KR100312800B1 - Improved method for the quantification of nucleic acid - Google Patents

Abstract

The present invention is related to an improved method for the quantification of nucleic acid, which can be performed with a minimal amount of nucleic acid amplification reactions. The method according to the invention for the quantification of analyte nucleic acid in a sample comprises the steps of: adding to the sample different respective amounts of different nucleic acid constructs, each construct being distinguishable from the analyte nucleic acid and capable of being co-amplified with the analyte nucleic acid; subjecting the sample to a nucleic acid amplification procedure, using amplification reagents capable of reacting with both the analyte nucleic acid and the nucleic acid constructs; detecting the relative amounts of amplificates derived from analyte nucleic acid and each nucleic acid construct; calculating the amount of analyte nucleic acid from said relative amounts. Each nucleic acid construct is different; the nucleic acid constructs can be distinguished from one another and from the analyte nuclic acid. The nucleic acid constructs do resemble each other, and the analyte nucleic acid, in that all are capable of reacting with the same amplification reagents.

Description

Method for Quantifying Nucleic Acids {IMPROVED METHOD FOR THE QUANTIFICATION OF NUCLEIC ACID}

The present invention relates to an improved method for quantifying nucleic acids. Nucleic acid quantification can be very important in the diagnostic field as well as in various research fields. Nucleic acid quantification is an important tool for understanding gene regulation and can also be used to monitor therapeutic effects. For example, the amount of a particular nucleic acid sequence present in a sample, such as human blood or other body fluids, may provide valuable information regarding the disease state of a person infected with, for example, a particular virus, and the effectiveness of treatment with a particular medication.

Various methods for quantitative amplification of various nucleic acids have been disclosed. WO 91/02817 describes methods for quantifying nucleic acids using polymerase chain reaction (PCR) as an amplification technique. The method includes adding a standard nucleic acid segment to a sample, which is capable of reacting with the same primers used to amplify unknown amounts of target nucleic acid present in the sample. The amount of each of the two PCR products is then amplified and measured and extrapolated to a standard curve to quantify the amount of target segment in the original sample. Standard curves are constructed by plotting the amount of standard segments produced in the polymerase chain reaction against various but known amounts of nucleic acid present prior to amplification.

The addition of other sequences that can be amplified simultaneously with the analyte nucleic acid to a sample containing the analyte nucleic acid is also described in Becker and Hahlbrock, Nucleic Acids Research, Vol. 17, Number 22. (1989). The method described by Becker and Halbrok contains several known amounts of internal standard material in which only one nucleotide contains a nucleic acid that is analyte and another (point mutant) nucleic acid sequence, and contains an unknown amount of nucleic acid that is analyte. Nucleic acid is quantified by adding it to a known volume of sample fraction and simultaneously amplifying by PCR with the nucleic acid to be analyzed. Thus, identical fractions of total RNA are "spiked" by a reduced amount of internal standard RNA.

By introducing one base change into the internal standard sequence, a specific restriction site is made and the variant sequence is cleaved with an appropriate restriction enzyme and the sample containing the amplified nucleic acid is then subjected to an electrophoretic gel. Nucleic acid is quantified by comparing the variant sequences (parts thereof) and the bands in the gel representing the nucleic acid being the analyte. One of the drawbacks of this method is that incomplete degradation of internal standards by restriction enzymes can yield inaccurate results in determining the amount of nucleic acid present.

Becker and Halbrock use internal standards as markers in the quantification method, where the analytes and markers are amplified non-competitively.

Methods for quantifying nucleic acids using the addition of a known number of molecules of nucleic acid sequence corresponding to a target nucleic acid to a sample containing an unknown amount of target nucleic acid sequence are also described in co-pending applicant's European publication under EP 525882. It has been described in a patent application. The method as described in EP 525882 is based on the principle of amplifying nucleic acid from a sample containing an unknown concentration of wild-type target nucleic acid with the addition of a well-known known amount of variant sequences. Amplification is carried out by a group of primers capable of hybridizing not only the variant sequence but also the target sequence. The competitive amplification method described in EP 525882 can be carried out using a fixed amount of sample and a series of diluted variant sequences or vice versa.

The method described above includes the step of adding a clearly defined standard sequence to the amplification mixture. In order to be able to obtain accurate and reliable measurements with the methods described above, many amplification reactions must be carried out as follows. The sample should be divided into known fractions of each fraction, with each known quantity of standard nucleic acid added. Or a standard curve from a series of dilutions with a method as described in WO 91/02817, which is somewhat cumbersome, and also because the actual amplification for the target nucleic acid and the amplification for the standard curve are performed separately. May be inaccurate.

The method described above is very laborious and time consuming because a large amount of amplification reactions must be performed to determine the amount of nucleic acid in an accurate and reliable manner. Therefore, a method of quantifying nucleic acids with less effort and less time is needed. The present invention provides such a method.

The present invention comprises the steps of adding to the sample different amounts of different nucleic acid constructs that can be distinguished from and co-amplified with the nucleic acid to be analyzed;

Treating the sample with a nucleic acid amplification procedure using an amplification reagent capable of reacting with the analyte nucleic acid and the nucleic acid construct;

Measuring a relative amount of the analyte nucleic acid and the amplification derived from each nucleic acid construct;

It provides a method for quantifying an analyte of a nucleic acid in a sample consisting of calculating the amount of the analyte of analyte from the relative amount.

In the method of the invention, several nucleic acid constructs are added to the sample.

