JP2019216720A - Control nucleic acids for multiple parameters - Google Patents

Abstract

To provide processes for amplifying and detecting control nucleic acids for multiple parameters.SOLUTION: Provided is a process for isolating and simultaneously amplifying a first and a second target nucleic acid that may be present in one or more fluid samples, the process comprising predetermined automated processes, such as immobilizing a nucleic acid containing a target nucleic acid, and a quantitative standard nucleic acid on a solid support material, purifying the nucleic acid by isolating the solid support material from the other material present in the fluid samples in a separation station, and amplifying the purified target nucleic acid and the quantitative standard nucleic acid and detecting a signal, the conditions for amplification and detection being the same for the first and second purified target nucleic acids as well as the quantitative standard nucleic acid.SELECTED DRAWING: None

Description

FIELD OF THE INVENTION The present invention belongs to the field of in vitro diagnostics. In this field, the invention relates in particular to the amplification of at least a first and a second target nucleic acid that may be present in at least one liquid sample, using an internal control nucleic acid for qualitative and / or quantitative purposes. .

BACKGROUND OF THE INVENTION In the field of molecular diagnostics, amplification of nucleic acids from a number of sources has become of considerable importance. Examples of diagnostic applications for nucleic acid amplification and detection include detection of viruses such as human papillomavirus (HPV), West Nile virus (WNV), or human immunodeficiency virus (HIV), hepatitis B virus (HBV) and / or type C Routine screening of blood donations for the presence of hepatitis virus (HCV). Furthermore, said amplification technique is suitable for the analysis of bacterial targets such as mycobacteria or cancer markers.

  The most prominent and widely used amplification technique is the polymerase chain reaction (PCR). Other amplification reactions include ligase chain reaction, polymerase ligase chain reaction, Gap-LCR, repair chain reaction, 3SR, NASBA, strand displacement amplification (SDA), transcription-mediated amplification (TMA) and Qβ amplification, among others.

  Automated systems for PCR-based analysis often utilize real-time detection of amplification products during the PCR process in the same reaction vessel. The key to such a method is the use of modified oligonucleotides with reporter groups or labels.

  It has been shown that amplification and detection of more than one target nucleic acid in the same container is possible. This method is commonly referred to as "multiplex" amplification and requires different labels to distinguish when real-time detection is performed.

  For qualitative (performance control) and / or quantitative (determination of the amount of target nucleic acid using a control as a reference), taking control of each amplification using a control nucleic acid of known sequence, Most desirable or even essential in the field of clinical nucleic acid diagnostics. Due to the diversity of diagnostic targets, especially including prokaryotic, eukaryotic and viral nucleic acids, and between different types of nucleic acids such as RNA and DNA, control nucleic acids are usually designed in a specific manner . Briefly, these controls usually resemble the target nucleic acid and serve as controls to mimic its performance during the process. This situation applies to both qualitative and quantitative assays. If multiple parameters are detected in a single or parallel experiment, different controls are used, usually resembling different target nucleic acids, such as Swanson et al. Stoecher et al. (Non-Patent Document 2) discloses a control nucleic acid in which a number of virus-specific competition controls are contained on the same DNA molecule.

  The present invention provides a controlled amplification method using different approaches that exhibit various advantages.

J. Clin. Microbiol., (2004), 42, pp. 1863-1868 J. Virol. Meth. (2003), 108, pp. 1-8

  It is an object of the present invention to provide a control nucleic acid for a number of parameters.

That is, the gist of the present invention is:
[1] A method for isolating and simultaneously amplifying first and second target nucleic acids which may be present in one or more liquid samples, wherein the method comprises:
a. adding a quantitative standard nucleic acid to each of the liquid samples, wherein the sequence of the quantitative standard nucleic acid is derived from a source different from the source of the liquid sample and differs from other nucleic acid sequences in the liquid sample. Is different, is derived from the plant genome, is mixed, and the quantitative standard nucleic acid has a concentration of 20 × LOD to 5000 × LOD;
b. the solid support material and the one or more liquid samples for one or more times and under conditions sufficient for the nucleic acid containing the target nucleic acid and the quantitative standard nucleic acid to be immobilized on the solid support material; Of combining together in a container
c. isolating the solid support material from other materials present in the liquid sample at the separation station.
d. purifying the nucleic acid at the separation station and washing the solid support material one or more times with a washing buffer;
e. an amplification reagent comprising a purified target nucleic acid and a purified quantitative standard nucleic acid and at least one different set of primers for each of said target nucleic acids and said quantitative standard nucleic acid in at least two reaction vessels Contacting in at least two reaction vessels for the amplification of at least the first and second target nucleic acids and the quantitative standard nucleic acid of
f. in the reaction vessel, the purified target nucleic acid and the purified quantitative standard nucleic acid, and the amplification reagent, for a time sufficient for an amplification reaction to be performed indicating the presence or absence of the target nucleic acid, and Incubate under conditions, amplify the first target nucleic acid instead of the second target nucleic acid in the first reaction vessel, and amplify the second target nucleic acid instead of the first target nucleic acid in the second reaction vessel Process
g. detecting and measuring a signal generated by the amplification product of the target nucleic acid and proportional to the concentration of the target nucleic acid, and detecting and measuring a signal generated by the quantitative standard nucleic acid;
h. comprises an automated step of determining one or more amounts of the target nucleic acid by using a quantitative standard nucleic acid as a reference, wherein the conditions for amplification and detection in steps e.-g. The same for the first and second purified target nucleic acids and the quantitative standard nucleic acid,
[2] the description of [1], wherein the presence of the amplification product of the quantitative standard nucleic acid indicates that amplification occurs in the reaction mixture even in the absence of the amplification product of one or more of the target nucleic acids; the method of,
[3] the method of [1] or [2], wherein the quantitative standard nucleic acid is a sheathed nucleic acid;
[4] the method of any of [1] to [3], wherein the melting temperature of the quantitative standard nucleic acid is 50 ° C to 90 ° C;
[5] the method of any one of [1] to [4], wherein the quantitative standard nucleic acid has a length of up to 500 bases;
[6] The method according to any one of [1] to [5], wherein the sequence of the quantitative standard nucleic acid has a GC content of 30% to 70%.

  According to the present invention, control nucleic acids for a number of parameters can be provided.

