precipitated from the pooled samples differ from those described above, it may be necessary to optimize the PEG and salt concentrations for these viruses.

With respect to step 5, it should be noted that resuspending in a large volume of lysis buffer is critical because this helps to remove the precipitant (PEG). If the volume is less than 1 ml, partial or complete inhibition of the PCR reaction may occur.

The conditions described in step 15 are particularly suitable for detecting HCV. When further testing for HCV is performed, the latter solution is added at twice the concentration. HC
If only V is tested, the pellet is resuspended directly in HCV sample diluent [50 mM Bicine, 100 mM potassium acetate, 7 mM manganese (II) acetate, 0.05% (w / v) sodium azide]. You may. This volume is much lower than the input sample volume (55 μl / 5 ml serum) than the volume (1 ml / 100 μl serum) in the standard AMPLICOR® monitoring method. As a result, an amount equivalent to a starting volume of 5 ml is used instead of 5 μl to start the PCR. This increases sensitivity by a factor of 1000 when using one sample, rather than a pool of many samples. Of course, when many samples are pooled, the sensitivity is reduced as compared to individual samples.

The method of Example 1 makes it possible to reproducibly detect about 100 copies of HCV nucleic acid in 5 ml of pooled plasma. HCV nucleic acid recovery rate is 70-100%
(Starting plasma containing 8000 copies of HCV nucleic acid per ml was first tested in a standard HCV Amplicor® test, and then diluted 400-fold after the sample was diluted. Based on the method tested)).

The assay of the present invention is particularly useful for any method for detecting HCV. High speed centrifugation (50,
000 g for 60 minutes), it was observed that the HCV virus particles in the pellet were not concentrated. Therefore, before dissolving the virus particles, polyethylene glycol (PE)
G), the NaCl precipitation method was performed as the first step. The optimal concentrations of PEG and NaCl required to achieve maximum recovery and convenient pellet resuspension were evaluated. The addition of 3% PEG and 145 mM NaCl to the plasma pool has been found to be optimal. 50 unselected donated blood 100
For 15 pools containing μl, a quantitative detection assay (AMPLICOR® H
The potential for inhibition of the PCR reaction was evaluated using a CV Monitor.

[0055]

[Table 1]

Table 1 shows the results of 15 pools tested with and without the addition of plasma containing known HCV RNA copy numbers. The table uses the following abbreviations: OD
Means optical density (Amplicor (AMPLIC)
OR) (registered trademark) HIV Monitor (HIV Moni)
tor)), IQS means internal quantitative standard, D
F means the dilution factor of the detection method. All 15 pools tested were HCV RNA negative (OD HCV <
0.2), and all pools (5,000-45 copies / 5 ml) containing a known number of HCV RNA copies were positive. 30 containing 750 donated blood plasma samples
In the PCR reaction, no inhibition of the PCR reaction was observed as indicated by the OD value of the internal HCV quantitative standard (IQS).

The sensitivity limit of the PEG method for the detection of HCV RNA in a 5 ml pool was evaluated using a quantitative detection method (Cobas Amplicor (AMPLIC)).
OR) HCV Test (HCV Tes)
t)). The sensitivity of this assay is about 100 HCV RNA copies / ml, which is at least 10 times higher than the quantitative assay.
Twice lower. FIG. 1 shows the percentage of HCV RNA positive pool according to the input amount of HCV RNA copy / pool. The HCV RNA-positive plasma used in the two experiments was quantified in duplicate at four dilutions, with an average HCV RNA level of 2
3,300 copies / ml (range, 16,340 to
30, 210). This patient was infected with HCV genotype 4h. HCV RN of 10 and 3.3 respectively
All duplicates (1 in 5 ml pool containing A copies)
0/10 and 14/14), 13/14 multiplex containing 1.1 copies, and 2/14 multiplex containing 0.37 copies are positive, multiplex with 0.12 copies None of the items were positive. In the 58 pools tested, the OD value of the internal control (IC) was 3.
Since it exceeded 0, no inhibition of the PCR reaction was observed. HIV-1 RNA was also added in each pool tested (1,000 copies / pool for the first experiment and 100 copies / pool for the second experiment). All 58 pools tested were positive. 1
Of the HIV-1 RNA copy of 2.056 to 0.219 (1/125 D
OD HIV in the range of F) is always clearly detected and P
No inhibition of the CR response was observed (OD IQS>
3.5).