Each nucleic acid construct is different and the nucleic acid constructs can be distinguished from each other and from the nucleic acid being analyte, but the nucleic acid constructs can react with each other and with the subject in that they can react using the same amplification reagents. It is similar to nucleic acid which is water. Amplification reagents, among others, mean one or more amplification primers that can hybridize to the analyte nucleic acid and nucleic acid construct. Other amplification reagents are conventional reagents used in the amplification procedure, such as nucleic acid polymerase, which is an essential enzyme used in other amplification techniques known in the art. The method according to the invention can be used with any kind of amplification procedure, for example the so-called polymerase chain reaction (PCR) as described in USP 4,683,195 and 4,683,202. Another method of nucleic acid amplification is "amplification based on nucleic acid sequence (NASBA)" as described in European patent application EP 0,329,822.

Nucleic acid constructs used in the method according to the invention are nucleic acid sequences similar to the nucleic acid to be analyzed in that they can be reacted with the same amplification reagent. Thus, each nucleic acid construct must comprise at least a sequence to which the nucleic acid amplification primers used can anneal, which sequences are also present in the nucleic acid to be analyzed. The nucleic acid constructs used in the method according to the invention can be distinguished from each other and from the nucleic acid being the analyte. This can be obtained by constructing a nucleic acid construct such that each construct contains a distinguishable non-repeat sequence that does not exist in the other nucleic acid construct or nucleic acid that is being analyzed. Preferably, nucleic acid constructs use random sequences whose distinguishable non-repeat sequences contain, for example, about 20 nucleotides, such that the length and nucleotide components of the construct and the analyte nucleic acid remain the same. In this detection method, after amplifying the analyte nucleic acid and the nucleic acid construct present in the sample, the various nucleic acid constructs can be measured separately by using different detection probes, wherein each probe is contained in the nucleic acid construct. Only one non-repeating sequence present can be recognized. Of course, there are other ways in which nucleic acid constructs can be constructed to have uniqueness. However, it is desirable that no mutation of the nucleic acid construct interfere with the amplification. Preferably, in the method according to the invention, the nucleic acid construct may be amplified with the same efficiency as the analyte nucleic acid, otherwise the amount of the analyte nucleic acid and the construct nucleic acid present in the sample after amplification. It is difficult to calculate accurately based on

Nucleic acid constructs are polynucleotides that can be prepared by a variety of methods known in the art. Nucleic acid constructs can be prepared, for example, by various recombinant DNA strategies. For example, after digesting the plasmid containing the analyte sequence with a restriction enzyme, the original sequence is removed and inserted into the analyte sequence by inserting a new sequence that can be prepared on a nucleic acid synthesizer or subcloned from another plasmid. New sequences can be introduced. Complete nucleic acid constructs can also be prepared using nucleic acid synthesizers.

Nucleic acid constructs must be added to the sample prior to subjecting the sample containing the nucleic acid of unknown analyte to the amplification procedure. Samples to which known amounts of various nucleic acid constructs are added must be of a predetermined dose so that the concentration of nucleic acid present in the sample can be calculated thereafter. The sample must contain a known dose or amount of material taken from the material whose nucleic acid amount is to be measured. When the concentration of nucleic acid present in a particular test solution must be measured, the sample must contain all nucleic acids that are separated from the known amount of test material under study.

Nucleic acid constructs may be added before or after the test solution under study (eg, human serum) is subjected to a nucleic acid isolation procedure. Methods for isolating nucleic acids have been disclosed and are known to those skilled in the art. Nucleic acid isolation procedures that can be used to prepare samples to be processed by the method according to the invention are described, for example, in Manatis et al., Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratory. Another method for treating nucleic acid containing samples is described in Boom et al., J. Clin. Microbiol. 28: 495-503, 1990.

In a preferred embodiment of the invention, the nucleic acid construct is added before the nucleic acid is separated from a certain amount of test solution. The loss of nucleic acid that can optionally occur during the isolation procedure in this method will be known from the final amount of nucleic acid, which is the analyte and the construct, present in the sample. From the results obtained, the amount of nucleic acid, an analyte, present in the original test solution under study can be directly calculated.

When nucleic acid constructs are added to a known amount of test solution prior to treatment of the test solution with the nucleic acid separation procedure, the nucleic acid construct for isolation control may be added to the sample after the nucleic acid separation procedure is performed. By adding an IC (separation control) sequence, it is possible to measure the efficiency of the nucleic acid isolation procedure. Thus, the initial value for each sample can be adjusted according to the separation efficiency. The isolation control construct may be a different sequence than the analyte and other nucleic acid constructs, for example because the construct contains non-repeat sequences, but preferably is amplified with the same efficiency. The IC can be considered as another “nucleic acid construct”, with the difference that the IC molecule is added after the nucleic acid isolation procedure is performed, whereas the nucleic acid construct in a preferred embodiment of the present invention provides a test solution for the nucleic acid separation procedure. Is added before treatment. As described above, after the correct amount of molecules of each nucleic acid construct is added, the test solution is treated with a nucleic acid separation procedure, and after the nucleic acid separation procedure is performed, the correct amount of IC molecules is added, thereby calculating the efficiency of the separation procedure. For example, when 100 molecules of a particular nucleic acid construct are added and the same amount of IC molecules is added, 100% separation efficiency will yield the same signal (after amplification) for the nucleic acid construct and IC. If the IC signal obtained is twice that of the signal obtained for the nucleic acid construct, the efficiency of the nucleic acid isolation procedure is only 50% (meaning that half of the original nucleic acid molecules were lost during the isolation procedure). This can reduce the risk of false negative test results.

One or more nucleic acid constructs are each added in various known amounts. Preferably, the nucleic acid construct is added in a range of amounts that differ from each other by a certain coefficient (eg, a coefficient of 10). For example, three nucleic acid constructs QA, QB and QA 10 ² molecules, QB QC if used may be added to the sample 10 ³ molecules, QC 10 ⁴ molecules.