FIG. 1 is a schematic depiction of the sample preparation workflow used in embodiments of the present invention. Arrows pointing down indicate the addition of components or reagents to each respective well of the deep well plate described above, and arrows pointing up indicate their respective removal. These actions are performed manually in steps 2, 3, 4, 21 and 22 and in the process head of the apparatus in steps 10, 14, 16, 18 and 24, and in steps 5, 6, 7, 11, 15 and In 19, it was done at the reagent head of the instrument. It should be understood that the volume used can be adjusted flexibly within the spirit of the invention, preferably at least up to about 30% of the disclosed value. In particular, for step 2, the sample volume is preferably variable, as is known to those skilled in the art, to account for different types of liquid samples that may require some starting material to obtain adequate results. It is. Preferably, the range is from about 100 ul to about 850 ul. More preferably, the volume is about 100 ul, about 500 ul or about 850 ul. Preferably, the volume in each container is adjusted to the same total volume by dilution in step 3. Preferably, as in the schematic shown in FIG. 1, the total volume is added up to about 850 ul. FIG. 2 is an increasing curve of amplification of target nucleic acids from HIV, HBV and CT performed on LightCycler480 (Roche Diagnostics GmbH, Mannheim, DE) described in Example 1. The "signal" shown on the y-axis is the normalized fluorescent signal. The x-axis indicates the number of each PCR cycle. HIV and HBV growth curves are shown, along with the corresponding internal control nucleic acid growth curves. The curves for each target nucleic acid are shown as solid lines, and the curves for control nucleic acids are shown as dotted lines. FIG. 2a is a qualitative HIV assay measured in a channel for detection of a target probe. FIG. 2b is a qualitative HIV assay measured in a channel for detection of a control probe. FIG. 2c is a quantitative HIV assay measured in a channel for detection of a target probe. FIG. 2d is a quantitative HIV assay measured in a channel for detection of a control probe. FIG. 2e is a quantitative HBV assay measured in a channel for detection of a target probe. FIG. 2f is a quantitative HBV assay measured in a channel for detection of a control probe. FIG. 2g is a CT assay measured in a channel for detection of a target probe. FIG. 3 is a perspective view of a processing plate. FIG. 4 is a perspective view of the processing plate from an opposite angle. FIG. 5 is a top view of the processing plate. FIG. 6 is a cross-sectional view along a long side surface of the processing plate. FIG. 7 is a partial view of a sectional view. FIG. 8 is a perspective view of the long side surface of the processing plate. 9a) -d) show different views of the second embodiment of the magnetic separation station. FIGS.10 (a)-(c) show a magnetic separation station holding a processing plate having a first type of magnet at the highest Z position and a second type of magnet at the lowest Z position. FIG. 2 shows a diagram of a first embodiment. FIG. 11 is a schematic diagram of an analyzer including various stations, modules or cells. FIG. 12 shows the analysis system of the present invention. FIG. 13 is the linearity of a quantitative HBV assay in EDTA plasma according to the data of Example 2. FIG. 14 is the linearity of a quantitative HBV assay in serum according to the data of Example 2. FIG. 15 is the linearity of a quantitative HCV assay in EDTA plasma according to the data of Example 2. FIG. 16 is the linearity of a quantitative HCV assay in serum according to the data of Example 2. FIG. 17 is the linearity of a quantitative HIV assay in EDTA plasma according to the data of Example 2.

DESCRIPTION OF THE INVENTION The present invention provides a method for the controlled amplification of at least first and second target nucleic acids that may be present in a liquid sample.

In a first aspect, the present invention relates to a method for isolating and simultaneously amplifying at least first and second target nucleic acids that may be present in one or more liquid samples, said method comprising:
a. adding an internal control nucleic acid to each of the liquid samples
b. transferring the solid support material and the one or more liquid samples in one or more containers for a time sufficient for the nucleic acid containing the target nucleic acid and the internal control nucleic acid to be immobilized on the solid support material. And the process of joining together under conditions
c. isolating the solid support material from other materials present in the liquid sample at the separation station.
d. a step of purifying the nucleic acid in the separation station and washing the solid support material one or more times with a washing buffer;
e. at least two reaction vessels comprising a purified target nucleic acid and a purified internal control nucleic acid, and one or more amplification reagents comprising at least one different set of primers for each of the target nucleic acids and the internal control nucleic acid. Contacting, wherein at least a first reaction vessel includes at least the first target nucleic acid, at least a second reaction vessel includes at least the second target nucleic acid, and a second target nucleic acid is the first target vessel. Not present in the reaction vessel
f. in the reaction vessel, the purified target nucleic acid and the purified internal control nucleic acid and the one or more amplification reagents are sufficient to cause an amplification reaction indicative of the presence or absence of the target nucleic acid to occur. Incubation time and conditions
g. detecting and measuring a signal generated by the amplification product of the target nucleic acid and proportional to the concentration of the target nucleic acid; andautomating a step of detecting and measuring a signal generated by the internal control nucleic acid, The amplification and detection conditions in steps d-g are the same for the at least first and second purified target nucleic acids and the internal control nucleic acid, and the sequence of the internal control nucleic acid is the at least first and second For the purified target nucleic acids.

  The present invention allows for the development of simultaneous assays for multiple parameters and / or nucleic acid types, while using the same internal control nucleic acid sequence for different parameters and / or nucleic acid types. Thus, the present invention contributes to reducing the overall complexity of the corresponding experiment at various levels. For example, the time and expense of designing and synthesizing or purchasing a large number of control nucleic acid sequences is saved because only the sequence of one internal control nucleic acid need be designed and added to each amplification mixture. One or more assays can be streamlined, reducing the risk of handling errors. Also, as more different control nucleic acid sequences are used simultaneously in one assay or in parallel assays performed under the same conditions, the regulation of each condition can become more complex. In addition, with one control suitable for multiple nucleic acids, the controls can be dispensed from one source, for example, to different containers containing the different target nucleic acids. Within the scope of the present invention, one control nucleic acid sequence may also serve as a qualitative control and a quantitative control.

  As a further advantage of the above-described method, the controls used in the present invention can be used to serve as controls for the amplification of various nucleic acids, so that in a possible subsequent experiment, specific biological samples for other nucleic acids may be used. Testing need not involve separate sample preparation procedures involving the addition of different internal control nucleic acids. Thus, once the internal control nucleic acid is added, other parameters can be tested in the same sample and under the same conditions.

  An internal control nucleic acid can be competitive, non-competitive or partially competitive.

  A competitive internal control nucleic acid competes with the target for the same primer because it possesses essentially the same primer binding site as the target. This principle results in a less sensitive assay, as they allow for a good mimicry of each target nucleic acid because of their similar structure and can reduce amplification efficiency with respect to one or more target nucleic acids.

  The non-competitive internal control nucleic acid has a different primer binding site than the target and thus binds to a different primer. The advantages of such a setting include, inter alia, the fact that the amplification events of one of the different nucleic acids in the reaction mixture can take place independently of one another without any competing effects. Thus, there is no adverse effect on the detection limit of the assay, as can occur in a competitive setting.

  Finally, in an amplification using a partial competition setting, each control nucleic acid and at least one target nucleic acid compete for the same primer, and at least one other target nucleic acid binds to a different primer.

  The fact that the method described above involves a different set of primers for each of the target nucleic acids and the internal control nucleic acid makes the method considerably flexible. In this non-competitive setting, the target-specific binding site does not need to be introduced into the control nucleic acid as in the competitive setting, avoiding the disadvantages of the competitive setting described above. In a non-competitive setting, the internal control nucleic acids have a sequence that is different from any of the target sequences so as not to compete with their primers and / or probes. Preferably, the sequence of the internal control nucleic acid is different from other nucleic acid sequences in the liquid sample. By way of example, if the liquid sample is from a human, the internal control nucleic acid preferably does not have any sequences that are endogenous in humans. Thus, sequence differences should be at least significant so that the primer and / or probe do not bind to each one or more endogenous nucleic acids under stringent conditions and the setting is not competitive. . To avoid such interference, the sequence of the internal control nucleic acid used in the present invention is preferably from a source different from that of the liquid sample. Preferably, the sequence is from a naturally occurring genome, preferably from a plant genome, more preferably from a grape genome. In a highly preferred embodiment, nucleic acids from naturally occurring genomes are mixed. As known in the art, "scrambling" means introducing some base mutation into a sequence. Preferably, the sequence of the internal control nucleic acid used in the present invention is substantially modified with respect to the naturally occurring gene from which it is derived.