The sensitivity limit of the PEG method for the detection of HIV-1 RNA in a 5 ml pool was evaluated using dilutions of HIV-1 RNA positive plasma (FIG. 2). Samples were quantified in duplicate at two dilutions and the average HIV RNA level was 40,000 copies / ml (range, 35,8
00 to 49,450). 300 and 100 HIVR
All of the 5 ml pool containing NA copies, and 33
Of the HIV-1 RNA copies were positive. HCV-1R at each pool tested
NA was also added (100 copies / pool) and all 15 pools tested were positive. No inhibition of the PCR reaction was observed (OD HIV-1 IQS> 3.0 and OD HCV IC> 3.7).

HC between pellet and supernatant after PEG precipitation
V RNA separation was assessed using a quantitative detection assay (Table 2). One individual (K) with established HCV infection and three pre-seroconversion individuals (S1, S2, S
Using four different plasma samples with known HCV RNA copy numbers from 4), the plasma 1
Added to 5 ml pool containing 00 μl. The HCV RNA concentration of the HCV RNA in the supernatant after PEG precipitation was assessed by standard methods (AMPLICOR® HCV Monitor) using 100 μl of supernatant. HCV
Total recovery of RNA (pellet + supernatant) was 53-132
% And the recovery of HCV RNA in the pellet was 4%.
It varied from 0 to 68%. Similar HCV RNA recovery was observed for the established HCV infection and pre-seroconversion samples. The genotype of the HCV strain used in sample K is 1a. The HCV genotypes of samples S1, S2, S4 are shown in Table 3.
Shown in In this experiment, sample S1 was used at 1/10 dilution.

[0060]

[Table 2]

To determine whether the presence of the complexing antibody affects the ability to precipitate the virus,
Pre-seroconversion sample (HCV RNA positive, anti-H
CV antibody negative) and analyzed in 5 ml pools. 8
Five 10-fold dilutions of the pre-seroconversion sample were used and added to a 5 ml pool.

[0062]

[Table 3]

Table 3 shows the number of positive pools according to the dilution of the sample before seroconversion. HCV RNA levels of the pre-seroconversion sample were measured in duplicate and were determined to be 1,250-762,000
It fluctuated in copies / ml. Using 100 μl of undiluted plasma sample, the three pre-seroconversion samples with the lowest HCV RNA levels (S3, S4, S5) are positive in duplicates and 多重 multiplex measurements are positive in 1/10 dilution (Input volume 10 μl / pool). All other pre-seroconversion samples were constantly detected at 1/100 dilution in duplicate. Based on the dilution analysis, the different HCV genotypes tested (1
a, 2a / c, 3a, 3c, 4c / d, 4h)
No difference in CV RNA detection limit was observed.

Example 2 Pool of Donated Blood Whole blood was collected from a blood donor in a 7 ml EDTA test tube (Transfusion Center, Geneva University Hospital (Geneva).
(University Hospital)), 1,
Plasma was separated by centrifugation at 000 g for 10 minutes at room temperature.
A pool of 50 donor blood was made using 2 ml of each plasma and the pool was centrifuged again at 1,000 g for 10 minutes. A 5 ml pool and an aliquot of each plasma were stored at -75 ° C within 4 hours. The test protocol was approved by the local ethics committee. HC with known RNA copy number
Dilutions of V or HIV-1 positive plasma were added to the pool to assess the sensitivity of the PCR-based pool assay.

[0065] seroconversion patient continuously sample from anti-HCV antibody-negative (Akishimu (Axsy
m) 3.0, Abbott Laboratories (Abbott)
Laboratories), Abbott Park (Abb)
ot Park), Ill.) and HCV RNA positive (AMPLICOR® H).
Plasma samples for the CV Test were selected from our -75 ° C plasma collection.

Sample Preparation The extraction of HCV RNA and HIV-1 RNA was performed according to the manufacturer's instructions (AMPLICOR® Monitor, Monitor), but with some modifications, A polyethylene glycol (PEG) precipitation step before
In order to increase the input amount of the extracted RNA during the PCR reaction, the reagent volume was changed. Briefly, 145 mM N
aCl and 3% PEG (PEG6000 solution, Fluka, Buchs, Switzerland)
In a 15 ml test tube (Falcon, Becton Dickinson)
n), Lincoln Par
k), New Jersey). Incubate the tubes on ice for 30 minutes, then
Centrifuged at 500 g for 30 minutes at 4 ° C. Remove the supernatant,
The pellet is again mixed with 2.5 ml of lysis reagent (Coba
s) Amplicor (registered trademark) H
CV) and maintained at room temperature for 10 minutes. After adding 2.6 ml of isopropanol, the tubes were centrifuged at 1,500 g for 15 minutes at room temperature. The pellet was resuspended in 1 ml of 70% ethanol and mixed in a 1.5 ml test tube (Sarstedt, Severe).
n), Switzerland) and centrifuged at 12,500 rpm for 5 minutes at room temperature. The ethanol was completely removed using a disposable Pasteur pipette and the test tube was opened for 15 minutes to remove traces of ethanol. Pellets 55
μl of HIV sample diluent (Amplicor (AMPLIC)
OR) (registered trademark) HIV Monitor (HIV Moni)
tor)).