Samples may also be divided into one or more reaction volumes, each of which may be added at known doses, with nucleic acid constructs in each of a range of amounts. Preferably, the same nucleic acid construct is added to each reaction dose. For example, when two reaction doses are used, a "low concentration range" reaction and a "high concentration range" reaction can be carried out. For example, when they duplicate the scope of the nucleic acid construct, in the low concentration range of the reaction capacity of the QA, QB, and, while that may be added in an amount as described above, the QC, the high concentration range of the reaction capacity, QA, QB and QC, respectively 10 ^4, 10 ⁵ and 10 ⁶ molecules are added. The ranges of amounts of nucleic acid constructs may overlap, but there may also be gaps between the low and high concentration ranges of nucleic acid constructs. The use of more than one reaction volume means that the amplification reaction should be carried out more than once, but the work can be done in comparison to the conventional methods for quantifying nucleic acids since the concentration of a very wide range of analytes can be quantified with a minimum reaction capacity. And time savings.

The advantage of using a range of amounts of non-overlapping nucleic acid constructs is that although the number of constructs used is reduced, a wide range of concentrations can still be quantified. If the analyte is present in an amount that falls within the gap between the ranges of the nucleic acid construct amounts, there is no direct comparison with the signal range derived from the nucleic acid constructs present in the sample or a certain amount of reaction, but the signal of the analyte nucleic acid. Since it is stronger than the signal in the low concentration range and weaker than the signal in the high concentration range, the amount of the analyte nucleic acid can be measured therefrom, and the data can be used to calculate the concentration.

When a range of nucleic acid constructs is added to one or more reaction doses using the method according to the present invention, the samples for which the concentration of the analyte nucleic acid has to be determined should be divided into several doses and the nucleic acid constructs at these doses. Each dose is then added and subjected to nucleic acid isolation procedures. Although each dose must be treated with a separate separation procedure, the variation in performance is corrected by the addition of nucleic acid constructs that are subjected to exactly the same treatment and treated with the same changes, so that any loss of analyte nucleic acid during the separation is corrected. No calibration is required. Of course, the total dose can be treated with a nucleic acid separation procedure and divided into several doses, after which a range of constructs will be added.

A problem that may arise when performing the amplification reaction is that the sample may be contaminated with nucleic acid molecules, for example, from other samples previously tested in the same laboratory. This can lead to false positive test results or, in the case of quantitative measurements, false results in the calculation of the number of nucleic acid molecules present in a sample.

If the sample tested by the method according to the invention is contaminated in the range of 10 to 100 molecules, the internal calibration line does not change, but the contamination is mainly composed of the analyte molecule and the amount of nucleic acid that is the analyte in the sample being tested. Less than 100 molecules will only affect the calculated amount of the nucleic acid molecule as analyte. Contamination with about 100 to 1000 molecules will slightly affect the internal calibration lines, and if the amount of the analyte molecule present in the sample is less than 1000 molecules, it will interfere with the calculation of the amount of the analyte nucleic acid molecule present in the sample. will be. Contamination above 1000 molecules will change calibration lines and calculated values.

Preferred embodiments of the present invention can be used to detect contamination of a sample with nucleic acid molecules from previously tested or other samples.

When the method according to the invention is used to test more than one sample, contamination can be detected if the same nucleic acid construct is used in all samples and the amount of each particular nucleic acid construct varies. For example, three if the nucleic acid construct is used may be a small addition of offering the following amount of the (Qa, Qb and Qc), the sample "A": Qa _{high, low medium} and Qc Qb, sample "B _"and the same in the construct _that Qa, Qb, while that may be added in an amount _of Qc, sample" C "there are added in an amount such as _{one, that} Qb, Qc _and Qa. Of course, the same composition of nucleic acid constructs can also be used in one or more samples, eg, in a series of ten samples, before converting to use another composition.

If three constructs are used, the nucleic acid constructs of the same composition can be made up to six variants before it must be used repeatedly. If four constructs are used, up to 24 variants can be made. Using nucleic acid constructs of different compositions in different samples may cause other compositions to be contaminated with nucleic acid material derived from the sample used for detection. Can be. Contamination will change the internal calibration line of the sample under study, i.e. the slope of the calibration line will change, and the correlation coefficient will also be lower than normal data to distinguish it as contamination.

If one of the constructs in each sample is used in an amount of zero (and the construct used in an amount of zero varies from sample to sample), very few contaminating molecules can be detected. If a particular construct added in an amount of zero is present in a detectable amount, it can be clearly seen that this particular sample is contaminated with material from the sample from which the other composition was chosen.

The detection of nucleic acid constructs and the analyte nucleic acid after amplification can be performed in a number of ways. As long as the measurable signal is generated differently depending on the amount of amplified nucleic acid (construction or analyte) it represents, many detection procedures known in the art can be used. Detection methods that can be used with the method according to the invention include, for example, detection methods using solid labeling factors such as gold or dye sol and detection methods based on aggregation, as well as enzymatic and luminescent (fluorescent, electrochemical luminescent, Phosphorescent) phenomenon.

The concentration of the analyte nucleic acid present in the sample can be calculated from the relative amounts of the analyte nucleic acid and the nucleic acid construct represented by the signal generated during the detection procedure.

For example, log (Q / A), which is the logarithm of the ratio between the signal representing the construct (Q) and the analyte (A) sequence, is log (Q, which is the logarithm of the amount of construct added to the sample. -Injection). If log (Q / A) is plotted as a function of Q (injection), a linear graph should be obtained, where the amount of the analyte nucleic acid can easily be obtained from the point where it intersects the X axis in a straight line. The amount of nucleic acid that is analyte should be near the range of the amount of nucleic acid construct added).