  Methods involving the above-described automated steps also exhibit various additional advantages.

  In the prior art, in multiplex assays performed in a single reaction vessel, it was difficult to limit the number of different target nucleic acids by the number of appropriate labels. For example, in real-time PCR assays, possible duplication of fluorochrome spectra has a significant effect on the performance of the assay (risk of false positive results, low accuracy, etc.). Therefore, each fluorophore must be carefully selected and spectrally separated sufficiently to ensure the desired performance of the diagnostic test. Typically, the number of different available fluorophores corresponds to the single digit number of fluorescent channels in a PCR device.

  In contrast, in the above method, the internally controlled amplification of at least the first and second target nucleic acids occurs in at least two different reaction vessels, and the signals in the different reaction vessels are detected independently of each other. As such, simultaneous amplification of a greater number of different target nucleic acids is possible. Further, embodiments within which the multiplex reaction occurs in one or more multiple reaction vessels, thereby increasing the number of targets that can be simultaneously amplified under the same conditions, are within the scope of the invention. In such embodiments, the internal control nucleic acid serves as a control for different target control nucleic acids in one container and various target nucleic acids in different containers.

  Accordingly, one preferred aspect of the present invention relates to the above method wherein at least two target nucleic acids are amplified in the same reaction vessel.

  In other cases, for example, depending on the sample and / or one or more target nucleic acids of interest, amplify the first target nucleic acid, but not the second target nucleic acid, in the first reaction vessel; and It may be preferable to amplify only the second target nucleic acid, but not the first target nucleic acid, in the second reaction vessel.

  Therefore, a further preferred embodiment of the present invention is the above method, wherein the first target nucleic acid is not present in the second reaction vessel.

  In particular, if the liquid sample is suspected of containing target nucleic acids from different organisms or even different organisms themselves, or if it is unclear whether different nucleic acids or organisms may be present in the sample, the first target nucleic acid The method described above, wherein the second target nucleic acid is from a different organism, is advantageous and is therefore a preferred embodiment of the present invention.

  A further preferred aspect of the present invention is the above method, wherein said first and / or second target nucleic acid is a non-viral nucleic acid.

  A preferred aspect of the present invention is the above-mentioned method, wherein the first and / or second target nucleic acid is a bacterial nucleic acid.

  As previously described, the above methods are useful for qualitative or quantitative control of amplification of at least a first and at least a second target nucleic acid.

  Qualitative detection of nucleic acids in biological samples is important, for example, in recognizing infection in an individual. Thereby, one important requirement for assays for the detection of microbial infections is that false-negative or false-positive results are almost inevitably yielding significant results in the treatment of each patient, thus avoiding such results. It is to be. Thus, particularly in PCR-based methods, a qualitative internal control nucleic acid is added to the detection mixture. The controls are particularly important for validating the test results. At least in the case of a negative result for each target nucleic acid, the qualitative internal control reaction needs to be reactive in the given setting, i.e. a qualitative internal control must be detected, otherwise the test It is assumed that it is not working. However, in a qualitative setting, the qualitative internal control need not necessarily be detected in the case of a positive result. For qualitative tests, it is particularly important that the sensitivity of the reaction is guaranteed and therefore tightly controlled. As a result, for example, the concentration of the qualitative internal control must be relatively low so that no qualitative internal control is detected even in the case of slight inhibition, so that the test is invalid.

  Accordingly, a preferred embodiment of the present invention provides a method as described above, wherein the presence of the amplification product of said internal control nucleic acid indicates that amplification has occurred in the reaction mixture even in the absence of an amplification product for one or more of said target nucleic acids. This is the method.

  On the other hand, it is often important to determine the amount of the nucleic acid in addition to simply detecting the presence or absence of the nucleic acid in the sample. As an example, the stage and severity of a viral disease can be assessed based on viral load. In addition, monitoring of any treatment requires information about the amount of pathogen present in the individual to assess the success of the treatment. For a quantitative assay, it is necessary to introduce a quantitative standard nucleic acid that serves as a reference for determining the absolute amount of the target nucleic acid. Quantitation can be performed either by taking a reference to an external calibration or by providing an internal quantification standard.

  In the case of external calibration, a standard curve is generated in a separate reaction using a known amount of the same or equivalent nucleic acid. The absolute amount of the target nucleic acid is subsequently determined by comparing the standard function with the results obtained with the analyzed sample. However, external calibration has the disadvantage that possible extraction procedures, its altered efficiency, and the presence of possible and often unpredictable agents that inhibit amplification and / or detection reactions are not reflected in the controls.

  This situation applies to any sample related effects. Thus, the sample may be judged negative due to unsuccessful extraction procedures or other sample-based factors, but the target nucleic acid to be detected and quantified is actually present in the sample.

For these and other reasons, an internal control nucleic acid added to the test reaction itself is advantageous. When functioning as a quantitative standard, the internal control nucleic acid has at least two functions in a quantitative test:
i) Monitor the validity of the reaction with the internal control nucleic acid.
ii) Since the internal control nucleic acid serves as a reference in the titer calculation, it controls the preparation and amplification process to compensate for the effects of the inhibition and allow for more accurate quantification. Thus, in contrast to a qualitative internal control nucleic acid in a qualitative test that must be positive only in a target negative reaction, a quantitative control nucleic acid in a quantitative test has two functions: a reaction control and a reaction calibration. Therefore, the quantitative control nucleic acid must be positive and valid in both target negative and positive reactions.

  Quantitative control nucleic acids also need to be suitable to provide a reliable reference for calculation of high nucleic acid concentrations. Therefore, the concentration of the internal quantification control nucleic acid needs to be relatively high.

Therefore, preferred aspects of the present invention include:
h. The method as described above, further comprising determining one or more amounts of said target nucleic acid.

  The above-described method, which is internally conjugated, requires a much shorter run time and the test is much simpler than performing the real-time PCR method used in the prior art. The method offers great advantages, for example in the field of clinical virology, since it allows the parallel amplification of several viruses in parallel experiments. The method is particularly useful for managing post-transplant patients requiring frequent virus monitoring. Thereby, the method facilitates economical diagnosis and contributes to the use of antiviral agents and reduced viral complications and hospitalization. This applies equally to the field of clinical microbiology. Generally, efficiency is increased with faster turnaround times and improved test flexibility. Consequently, this may reduce the number of tests required by the patient to make a diagnosis and may reduce the length of hospital stay (e.g., if diagnosis can be provided earlier, patients requiring antimicrobial therapy Receive the therapy sooner and recover faster). Also, the cost of adjuvant therapy (eg, intensive care for delayed diagnosis of sepsis) is lower, as patients exhibit lower morbidity. Providing negative results sooner may have important implications for over-prescribing antibiotics. For example, if the test results obtained by the method according to the invention can rule out the pathogen sooner than a standard real-time PCR method, the clinician is not forced to use empirical antibiotics. Alternatively, if empirical antibiotics are used, the duration of each treatment may be shorter.