Increased HCV RNA and HIV-1 RNA
Width and detection For the HCV PCR reaction, add 25 μl of extracted sample to 2
5 μl of HCV sample diluent (2 ×) (containing 2 × internal control (IC)) was mixed with 50 μl of HCV master mix. The amplification cycle and detection of HCV RNA
Cobas (C) offers complete automation of the PCR and detection steps
obas) performed according to the manufacturer's instructions of the device. H
For the IV-1 PCR reaction, 25 μl of the extracted sample was mixed with 25 μl of the HIV sample diluent and 50 μl of the HIV master mix. PCR is performed by Amplicon (AM
PLICOR® HIV Monitor (HIV
Monitor) and the number of amplification cycles was increased from 36 to 40 using the manufacturer's instructions. An internal HIV quantitation standard (IQS) was added to the lysis reagent (6.6 μl IQS per sample). This allows for adjustment of the recovery of RNA during the purification process.

The optical density (OD) of the HCV detection exceeds 0.2, the OD of the IC exceeds 0.2, and the O
When D exceeds 0.2 and IQS OD exceeds 0.3,
The sample was considered positive.

HCV RNA and HIV-1 R of each sample
The quantification of the NA level is determined by Amplico (AMPLICO)
R) (Monitor) (Roche) according to the manufacturer's instructions.

HCV Genotypes HCV genotypes and subtypes are
After amplification on the LICOR® HCV system, a commercially available assay (INNO-LiPA HC
V II, Innogenetics
sN. V. ), Zindrecht (Zwijndrec)
t), Belgium).

[Brief description of the drawings]

FIG. 1 shows the limit of detection of HCV RNA in pooled plasma samples containing decreasing amounts of HCV RNA copies.

FIG. 2 shows the limit of detection of HIV-1 RNA in pooled plasma samples containing decreasing amounts of HIV RNA copies.

Claims (13)

[Claims] Claims: 1. The sensitivity of a diagnostic test for the detection of nucleic acids of an infectious substance in a pooled sample of liquid biological material, characterized in that the nucleic acids are selectively precipitated by adding a precipitant. A preferred precipitant has a molecular weight range of 1,000 to 10,000 and a final concentration of 1
Such a method, wherein the method is ~ 5% (w / v) polyethylene glycol. 2. The precipitant has a molecular weight range of 7,000 to 9,000 and a final concentration of 2.0 to 4.0 in the pooled sample.
The method according to claim 1, wherein 0% (w / v) polyethylene glycol, preferably a final concentration of about 2.8-2.9% (w / v) PEG6000. 3. Monovalent or bivalent to aid precipitation of nucleic acids.
A process according to claim 1, wherein a valent salt is added, preferably a monovalent salt. 4. The method according to claim 3, wherein the monovalent salt is NaCl or KCl, preferably wherein the salt is added to a final concentration of 200-450 mM, most preferably 270-325 mM. 5. The method according to claim 1, wherein the precipitation is performed at a temperature of less than 5 ° C. 6. The method according to claim 1, comprising co-precipitation of a plurality of types of virus. 7. The method according to claim 7, wherein the material is a chaotropic substance.
7. The method according to any one of the preceding claims, wherein lysis of the infectious matter is followed. 8. The process according to claim 7, wherein an additional purification step follows, preferably a precipitation step using isopropanol or another C ₁ -C ₆ alcohol. 9. To increase the sensitivity of detecting nucleic acids of an infectious object in a pooled sample of liquid biological material, or to increase the detection sensitivity of each object in various biological samples. Use of the method according to any one of claims 1 to 8 in a diagnostic test. 10. A kit for measuring the presence of nucleic acid of an infectious agent in a pooled sample of liquid biological material, preferably a viral nucleic acid in a blood sample (plasma or serum). Such a kit comprising a solution of polyethylene glycol as defined in 1 or 2, and preferably a solution of other reagents required to detect the presence of nucleic acid (eg, a sequence-specific oligonucleotide probe). 11. A monovalent or divalent salt, preferably Na
The kit of claim 10, further comprising a solution of Cl or KCl. 12. The kit according to claim 10, further comprising a solution of guanidine thiocyanate in a suitable buffer. 13. The kit according to any one of claims 10 to 12, further comprising reagents necessary for performing an amplification reaction and preferably a detection step.