Another embodiment of the present invention includes a method for quantifying a nucleic acid to be analyte in a panel of test solutions, including diluting a predetermined amount of test solution to a known coefficient. Samples may be taken from the diluted test solution containing the amount of nucleic acid that is analyte expected to fall within a certain range. When the amount of analyte nucleic acid present in each panel of test solutions has to be determined, the entire panel can be prediluted to a concentration expected to fall within this range. The preliminary dilution should be carried out so that the majority of the samples are at concentrations within this range and the remaining samples are all at or below this range, or all at or above the range. After the dilution step, the entire panel is treated with the method according to the invention as described above, wherein the amount of nucleic acid construct added to the sample should fall within this range.

The remaining test solution taken from the panel below (or above) the above range can be retested with other adjusted dilutions or undiluted samples. The advantage of this method is more than that required for the high / low concentration range described above. Less amplification reactions are required.

Another embodiment of the method for quantifying an analyte nucleic acid present in a sample according to the present invention comprises adding to the sample an amount of a nucleic acid construct that is estimated to be within the same range as the expected amount of the analyte nucleic acid present in the sample. Treating the sample with a nucleic acid amplification procedure using an amplification reagent that can react with both the nucleic acid and the nucleic acid construct to be analyzed, and the relative amounts of the amplification derived from the nucleic acid and the nucleic acid construct as the analyte. Detecting, and estimating the amount of nucleic acid that is an analyte present in the sample from this relative amount. After this calculation, the second sample taken from the same test solution can be processed by the method according to the present invention, wherein the amount of the nucleic acid construct added is equal to the estimated amount of the nucleic acid as the analyte present in the first sample. Use in the same range.

The advantage of this procedure is that it can reduce the range of the amount of construct used, and thus increase the accuracy of the method and will degrade the amplification reaction that must be carried out further.

Description of the Drawings

FIG. 1A shows the injection and amplification amounts of the nucleic acid construct and sample (analyte) nucleic acid used in Experiment 1 as described in Example 1. FIG.

FIG. 1B shows the ratio of the construct signal and sample signal plotted on a logarithmic scale relative to the amount of injection construct copy number as used in Experiment 1 described in Example 1. FIG. The amount of nucleic acid as analyte is shown on the x axis with respect to the vertical line in the graph.

FIG. 2A shows the injection amount and amplification amount of the nucleic acid construct and the sample (analyte) nucleic acid in Experiment 2 as described in Example 1. FIG.

2B shows the ratio of the construct signal and sample signal plotted in logarithmic scale relative to the amount of injection construct copy number as used in Experiment 2 described in Example 1. FIG. The amount of nucleic acid as analyte is shown on the x axis with respect to the vertical line in the graph.

FIG. 3A shows the injection amount and amplification amount of the nucleic acid construct and the sample (analyte) nucleic acid in Experiment 3 as described in Example 1. FIG.

FIG. 3B shows the ratio of construct signal and sample signal plotted in logarithmic scale to the amount of injection construct copy number as used in Experiment 3 described in Example 1. FIG. The amount of nucleic acid as analyte is shown on the x axis with respect to the vertical line in the graph.

4A shows the injection amount and amplification amount of the nucleic acid construct and the sample (analyte) nucleic acid in Experiment 4 as described in Example 1. FIG.

FIG. 4B shows the ratio of the construct signal and sample signal plotted in logarithmic scale to the amount of injection construct copy number as used in Experiment 4 described in Example 1. FIG. The amount of nucleic acid as analyte is shown on the x axis with respect to the vertical line in the graph.

FIG. 5A shows the injection amount and amplification amount of the nucleic acid construct and the sample (analyte) nucleic acid in Experiment 5 as described in Example 1. FIG.

FIG. 5B shows the ratio of construct signal and sample signal plotted on a logarithmic scale to the amount of injection construct copy number as used in Experiment 5 described in Example 1. FIG. The amount of nucleic acid as analyte is shown on the x axis with respect to the vertical line in the graph.

6 shows the dynamic range of a single in vitro Q-NASBA. As quantitative infusions, WT-RNA molecules generated in vitro of 10 ^1.44 , 10 ^2.83 , 10 ^4.23 and 10 ^4.83 were used. Quantification of all WT-RNA amounts was performed 8-10 times. Q _A , Q _B and Q _C RNA were used in amounts of 10 ⁴ , 10 ³ and 10 ² RNA molecules, respectively.

Figure 7 shows a schematic flow chart of a single in vitro Q-NASBA with the addition of internal standard RNAs Q _A , Q _B and Q _C prior to separating nucleic acids with lysis buffer.

The invention is further illustrated by the following examples:

Example 1

The following examples are theoretical examples of quantitative and continuous detection assays carried out according to the method of the invention. From this experiment it is possible to show how the analysis according to the invention is carried out as well as the results that can be produced.

The five experiments that can be performed by the method according to the present invention are summarized below, which vary in the range of the amount of the analyte to be injected and the amount of nucleic acid construct added prior to amplification.

The analyte injection amount and the construct injection amount are as shown in the following table (Table 1).

As can be seen from this table, the first three theoretical experiments added three constructs (QA, QB, and QC, respectively), and the two amplification reactions were carried out in the "low concentration" construct and in the "high concentration range". It was carried out in the amount of sacrifice. In this experiment, the low and high concentration ranges overlap. The overlapping points of the two ranges can be used to compare the amplification (if the amplification is performed in the same way, the signal generated for the overlapping amount of constructs in both ranges must be the same). Experiments 4 and 5 also used construct amounts in two concentration ranges, but did not overlap the low and high concentration ranges. As a result, the amount of construct used per amplification reaction can be reduced but the same total range of amounts will still be quantified.