With respect to designing a particular test based on the method of the present invention, one of ordinary skill in the art will not particularly be limited to the following advantages:
Reduced software complexity (results in reduced risk of programming errors)
Focusing assay development efforts on chemistry optimization instead of chemistry and instrument control parameters.A single process is always used, and hardware can be optimally designed to implement this protocol. Significantly increases the reliability of the system.Those skilled in the art performing the above internally symmetric methods provide the flexibility to perform many different assays in parallel as part of the same method Benefit from cost savings.

  In the sense of the present invention, "purification", "isolation" or "extraction" of a nucleic acid refers to the following: Before a nucleic acid can be analyzed in a diagnostic assay, for example by amplification, the nucleic acid typically comprises There is a need to purify, isolate or extract from a biological sample containing a complex mixture of components. Often, for the first step, a process is used that allows the concentration of the nucleic acid. To release the contents of the cell or virus particle, the cell or virus particle can be treated with an enzyme or chemical to dissolve, degrade or denature the cell wall or virus particle. This process is commonly referred to as lysis. The resulting solution containing such a dissolved substance is called a lysis solution. A problem often encountered during lysis is that other enzymes that degrade the component of interest, such as deoxyribonucleases or ribonucleases that degrade nucleic acids, come into contact with the component of interest during the lysis procedure. These degrading enzymes may be present outside the cells or may be spatially separated into different cell compartments before lysis. As the dissolution occurs, the target component is exposed to the degrading enzyme. Other components released during this process can be, for example, endotoxins belonging to the family of lipopolysaccharides that are toxic to cells, which can cause problems for products intended for use in human or animal therapy.

  There are various means to address the above issues. Where it is intended to release nucleic acids, it is common to use chaotropic agents such as guanidinium thiocyanate or anionic, cationic, zwitterionic or nonionic surfactants. It is also advantageous to use the aforementioned enzymes or proteases that rapidly degrade unwanted proteins. However, this can create another problem in that the substances or enzymes can interfere with reagents or components in subsequent steps.

  Enzymes that can be advantageously used in such lysis or sample preparation processes described above are those that cleave amide bonds in protein substrates and are classified as proteases or (interchangeably) peptidases (Walsh, 1979, Enzymatic Reaction Mechanisms. See WH Freeman and Company, San Francisco, Chapter 3.). Proteases used in the prior art include alkaline protease (WO 98/04730) or acidic protease (US 5,386,024). Proteases widely used for sample preparation in the isolation of nucleic acids in the prior art are active near neutral pH and are derived from Tritirachium album, which belongs to the family of proteases known to those skilled in the art as subtilisins. (See, eg, Sambrook J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989). The enzyme esperase (EP 120 753), a potent protease that maintains its activity both at high alkalinity and at high temperatures, is particularly advantageous for use in the lysis or sample preparation process described above.

  In the sample preparation step after the dissolution step, the target component is further concentrated. Where the non-protein component of interest is, for example, a nucleic acid, the nucleic acid is usually extracted from a complex lysis mixture before being used in a probe-based assay.

Several methods are available for nucleic acid purification:
-Sequence-dependent or organism-specific methods, for example:
・ Affinity chromatography ・ Hybridization to immobilized probe
-Sequence independent or physicochemical methods, for example:
・ Liquid-liquid extraction using phenol-chloroform ・ Precipitation using pure alcohol ・ Extraction using filter paper ・ Extraction using micelle forming agent such as cetyltrimethylammonium bromide ・ Immobilized intercalation dye, Examples include binding to acridine derivatives, adsorption to silica gel or diatomaceous earth, and adsorption to magnetic glass particles (MGP) or organo-silane particles under chaotropic conditions.

  While other surfaces are possible, adsorption of nucleic acids to glass surfaces is of particular interest for purification purposes. A number of approaches have recently been proposed for isolating nucleic acids from their natural environment by using the binding behavior of nucleic acids to glass surfaces. When an unmodified nucleic acid is the target, direct binding of the nucleic acid to a substance having a silica surface is preferred, especially since the nucleic acid does not need to be modified and can bind even a natural nucleic acid. These methods are described in detail in various documents. USA 76 (1979) 615-9, for example, the binding of nucleic acids from agarose gels to ground flint glass in the presence of sodium iodide. Is proposed. Purification of plasmid DNA from bacteria on glass dust in the presence of sodium perchlorate is described in Marko M. A. et al., Anal. Biochem. 121 (1982) 382-387. DE-A 37 34 442 describes the isolation of single-stranded M13 phage DNA on glass fiber filters by precipitation of phage particles using acetic acid and lysis of phage particles using perchlorate. The nucleic acids bound to the glass fiber filters are washed and then eluted with a methanol-containing Tris / EDTA buffer. A similar procedure for purification of DNA from lambda phage is described in Jakobi R. et al., Anal. Biochem. 175 (1988) 196-201. The technique requires the selective binding of nucleic acids in a chaotropic salt solution to a glass surface and the separation of nucleic acids from contaminants such as agarose, proteins or cellular debris. To separate the glass particles from contaminants, the particles can be centrifuged or the liquid can be drawn off with a glass fiber filter. However, this is a limiting step that prevents using the technique to process large samples. The use of magnetic particles to immobilize nucleic acids after precipitation by the addition of salts and ethanol is more advantageous, for example Alderton RP et al., S., Anal.Biochem. 201 (1992) 166-169 and PCT GB 91/00212. In this approach, nucleic acids aggregate together with the magnetic particles. Aggregates are separated from the original solvent by applying a magnetic field and performing a washing step. After one washing step, the nucleic acids are dissolved in Tris buffer. However, this approach has the disadvantage that the precipitation is not selective for nucleic acids. Rather, various solids and dissolved substances also aggregate. Consequently, this approach cannot be used to remove significant amounts of any inhibitors of a particular enzymatic reaction that may be present. Magnetic porous glasses comprising magnetic particles in a porous specific glass matrix and coated with a streptavidin-containing layer are also commercially available. This product can be used to isolate a biological material, such as a protein or nucleic acid, where the biological material has been modified to covalently bind to biotin in a complex preparation step. Certain magnetizable adsorbents have been found to be very effective and suitable for automated sample preparation. Ferrimagnetic and ferromagnetic and superparamagnetic dyes are used for this purpose. The most preferred magnetic glass particles and methods of using them are those described in WO 01/37291. In the context of the present invention, the method according to R. Boom et al. (J Clin Microbiol. 28 (1990), 495-503) is particularly useful for the isolation of nucleic acids.

  The term "solid support material" includes any of the solid materials described above with respect to immobilization of nucleic acids, such as magnetic glass particles, glass fibers, glass fiber filters, filter paper, etc., wherein the solid support material is Not limited.

  Accordingly, a preferred aspect of the present invention is the method as described above, wherein the solid support material comprises one or more materials selected from silica, metals, metal oxides, plastics, polymers and nucleic acids. In a highly preferred embodiment of the present invention, the solid support material is a magnetic glass particle.

  In the context of the present invention, "immobilization" means to capture an object, for example a nucleic acid, in a reversible or irreversible manner. In particular, "immobilized on a solid support material" refers to binding an object or objects to a solid support material to separate the object or objects from any surrounding medium, at a later time, It means that it can be recovered from the solid support material, for example, by separation. In this context, "immobilization" may include, for example, the adsorption of nucleic acids to a glass surface or other suitable surface of a solid substance as described above. Further, by binding to a capture probe, the nucleic acid can be specifically "immobilized", wherein the nucleic acid is bound by base pairing to an essentially complementary nucleic acid bound to a solid support. In the latter case, such specific immobilization results in predominant binding of the target nucleic acid.