The amplification rates of Q-RNA (construction) and wild-type RNA are identical because the RNAs are the same size, differing only in the sequence of 20 random nucleotides, and the same primers and enzymes are used for amplification. Thus, the initial ratio of each construct RNA amount and wild type RNA amount will not change during amplification. Amplification Following the detection procedure, the mixture of each amplification was divided into four analytes to detect wild type, QA, QB or QC RNA amplifications (the mixtures of amplifications of experiments 4 and 5 were split into only three analytes). do).

If the log ratio of the construct signal and wild type signal is plotted against the log value of the dose of construct RNA, a straight line is expected. From this straight line the amount of wild type RNA can be calculated. The results obtained by the above experiments are shown in FIGS. 1 to 5. The first graph (a) of each figure shows the amount of the various constructs before and after amplification, while also showing the amount of nucleic acid being analyte.

The second graph (b) of each figure shows a graph in which the log ratio of the estimated construct signal and the analyte signal (sample signal) is expressed as a function of the log value of the injection amount of the construct. From this graph, the injection amount of the nucleic acid molecule as an analyte can be derived, i.e., calculated from the logarithm of the injection value (shown on the horizontal axis) belonging to the point on the vertical axis whose log ratio is 1.

Example 2

A mixture of quantified amounts of construct HIV-1 gag1 RNA transcripts comprising three nucleic acid constructs QA, QB, and QC, each containing 200, 2000, and 20000 copies, is prepared. Quantified amounts of five different wild type HIV-1 gag1 transcripts, ie 200000, 20000, 2000, 200, 20 copies and control, respectively, are mixed with a mixture of Q construct RNA. Six amplification reactions were performed according to standard protocol (T. Kievits et al.) with one sample for each amount of wt-RNA.

5 μl from each amplification was diluted with 100 μl of TBE buffer (90 mM Tris-borate, 1 mM EDTA, pH8.4). 5 μl from each dilution, 3 pmol of biotin-oligo and 3 trimol of several tri (2,2′-bipyridine) ruthenium II chelate-labeled oligonucleotides (each a wild-type RNA amplification or construct (QA) , QB or QC)), and specific streptavidin coated magnetic dynal beads M280 20 Was added to a test tube containing 15 μl of the mixture dissolved in 6.67 × SSC buffer (0.75 M NaCl, 0.075 M sodium citrate pH7-8).

Four hybridization assays for detecting WT, QA, QB or QC were performed on one amplifier. The mixture was hybridized at 41 ° C. for 30 minutes and mixed every 10 minutes. 300 μl of assay buffer for electrochemiluminescent ECL detection was added and mixed using IGEN's ORIGEN 1.5 detection system. Actual detection in the ORIGEN 1.5 detection system was performed according to the manufacturer's protocol.

Example 3

This example relates to a Q-NASBA assay in which three Q-RNA internal standard spiked in a WT-RNA sample in amounts of 10 ⁴ , 10 ³ , 10 ² molecules. Three internal standard RNA molecules use ECL labeled probes specific for the 20 nucleotide random sequence (Van Gemen et al. J. Virol. Methods. 43, 177-188, 1993) specific to each internal standard. By distinguishing them. The ratio of NASBA amplified internal standard to WT-RNA was measured using an ECL detection instrument.

ECL is based on chemiluminescent markers that luminesce on the surface of the electrode (Blackburn et al., Clin. Chem. 37, 1534-1539, 1991). The detection of the signal can be quantified in the dynamic range of grade 5 or higher using a specifically developed detection instrument. ECL techniques have been applied to detect amplified nucleic acids using ECL labeled oligonucleotides in hybridization assays (Kenten, J. H, et al. Clin. Chem. 38, 873-879, 1992). Since the inactivation of WT, Q _A , Q _B and Q _C ECL probes is known, the ratio of WT, Q _A , Q _B and Q _C NASBA amplified RNA can be measured from the signal ratio of each probe.

Initial amounts of WT injected RNA were read from the ratio of WT signal to Q _A , Q _B and Q _C signals in an assay based on ECL beads.

The method by which the Q construct was prepared and analyzed was described in the "Materials and Methods" section of this example.

A single in vitro Q-NASBA protocol was used with ECL detection to quantify the various amounts of WT-RNA produced in vitro. Three different assays were performed using 10 ^1.44 , 10 ^2.83 , 10 ^4.23 and 10 ^4.83 initial WT-RNA molecules as injections, respectively. Using an ECL bead based assay, Q-NASBA was quantified by mixing WT-RNA with 10 ⁴ Q _A RNA molecules, 10 ³ Q _B RNA molecules, and 10 ² Q _C RNA molecules in a single NASBA amplification. Eight to ten runs were performed on the amount of WT-RNA. The quantitative results (mean ± SD) performed were 10 ^{1.54 ± 6.23} , 10 ^{2.68 ± 0.21} , 10 ^{4.16 ± 0.20} , 10 10 ^.44 , 10 ^2.83 , 10 ^4.23 and 10 ^4.83 , respectively, using initial WT-RNA molecules as injections. 10 ^{4.81 ± 0.23} . The analysis results performed are shown in FIG. As can be seen from this figure, the WT-RNA amount was reliably quantified to 1/10 of the lowest Q-RNA amount used in one in vitro quantification protocol.

Materials and methods

Plasmid and RNA Synthesis

Genetic and recombinant DNA techniques followed standard procedures (Sambrook, J. et al., Molecular Cloning, A laboratory Manual, Cold Spring Harbor, NY: Cold Spring Harbor Lab., 1989, Ed. 2nd edition). Plasmid pGEM3RAN (Van Gemen et al., 1993) with 22 random nucleotide sequences (pos. 1429-1451 HIV-I pv22 sequence: Muesing, M.A. et al., Nature 313, 450-458, 1985) This was used to delete portions of the HIV-1 cloned sequence (pos 1691 to 2105 HIV-1pv22) that were not associated with quantitative NASBA amplification and AccI sites within multiple cloning sites. The random sequences in this plasmid were replaced with 5'ATG.CAA.GGT.CGC.ATA.TCA.GTA.A3 'or 5'ATA.AGC.ACG.TGA.CTG.AGT.ATG.A3', respectively, to pGEM3Q _B δ gag 3 and pGEMQ _C δ gag 3 were constructed. Plasmid pGEM3RAN was renamed pGEM3Q _A. PGEM 3p24 (WT-RNA), pGEM3Q _A (Q _A -RNA), pGEM3Q _B δ gag 3 (Q _B -RNA) and pGEM3Q _C δ gag 3 (Q _C -RNA) using SP6 RNA polymerase in vitro RNA was constructed from (Sambrook, 1989). re-cloned inserts of pGEM3p24 and pGEM3Q _A into the vector pGEM4 cloned made pGEM4p24 and pGEM4Q _A. RNA was prepared in vitro using this plasmid using T7 RNA polymerase (Sambrook, 1989). The length of the in vitro RNA is 1514 nucleotides for plasmids pGEM3p24 (WT-RNA) and pGEM3Q _A (Q _A RNA) and plasmids pGEM3Q _B δgag3 (Q _B -RNA) and pGEMQ _C δ gag 3 (Q _C -RNA) In this case 1090 nucleotides. This RNA was treated with DNase to remove plasmid DNA and purified on anion exchange column (Qiagen). In vitro RNA was quantified by spectroscopic analysis and diluted with water to the desired concentration. All RNA solutions were stored at -20 ° C.

Nucleic acid isolation

Nucleic acid was isolated from plasma according to the method of Boom, R., et al., J. Clin. Microbiol. 28, 495-503, 1990: Van Gemen et al. 1993. 100 μl of plasma nucleic acid was finally resuspended in 100 μl water and stored at −70 ° C.

NASBA

All enzymes were purchased from Pharmacia except AMV-reverse transcriptase from Seikagaku. BSA was purchased from Boehringer Mannheim. NASBA reaction mixture [final concentration in 25 μl reaction mixture: 40 mM Tris, pH8.5, 12 mM MgCl ₂ , 42 mM KCl, 15% v / v DMSO, 1 mM each dNTP, 2 mM each NTP, 0.2 Primer 1: 5'AAT.TCT.AAT.ACG.ACT. CAC.TAT.AGG.GTG.CTA.TGT.CAC.TTC.CCC.TTG.GTT.CTC.TCA, 0.2 Primer 2: 5'AGT.GGG.GGG.ACA.TCA.AGC.AGC.CAT.GCA.AA, 0.2 to 2 μl wild type RNA and 2 μl in vitro Q-RNA (Kievits, T. et al. J. Virol 35, 273-286, 1991; Van Gemen et al. 1993)] 23 μl was incubated at 65 ° C. for 5 minutes to remove stability of the secondary structure of the RNA and then cooled to 41 ° C. to anneal the primers. I was. Here is the enzyme mixture (0.1 Amplification was initiated by adding 2 μl / μl BSA, 0.1 unit RNase H. 40 unit T7 RNA polymerase and 8 units AMV-reverse transcriptase). The reaction was incubated at 41 ° C. for 90 minutes. Two negative controls were added per quantitative reaction.

Detection reaction based on enzymatic beads

Bead-based enzymatic assays were developed in the above quantitative protocol (Van Gemen et al. 1993) to detect and measure the ratio of NASBA amplified WT and Q _A RNA. 2.8 coated with streptavidin 100 μl of polystyrene paramagnetic beads (Dynal Inc, Great Neck, NY, USA) was washed twice with 200 μl 1 × PBS, 0.1% BSA and resuspended in 1 × PBS, 0.1% BSA. The washed beads were incubated with 300 pmol of HIV-1 specific biotinylated capture probe (5′TGT, TAA, AAG, AGA.CCA.TCA.ATG.AGG.A) for 1 hour at room temperature followed by 200 μl 5 Wash once with SSPE, 0.1% SDS and wash once with 200 μl 1 × PBS, 0.1% BSA. Beads were resuspended in 100 μl 1 × PBS, 0.1% BSA. 5 μL beads, 5 μL NASBA reaction mixture and 50 μL hybridization buffer (5 × SSPE, 0.1% SDS. 0.1% blocking reagent, 10 μg / ml salmon sperm DNA) were incubated at 45 ° C. for 30 minutes. The beads were washed twice with 100 μl 2 × SSC, 0.1% BSA, then 5 × 10 ⁻⁷ Incubated with mol WT or Q detection oligonucleotide probes, of which 10% was HRP labeled for 30 minutes at 45 ° C. in 50 μl hybridization buffer.

Bead-trapping oligonucleotide-NASBA amplified WT RNA or QA RNA detection probe complexes were washed with 100 μl 2 × SSC, once with 0.1% BSA, once with 100 μl TBST, and twice with 100 μl TBS. 100 μl chromogenic substrate (TMB / peroxide solution) was then added to the beads and incubated for 3 minutes at room temperature. The color reaction was stopped by adding 50 µl of 250 mM oxalate. Absorbance of the 150 μl chromogenic reaction was read at 450 nM with a microplate reader (Micro SLT 510, Organon Teknika, Boxtel, The Netherlands). Absorbance values were corrected for the base signal (ie, negative control) and the signal was calculated as% of the signal exhibited by the amplified WT RNA or QA RNA, respectively.