  Following purification or isolation of the nucleic acid, including the target nucleic acid, from natural surroundings, analysis can be performed, for example, by co-amplification as described above.

  In the sense of the present invention, "simultaneously" means that two operations, for example amplifying the first and second or more nucleic acids, are performed at the same time under the same physical conditions. In one embodiment, the simultaneous amplification of at least the first and second target nucleic acids is performed in one container. In another embodiment, the co-amplification is performed simultaneously with at least one nucleic acid in one container and at least a second nucleic acid in a second container, especially under the same physical conditions with respect to temperature and incubation time. Wherein the internal control nucleic acid described above is present in each of the containers.

  A “first target nucleic acid” and a “second target nucleic acid” are different nucleic acids.

  A “fluid sample” is any liquid substance that can be subjected to a nucleic acid-targeted diagnostic assay, preferably from a biological source. Preferably, the liquid sample is derived from a human and is a body fluid. In a preferred embodiment of the invention, the liquid sample is human blood, urine, sputum, sweat, swabs, pipetable feces or spinal fluid. Most preferably, the liquid sample is human blood.

  The term "reaction vessel" refers to, but is not limited to, a tube or microwell, deep well or other type of multiwell plate in which a reaction occurs for the analysis of a liquid sample, such as, for example, a reverse transcription reaction or a polymerase chain reaction. Including wells of the plate. The outer boundaries or walls of such vessels are chemically inert so as not to interfere with the analytical reactions taking place therein. Preferably, the nucleic acid isolation described above is also performed in a multiwell plate.

  In this context, a multi-well plate in an analysis system allows for parallel separation and analysis or storage of multiple samples. Multi-well plates can be optimized for maximum liquid uptake or maximum heat transfer. Preferred multiwell plates for use in the context of the present invention are optimized for incubation or separation of analytes in an automated analyzer. Preferably, the multi-well plate is constructed and arranged to contact magnetic devices and / or heating devices.

Said preferred multi-well plates, interchangeably referred to as "treatment plates" in the context of the present invention, are:
-Includes a top surface that includes a number of containers with openings at the top arranged in rows. The container includes a top part, a center part and a bottom part. The upper part is connected to the upper surface of the multi-well plate and includes two long sides and two short sides. The central part is a substantially rectangular cross section with two long sides and two short sides,
-Two opposing short side walls and two opposing long side walls, and
-Having a base, said base including openings constructed and arranged to place a multi-well plate in contact with said magnetic device and / or heating device.

  In a preferred embodiment of the multi-well plate, adjacent vessels in a row are connected on the substantially rectangular long side.

  Preferably, the multi-well plate includes a contiguous space located between adjacent rows of containers. The continuous space is constructed and arranged to accommodate a plate-type magnetic device. In a preferred embodiment, the bottom part of the container comprises a spherical bottom. In a more preferred embodiment, the bottom part of the container comprises a conical part located between the central part and the spherical bottom.

  In a preferred embodiment, the top surface comprises a rib, said rib surrounding the opening of the container. Preferably, one short side of the upper part of the container includes a recess, the recess including a curved surface extending from the rib to the inside of the container.

  Further, in a preferred embodiment, the container includes a rounded inner shape.

  For fixation to a processing or incubation station, the base preferably includes a rim including a recess. Latch clips on the analyzer station may engage the recess to secure the plate on the station.

  In a preferred embodiment, the container comprises an essentially constant wall thickness.

  The preferred processing plate (101) in the context of the present invention is a one-component plate. Its upper surface (110) contains a number of containers (103) (FIGS. 5, 6). Each container has an opening (108) at the top and is closed at the bottom end (112). The upper surface (110) is preferably elevated relative to the upper surface (110) and includes a rib (104) surrounding the opening (108) of the container (103). This prevents contamination of the contents of the container (103) by droplets that may fall on the upper surface (110) of the plate (101). A diagram of a preferred processing plate is shown in FIGS.

  The footprint of the processing plate (101) preferably includes a base length and width corresponding to the ANSI SBS mounting surface type. More preferably, the length is 127.76mm +/- 0.25mm and the width is 85.48mm +/- 0.25mm. Thus, the plate (101) has two opposing short side walls (109) and two opposing long side walls (118). The processing plate (101) includes a form locking element (106) for interacting with the pilot (500, FIG. 12). The processing plate (101) can be quickly, safely and quickly grabbed, moved and positioned while maintaining the correct orientation and position. Preferably, the form-locking element (106) for gripping is located in the upper central part, preferably in the upper central third of the processing plate (101). This has the advantage that possible distortions of the processing plate (101) have only a small effect on the form-fixing element (106) and that the handling of the plate (101) is more robust.

  Preferably, the processing plate (101) includes hardware identifiers (102) and (115). The hardware identifiers (102) and (115) are specific to the processing plate (101) and are different from the hardware identifiers of other consumables used in the same system. The hardware identifier (102, 115) preferably comprises a ridge (119) and / or recess (125) on the side wall of the consumable, wherein the pattern of the ridge (119) and / or recess (125) is specific. Type of consumables, preferably processing plate (101). This unique pattern is also referred to herein as a unique “surface geometry”. The hardware identifiers (102, 115) ensure that the user can load only the processing plate (101) in the appropriate direction and into the appropriate stacker location on the analyzer. The side of the processing plate (101) contains the guiding elements (116) and (117) (FIGS. 3, 4). They prevent tilting of the processing plate (101). The guide element (116, 117) allows the user to load the processing plate (101) into the analyzer using the guide element (116, 117) as a stack, and then vertically move the plate in a stacker in the apparatus without tilting the plate. To be able to move.

  The central part (120) of the container (103) has a substantially rectangular cross section (FIGS. 6, 7). They are separated along a generally rectangular long side (118) by a common wall (113) (FIG. 3). The row of containers (103) formed thereby has the advantage that instead of the available space being limited, it has a large volume, preferably 4 ml. Another advantage is that manufacturing is very economical due to the essentially constant wall thickness. A further advantage is that a high stability of the shape can be obtained since the containers (103) are reinforced with each other.

  A continuous space (121) is located between the rows of containers (103) (FIGS. 6, 7). The space (121) may house a magnet (202, 203) or a heating device (128) (FIG. 11). These magnets (202, 203) and heating device (128) are preferably solid devices. Thus, the magnetic particles (216) contained in the liquid (215), which can be retained in the container (103), cause the magnet (202, 203) to move closer to the container (103) ) Can be separated from the liquid (215) by applying a magnetic field. Alternatively, the contents of the container (103) may be incubated at a controlled high temperature when the processing plate (101) is placed on a heating device (128). Since the magnet (202, 203) or the heating device (128) can be solid, a high energy density can be achieved. The generally rectangular shape (FIG. 10) of the central part (120) of the container (103) also enhances the shape of the container by optimizing the contact surface between the container (103) and the magnet (202) or the heating device (128). Energy transfer to the container (103) is enhanced because the contact between the wall (109) and the flat shaped magnet (202) or the heating device (128) is optimized.