Detection reaction based on ECL beads

5 μL of NASBA amplified RNA (WT, Q _A , Q _B and Q _C ) diluted 20-fold with water was added 20 μL of HIV-1 specific biotinylated capture probe (see detection reaction based on enzymatic beads) (3.3 pmol), WT, Q _A , Q _B or Q _C NASBA specific ECL (tris [2,2-bipyridine] ruthenium [II] complex) labeled oligonucleotide probes 3.3 pmol and strep in 5 x SSC Triavidine Coated Magnetic Beads 20 (2 μl) and incubated at 41 ° C. for 30 minutes. Test tubes were mixed by vortexing every 10 minutes during incubation. Then 300 μl of TPA solution (100 mM tripropylamine, pH = 7.5) was added and the hybridization mixture was placed in an Origen 1.5 ECL detection instrument (Organon Teknika, Boxtel, The Netherlands).

Example 4

Nucleic acid isolation efficiency can affect the results of WT-RNA quantification. To avoid the effects of nucleic acid loss during separation, Q _A , Q _B and Q _C RNA can be added before or during step 1 of nucleic acid separation (FIG. 7). This principle was tested using 10 ⁴ molecules of HIV-1 isolated from in vitro generated WT-RNA and in vitro cultured virus stock solution. In vitro generated WT-RNA and HIV-1 virus stock RNA were re-separated during the first step of nucleic acid isolation (ie in lysis buffer) with Q _A , Q _B , and Q _C RNA added and without addition.

The method used is as described in the "Materials and Methods" section of Example 3.

When the first isolated WT-RNA and HIV-1 viral stock RNA were quantified by addition of Q _A , Q _B and Q _C during amplification (ie, control quantification), the result was in vitro generated WT-RNA and HIV. 3.3 × 10 ⁴ and 2.1 × 10 ⁴ for the −1 virus stock RNA, respectively. If Q _A , Q _B and Q _C RNA were added prior to the re-separation procedure, the re-isolated RNA was quantified and 2.5 x 10 ⁴ and 1.5 x 10 for in vitro generated WT-RNA and HIV-1 viral stock RNA, respectively. ⁴ was shown. However, when Q ^A , Q ^B and Q ^C RNA were added after the re-separation procedure, when the re-isolated RNA was quantified, the result was 4.7 x for each of the in vitro generated WT-RNA and HIV-1 virus stock RNA, respectively. 10 ³ and 2.0 x 10 ³ . This indicates that the RNA reisolation efficiency is about 10%. However, addition of Q _A , Q _B and Q _C prior to re-separation yielded the same RNA quantification as control quantification due to the constant ratio of WT: Q _A : Q _B : Q _C RNA regardless of the absolute amount of isolated nucleic acid.

Table 2 shows the results.

a. Q _A , Q _B and Q _C were added as 10 ² , 10 ³ and 10 ⁴ RNA molecules, respectively.

b. Control quantification not involving nucleic acid reisolation.

Example 5

The method according to the present invention was compared to the above-described Q-NASBA protocol (Van Gemen et al. 1993; Jurriaans et al., Submitted, 1993) in which only one Q-RNA was used and a dynamic range of 5 log was obtained. If necessary, six or more amplification reactions per clinical (wild-type) sample are needed, one positive WT control with no internal standards added and five reactions with increasing amounts (10 ² to 10 ⁶ ) of internal standard RNA molecules. It becomes

The number of amplification reactions can be reduced when using the methods described herein where various distinguishable internal standards are spiked in one amplification reaction.

In contrast, when only one Q-RNA (Q _A ) is used with an enzyme-labeled probe, the initial WT-RNA concentration is determined by using different concentrations of Q-RNA for different amplification reactions. It must be inferred from the ratio of the signals.

The two methods were compared using a plasma system and model system of HIV-1 infected individuals.

_A single in vitro Q-NASBA using Q _A , Q _B and Q _C RNA was used to analyze 0.1 ml plasma samples from three asymptomatic HIV-1 infected individuals, thereby using the above-described Q- using six amplification reactions per quantitative assay. Comparison with NASBA protocol (Van Gemen et al., 1993). In the same experiment, the differences between the addition of Q _A , Q _B and Q _C before and after nucleic acid separation were re-studied (Table 3). In the case where the WT-RNA is quantified by a single in vitro Q-NASBA protocol, the WT-RNA amount can be reliably quantitated up to 1/10 the amount of the Q-RNA lowest used in a single in vitro quantification protocol.

Q _A , Q _B and Q _C (6 each 10 ⁵ , 6 10 ⁴ and 6 10 ³ ) must be added to 0.1 ml plasma (when added to lysis buffer), so the lower reliable range of quantification is 6 per 0.1 ml plasma. 10 ² RNA copies. Addition of Q _A , Q _B and Q _C (10 ⁴ , 10 ^3, and 10 ² , respectively) to the isolated nucleic acid was reliable for 10 ³ RNA copies per 0.1 ml plasma when 1 μl of the plasma equivalent of nucleic acid was used for amplification. A lower quantitative range is provided. In both cases the WT-RNA amount can be reliably quantified up to 1/10 of the lowest Q-RNA used in a single in vitro quantification protocol. The protocol using six amplification reactions per quantitative assay with the lowest internal standard Q-RNA amount of 10 ² provides a reliable lower quantification of 10 ⁴ RNA copies per 0.1 ml plasma when nucleic acids of 1 μl plasma equivalent are used for amplification. Provide a range. This result showed that the nucleic acid separation efficiency varied between 100% (Patient 1) and 50% (Patient 2) in this particular experiment. Patient 3 was always below the reliable quantitative range, but this range was the most accurate (ie lowest range) for a single in vitro Q-NASBA assay with Q _A , Q _B and Q _C added to lysis buffer prior to nucleic acid isolation (Table 3).