  In the region of the conical bottom (111) of the container, the space (121) is much clearer and can accommodate further magnets (203). The combination of a large magnet (202) in the upper region of the vessel and a small magnet (203) in the conical region allows for the separation of magnetic particles (216) in a large or small volume of liquid (215). Thus, the small magnet (203) makes it easier to sequester the magnetic particles (216) during pipetting of the eluate. This allows the eluate to be pipetted with minimal loss by reducing the dead volume of the magnetic particle (216) pellet. Furthermore, the presence of magnetic particles (216) in the eluate to be transferred is minimized.

  At the upper end of the container (103), one of the short side walls (109) of the container (103) includes a reagent inflow channel (105) extending to a peripheral rib (104) (FIGS. 3, 4, 7). The reagent is pipetted into the reagent inlet channel (105) and flows from the channel (105) into the container (103). Thus, contact between the pipette needle or tip (3, 4) and the liquid contained in the container is prevented. In addition, splashing caused by liquid dispensed directly to another liquid (215) contained in the container (103), which can cause contamination of the pipette needle or tip (3, 4) or the adjacent container (103), is prevented. It is. Continuous pipetting of the largest amount of another reagent into the reagent inflow channel (105) after a small amount of reagent ensures that only the small amount of reagent flows completely into the container (103). It becomes. Thus, pipetting of small amounts of reagent is possible without compromising the accuracy of the tests performed.

  On the inside, at the bottom (111, 112) of the container, the shape becomes conical (111) and the end has a spherical bottom (112) (FIGS. 6, 7). The inner shape (114) of the container, including the rectangular central part (120), is round. The combination of the spherical bottom (112), round inner shape (114), conical part (111) and fine surface of the vessel (103) facilitates effective separation and purification of analytes in the processing plate (101) Cause favorable fluidics. The spherical bottom (112) allows for essentially complete use of the separated eluate and reduced dead volume, which reduces reagent carryover or sample cross-contamination.

  A rim (129) at the base of the processing plate (101) has a recess (107) for connection with a heating device (128) or a latch clip (124) or an analyzer (126) on the processing station (201). Included (FIGS. 5 and 9). The connection of the latch clip (124) and the recess (107) allows for the installation and securing of the processing plate (101) on the processing station (201). Due to the presence of the recess (107), a substantially perpendicular latching force acts on the processing plate (101) with respect to the base (129). Thus, only small lateral forces can occur. Thereby, the occurrence of distortion is reduced, so that the deformation of the processing plate (101) is reduced. The vertical latching force also counteracts any deformation of the processing plate (101), resulting in a more accurate placement of the spherical bottom (111) in the processing station (201). In general, a precise interface between the processing plate (101) and the processing station (201) or heating device (128) in the analyzer reduces dead volume and further reduces the risk of sample cross-contamination.

  A "separation station" is a device or component of an analytical system that allows the isolation of a solid support material from other materials present in a liquid sample. Such separation stations may include, but are not limited to, for example, centrifugation, racks with filter tubes, magnets or other suitable components. In a preferred embodiment of the invention, the separation station includes one or more magnets. Preferably, one or more magnets are used for the separation of magnetic particles, preferably magnetic glass particles, as a solid support. For example, if a liquid sample and a solid support material are combined together in a well of a multi-well plate, one or more magnets included in the separation station contact the liquid sample itself, for example, by introducing a magnet into the well. Alternatively, the one or more magnets may be approached to the outer wall of the well to attract the magnetic particles and subsequently separate the magnetic particles from the surrounding liquid.

  In a preferred embodiment, the separation station is a device that includes a multiwell plate that includes a container having an opening in the top surface of the multiwell plate and a closed bottom. The container includes a top part, a center part and a bottom part, wherein the top part is connected to the top surface of the multiwell plate and preferably comprises two long sides and two short sides. The central part has a substantially rectangular cross section with two long sides, said containers being arranged in rows. Between two adjacent rows, at at least two Z positions, there is a continuous space for selectively contacting the side wall with at least one magnet mounted on the fixture. The device further includes a magnetic separation station that includes at least one fixture. The fixture includes at least one magnet that produces a magnetic field. There is a movement mechanism for vertically moving at least one said fixture including at least one magnet between at least the first and second positions with respect to the container of the multi-well plate. Preferably, the at least two Z positions of the container include a side wall and a bottom part of the container. The magnetic field of the at least one magnet preferably attracts magnetic particles to an inner surface of a container adjacent to the at least one magnet when the at least one magnet is in the first position. The effect of the magnetic field is less when the at least one magnet is in the second position than when the at least one magnet is in the first position. Preferably, the fixture including the at least one magnet includes a frame. The container has the preferred features as described above in the context of a multiwell plate / processing plate. One such preferred feature is that at least a portion of the container has a substantially rectangular cross section orthogonal to the axis of the container.

  In the first position, the at least one magnet is adjacent to the part of the container. Adjacent is understood to mean either proximal, such as exerting a magnetic field on the contents of the container, or in physical contact with the container.

  The separation station includes a frame for receiving the multiwell plate, and a latch clip for attaching the multiwell plate. Preferably, the separation station includes two types of magnets. This preferred embodiment is described further below.

  A second preferred embodiment is described below, which includes a spring that applies pressure to the frame containing the magnet so that the magnet is pressed against the container of the multiwell plate.

  Preferably, the first magnet is constructed and arranged to interact with a container of a multi-well plate to apply a magnetic field to a volume of liquid containing magnetic particles held in the container. The second magnet is preferably constructed and arranged to interact with a container of a multiwell plate to apply a magnetic field to a small volume of liquid containing magnetic particles held in the container. The first and second magnets can be moved to different Z positions.

  Said separation station is a further method of isolating and purifying nucleic acids and is useful in the context of the present invention. The method includes the step of binding nucleic acids to magnetic particles in a container of a multiwell plate. The container includes a top opening, a center part and a bottom part. Moving a magnet from a second position to a first position when a majority of the liquid is located above a field portion where the conical part of the container replaces a central part having a rectangular shape; and At one location, the bound material is then separated from the unbound material contained in the liquid by applying a magnetic field to the central part and, optionally, a further magnetic field to the bottom part of the vessel. The magnetic particles can optionally be washed with a washing solution. If the majority of the liquid is located below where the conical part of the container replaces the central part having a rectangular shape, a small amount of liquid is applied by selectively applying a magnetic field to the bottom part of the container. Separated from magnetic particles.

  A magnetic separation station for separating nucleic acids bound to magnetic particles is also useful in the context of the present invention, wherein the separation station is a multi-stage for applying a magnetic field to a large volume of liquid containing magnetic particles held in the container. A first magnet constructed and arranged to interact with the well plate container, and interacts with the multi-well plate container to apply a magnetic field to a small volume of liquid containing magnetic particles retained in the container. And a second magnet constructed and arranged as described above, wherein the first and second magnets can be moved to different Z positions. Preferred embodiments of the magnetic separation station are described herein.

  A first preferred embodiment of the separation station (201) useful in the present invention is described below. A first preferred embodiment of the separation station (201) comprises at least two types of magnets (202, 203). The first long type magnet (202) is constructed and arranged to fit in the space (121) of the processing plate (101). Thus, the magnet (202) applies a magnetic field to the liquid (215) in the container (103) to isolate the magnetic particles (216) inside the container wall. Accordingly, when a large amount of the liquid (215) is present, the liquid (215) can be separated from the magnetic particles (216) inside the container (103) and any substance bonded thereto. The magnet (202) has an elongated structure and is constructed and arranged to interact with the substantially rectangular central part (120) of the container. Thus, the magnet (202) is used when most of the liquid (215) is located above where the conical part (111) of the container (103) replaces the central part (120) having a rectangular shape. Is done. As shown in FIG. 40, the preferred construction of the magnet (202) is that the fixture (204, 204) includes a magnet (202) that fits into the space (121) between the rows of vessels (103) in the processing plate (101). 204a). Another preferred embodiment of the magnet (202) includes a magnet (202) arranged on a fixture (204, 204a). The magnet (203) of the preferred separation station (201) is smaller and can interact with the conical part (111) of the container (103). This is shown in FIG. Preferably, the magnet (203) is arranged on the base (205) and can be moved to the space (121) of the processing plate (101). Each magnet (202, 203) is preferably constructed to interact with two containers (103) in two adjacent rows. In a preferred embodiment, the processing plate (101) has six rows of eight containers (103). A separation station (201) that can interact with the preferred processing plate (101) has three fixtures (204, 204a) containing magnets (202) and four bases (205) containing magnets (203). . Also included are embodiments in which the separation station has four magnetic fixtures (204, 204a) including magnets (202) and three magnetic bases (205) including magnets (203).

  The magnets (202, 203) are movable. The separation station (201) includes a mechanism for moving the fixtures (204, 204a) and the base (205). All fixtures (204, 204a) move together because they are interconnected by a base (217). All magnets (203) are linked to one base (218) and therefore move in concert. The mechanism for moving the magnetic plates (202) and (203) is constructed and arranged to move the two magnetic plates (202, 203) to a total of four terminal positions.

  40a-c, the magnet (203) is located proximal to the conical part of the container (103) of the processing plate (101). This is the highest position of the magnet (203) and is the separation position. In this figure, the magnet (202) is located at the lowest position. When they are in this position, the magnet does not participate in the separation.

  In the preferred embodiment shown in FIG. 10, the base (217) of the magnet (202) is connected to a positioning wheel (206). The base (217) includes a bottom end (207) that is in flexible contact with the connecting element (208) by the moving element (209). The moving elements are constructed and arranged to move the connecting element (208) along one of the rails (212) from one side to the other. The moving element (209) is fixed to the connecting element (208) by a pin (220). The connecting element (208) is fixed to the positioning wheel (206) with screws (210). The connecting element (208) is also connected to the shaft (211). Said connecting element (208) is preferably a rectangular plate. As the positioning wheel (206) moves eccentrically about the axis (211), the screw (210) moves from a point above the eccentric axis to a point below the eccentric axis and moves. The bottom end (207) of the base (204) to which the element (209) and the magnet (202) are attached is moved from the highest position to the lowest position. The base (218) rests on the bottom part (219) and is connected at its lower end to a moving element (214), preferably a wheel, by means of a pin (213) and interacts with a positioning wheel (206). When positioning wheel (206) rotates about axis (211), wheel (214) moves along with positioning wheel (206). When the wheel (214) is located at a position where the distance between the positioning wheel (206) and the axis (211) is short, the magnet (203) is at its lowest position. When the wheel (214) is located where the distance between the positioning wheel (206) and the axis (211) is greatest, the magnet (203) is at its highest position. Thus, in a preferred embodiment of the first embodiment of the separation station, the position of the magnet (203) is controlled by the shape of the positioning wheel (206). As the moving element (209) moves along the center rounded upper or lower part (212a) of the rail (212), the smaller magnet (203) will move up and down. When the moving element (209) is located on the side surface (212b) of the bottom end (207) and moves up and down, the magnet (202) moves up or down. The positioning wheel can be rotated by any motor (224).

  In a preferred embodiment, the base of the separation station (222) and the base (218) of the magnet (203) to ensure that the magnet (203) moves to the lowest position as it moves downward. The spring (225) is attached to.

  As used herein, the term "pin" relates to any securing element, including a screw or pin.

  In a second preferred embodiment, the separation station (230) comprises at least one fixture comprising at least one magnet (232), preferably as many magnets as the number of vessels (103) in the row (123). (231). Preferably, the separation station (230) includes as many fixtures (231) as the number of rows (123) of the multi-well plate (101) described above. More preferably, six fixtures (231) are mounted on the separation station (230). At least one magnet (232) is mounted on one fixture (231). Preferably, the number of magnets (232) is the same as the number of containers (103) in one row (123). Most preferably, eight magnets (232) are mounted on one fixture (231). Preferably, one type of magnet (232) is included in the fixture (231). More preferably, the magnet (232) is mounted on one side towards a container that interacts with the magnet.

  The fixture (231) is mounted on the base (233). Preferably, the load is flexible. The base (233) includes a spring (234) mounted thereon. The number of springs (234) is at least one per fixture (231) placed on the base (233). The base further includes a round groove (236) that limits the movement of the spring and consequently the movement of the fixture (231) including the magnet (232). Preferably, any one of said springs (234) is constructed and arranged to interact with fixture (231). More preferably, said spring (234) is a yoke spring. The horizontal movement of the fixture (231) is controlled by the interaction. Further, the separation station (230) includes a frame (235). The base (233) having the fixture (231) is connected to the frame (235) by the above-described moving mechanism for the magnet (232) of the first embodiment.

  Preferably, the base (233) and the fixture (231) are constructed and arranged to move vertically (in the Z direction).

  The multi-well plate (101) described above is inserted into the separation station (230). The fixture (231) including the magnet (232) is moved vertically. Thus, any one of the fixtures (232) moves into the space (121) between the two rows (123) of the container (103). The vertical movement causes the magnet (232) mounted on the fixture (231) to come into contact with the container (103). The Z position is selected depending on the volume of the liquid (215) in the container (103). In large quantities, the magnet (232) contacts the container (103) at a central position (120) where the container (103) is substantially rectangular. With a small amount of liquid (215) where most of the liquid (215) is located below the central part (120) of the container (103), the magnet (232) is preferably a conical part (111) of the container (103). Contact with

  A spring is attached to the base (233) of any one of the frames (231) (FIGS. 9a) and b)). The spring presses the magnet (232) against the container (103). This ensures contact between the magnet (232) and the container (103) during magnetic separation. Preferably, the magnet (232) contacts the container (103) at a side wall (109) located below the inlet (105). This has the advantage that the liquid added by pipetting flows over the isolated magnetic particles, ensuring that the particles are resuspended and that all samples in all vessels are treated identically. I do.

  In this embodiment, when different levels of liquid (215) are contained in the container (103) of the multi-well plate (101), the liquid (215) contained in the above-described multi-well plate (101) and the magnetic particles ( Particularly suitable for the separation of 216).

  A "wash buffer" is a liquid specifically designed to remove unwanted components in a purification procedure. Such buffers are well-known in the art. In the context of nucleic acid purification, a wash buffer is suitable for washing the solid support material to separate immobilized nucleic acids and any undesirable components. For example, the wash buffer may include ethanol and / or chaotropic agents in the buffered solution, or may include a solution at an acidic pH without the ethanol and / or chaotropic agents described above. Often, a wash solution or other solution is provided as a stock solution that needs to be diluted before use.