Example 6

Finally, we tested the reproducibility of a single in vitro Q-NASBA in which Q _A , Q _B and Q _C were added to lysis buffer prior to isolation of nucleic acids using HIV-1 virus mother liquor cultured in vitro. The amount of particles was quantified by electron microscopy (Layne, SP et al., Virology. 189. 695-714, 1992). 2.9 per ml 10 ¹⁰ HIV-1 virus mother liquor containing virus particles was diluted 10,000-fold with water and 100 μl of diluted virus stock solution was added to Q _A , Q _B and Q _C (6 each). 10 ⁵ , 6 10 ⁴ and 6 10 ³ ) resulted in the measurement of 4.35 × 10 ¹⁰ RNA molecules per ml. The results are shown in Table 4. The mean ± SD of RNA quantification was 10 ^{10.64 ± 0.05} , indicating that the accuracy of a single in vitro Q-NASBA is within 0.1 log when quantifying virus stocks cultured in vitro. (The following procedure is as described in the Materials and Methods section of Example 3).

As can be seen from Table 4, using the method according to the present invention, the accuracy of the assay within 0.1 log can be obtained when quantifying HIV-1 RNA in virus mother culture cultured in vitro. From this result, the difference in the amount of HIV-1 RNA of 0.4 log or more can be reliably measured.

Q _A , Q _B and Q _C (6 each 10 ⁵ , 6 10 ⁴ and 6 10 ³ ) was added to 100 μl of virus mother liquor at 10,000 dilution in water.

a. 2 times amplification

b. 3 times amplification

The mean value ± SD of all doses is 10 ^{10.64 ± 0.05} (4.35 × 10 ¹⁰ ) RNA molecules per ml.

Claims (17)

Adding to the sample different amounts of various nucleic acid constructs that are distinguished from the nucleic acid being analyte but can be co-amplified with the nucleic acid; Treating the sample with a nucleic acid amplification procedure using an amplification reagent capable of reacting with both the analyte and the nucleic acid construct; Measuring the relative amounts of the analyte nucleic acid and the amplification derived from each nucleic acid construct; A method for quantifying an analyte of a nucleic acid in a sample comprising calculating an amount of nucleic acid to be analyte from the relative amount. The method of claim 1, Wherein said nucleic acid construct has the same sequence as the nucleic acid being analyte except for the distinguishable non-repeating sequence present in each nucleic acid construct. The method of claim 2, Wherein said distinguishable non-repeat sequence is a random sequence comprising about 20 nucleotides. The method according to any one of claims 1 to 3, Wherein the nucleic acid construct is added in a range of amounts that vary from one another by a predetermined coefficient. The method according to any one of claims 1 to 3, Wherein the nucleic acid construct is added in a range of amounts different from each other by a factor of ten. The method of claim 1, The sample is prepared by subjecting a known amount of test solution to a nucleic acid separation procedure, wherein a nucleic acid construct is added to the test solution prior to treating the test solution with the nucleic acid separation procedure. The method of claim 6, Treating the test solution with a nucleic acid separation procedure and then adding a known amount of the control sequence to the sample. The method according to any one of claims 1 to 3, And dividing the sample into one or more reaction doses, each adding a nucleic acid construct in a range of amounts. The method of claim 8, The same nucleic acid construct is added to each reaction dose. The method of claim 8, And the amounts of said range do not overlap. A nucleic acid that is an analyte present in one or more samples, characterized in that the same nucleic acid construct is used for all samples and the amount of this particular nucleic acid construct is varied, respectively, in the treatment of each sample by the method of claim 1. Quantitative method of The method of claim 11, Wherein one of the nucleic acid constructs added in each sample is used in an amount of zero. Diluting a predetermined amount of test solution by a known factor and taking a sample from the diluted test solution containing the amount of nucleic acid that is an analyte that is estimated to be within a certain range, And treating said sample by the method of claim 1, wherein the amount of nucleic acid construct added to said sample is within said range. The method of claim 13, Determining a sample containing nucleic acid that is an analyte in an amount less than the amount of the range, And treating the test solution taken from the determined sample by the method of claim 13, wherein the amount of the test solution is diluted by a lower coefficient. The method of claim 13, Determining a sample containing a nucleic acid that is an analyte in an amount greater than the amount in the range, And treating the test solution taken from the determined sample by the method of claim 13, wherein the amount of the test solution is diluted by a higher factor. Adding to the sample an amount of a nucleic acid construct that is estimated to be within the same range as the expected amount of nucleic acid, the analyte present in the sample, Subjecting the sample to a nucleic acid amplification procedure using an amplification reagent capable of reacting with both the analyte nucleic acid and the nucleic acid construct, Measuring a relative amount of the analyte nucleic acid and an amplification derived from the nucleic acid construct, Estimating the amount of nucleic acid that is analyte present in the sample from the relative amount, Treating the second sample derived from the same test solution by the method of claim 1, wherein the amount of the nucleic acid construct to be added is an amount within the same range as the estimated amount of the nucleic acid to be analyte present in the first sample. A method for quantifying an analyte nucleic acid in a sample, which is characterized by the above-mentioned. A plurality of nucleic acid constructs, amplification reagents capable of reacting with the nucleic acid construct and the analyte nucleic acid, a detection probe for the amplified analyte nucleic acid, and several detection probes each specific for the various amplified nucleic acid constructs. A test kit for quantifying nucleic acid in a sample according to the method of claim 1.