  Washing in the method of the invention requires somewhat stronger contact between the solid support material and the nucleic acids immobilized thereon and the wash buffer. This can be accomplished in a variety of ways, such as by shaking the wash buffer and solid support material in or with one or more respective vessels. Another advantageous method is to aspirate and dispense the suspension containing the wash buffer and the solid support material one or more times. The method is preferably performed using a pipette, wherein said pipette preferably comprises a disposable pipette tip for aspirating and redispensing said suspension. Such pipette tips can be used several times before disposal and replacement. Disposable pipette tips useful in the present invention preferably have a volume of at least 10 μl, more preferably at least 15 μl, more preferably at least 100 μl, more preferably at least 500 μl, more preferably at least 1 ml, even more preferably about 1 ml. The pipette used in the context of the present invention may be a pipetting needle.

  Accordingly, a preferred aspect of the present invention is the above method, wherein said washing in step d comprises aspirating and dispensing a washing buffer comprising a solid support material.

  For further processing of the isolated nucleic acid, it may be advantageous to separate the nucleic acid from the solid support material before subjecting it to amplification.

  Therefore, a preferred aspect of the present invention is the above-described method, wherein the method further comprises, in step d, washing the solid support material and then eluting the purified nucleic acid from the solid support material with an elution buffer.

  An "elution buffer" in the context of the present invention is a liquid suitable for separating nucleic acids from a solid support. Such a liquid may be, for example, sterile water or an aqueous salt solution such as Tris buffer or HEPES, such as Tris HCl, or other suitable buffers known to those skilled in the art. The pH value of such an elution buffer is preferably alkaline or neutral. The elution buffer may include additional components such as, for example, a chelating agent such as EDTA to stabilize the isolated nucleic acid by inactivation of the degrading enzyme.

  Preferably, the elution is performed at an elevated temperature, so a preferred embodiment of the present invention is a method as described above, wherein step d is performed at a temperature from 70C to 90C, more preferably at a temperature of 80C.

  An "amplification reagent" in the context of the present invention is a chemical or biochemical component that allows amplification of a nucleic acid. Such reagents include, but are not limited to, nucleic acid polymerases, buffers, mononucleotides such as nucleoside triphosphates, oligonucleotides such as oligonucleotide primers, salts and their respective solutions, detection probes, dyes, and the like.

  As known in the art, a "nucleoside" is a combination of a base and a sugar. The base portion of the nucleoside is usually a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines.

  "Nucleotides" are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be attached to either the 2'-, 3'- or 5'-hydroxyl moiety of the sugar. Nucleotides are the monomer units of "oligonucleotides" which are more generally described as "oligonucleotide compounds" or "polynucleotides" and may be more generally described as "polymer compounds". Another general expression mentioned above is deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

  According to the present invention, an `` oligomeric compound '' is a `` monomer unit '' which may be a nucleotide alone or a non-natural compound (see below), more specifically a modified nucleotide (or nucleotide analog) or a non-nucleotide compound A compound consisting of "monomer units" which may be used alone or in combination.

  "Oligonucleotides" and "modified oligonucleotides" (or "oligonucleotide analogs") are a subgroup of oligomeric compounds. In the context of the present invention, the term "oligonucleotide" refers to a component formed by a plurality of nucleotides as its monomer unit. The phosphate groups generally refer to those that form the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3'-5 'phosphodiester linkage. Oligonucleotides and modified oligonucleotides useful in the present invention (see below) are described primarily in the art and can be synthesized as known to those skilled in the art. Methods for preparing oligomeric compounds of specific sequence are known in the art and include, for example, cloning and restriction of appropriate sequences, and direct chemical synthesis. Chemical synthesis methods include, for example, the phosphotriester method described in Narang SA et al., Methods in Enzymology 68 (1979) 90-98, and Brown EL, et al., Methods in Enzymology 68 (1979) 109-151. The disclosed phosphodiester method, the phosphoramidite method disclosed in Beaucage et al., Tetrahedron Letters 22 (1981) 1859, and the H-type disclosed in Garegg et al., Chem. Scr. 25 (1985) 280-282. Mention may be made of the phosphonate method and the solid support method disclosed in US 4,458,066.

  In the above method, the oligonucleotide may be chemically modified, ie, the primers and / or probes comprise modified nucleotides or non-nucleotide compounds. The probe or primer is then a modified oligonucleotide.

  A "modified nucleotide" (or "nucleotide analog") differs from a naturally occurring nucleotide by some modification, but still retains the base, pentofuranosyl sugar, phosphate moiety, base-like moiety, pentofurano Consisting of a sugar sugar-like moiety and a phosphate-like moiety or a combination thereof. For example, if a label can be attached to the base portion of the nucleotide, a modified nucleotide is obtained. Natural bases in nucleotides can also be replaced by, for example, 7-deazapurine, resulting in similarly modified nucleotides.

  “Modified oligonucleotides” (or “oligonucleotide analogs”), which belong to another specific subgroup of oligomeric compounds, have one or more nucleotides and one or more modified nucleotides as monomer units. Thus, the term "modified oligonucleotide" (or "oligonucleotide analog") refers to a structure that functions substantially similarly to an oligonucleotide and may be used interchangeably in the context of the present invention. From a synthetic point of view, modified oligonucleotides (or oligonucleotide analogs) can be made by chemical modification of the oligonucleotide, for example by appropriate modification of the phosphate backbone, ribose units or nucleotide bases (Uhlmann and Peyman, Chemical Reviews 90 ( 1990) 543; Verma S., and Eckstein F., Annu. Rev. Biochem. 67 (1998) 99-134). Exemplary modifications include phosphorothioate, phosphorodithioate, methylphosphonate, phosphortriester or phosphoramidate internucleoside linkage instead of phosphodiester internucleoside linkage; deaza- or azapurine instead of natural purine and pyrimidine bases And -pyrimidine, a pyrimidine base with a substituent at position 5 or 6; a purine base with a substituent at position 2, 6 or 8 or modified at position 7 as 7-deazapurine; alkyl-, alkenyl-, alkynyl- or A base having an aryl moiety, for example, a lower alkyl group such as methyl, ethyl, propyl, butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or an aryl group such as phenyl, benzyl, naphthyl; 'Substituent at position Sugar to; or carbocyclic or acyclic sugar analogs. Other modifications consistent with the spirit of the invention are known to those skilled in the art. It is best described that such modified oligonucleotides (or oligonucleotide analogs) are functionally interchangeable with natural oligonucleotides, but structurally different. More particularly, exemplary modifications are disclosed in Verma S., and Eckstein F., Annu. Rev. Biochem. 67 (1998) 99-134 or WO 02/12263. Also, modifications can be made to link nucleoside units through groups that are alternatives to internucleoside phosphate or sugar phosphate linkages. Such linkages include those disclosed in Verma S., and Eckstein F., Annu. Rev. Biochem. 67 (1998) 99-134. When a nucleoside unit is linked using a means other than a phosphate bond, such a structure is also described as “oligonucleoside”.

  "Nucleic acid" and "target nucleic acid" are polymeric polymeric compounds of nucleotides known to those of skill in the art. “Target nucleic acid” is used herein to indicate a nucleic acid in a sample to be analyzed, ie, to determine its presence, absence, and / or its amount in the sample.

  The term `` primer '' is used herein as known to those of skill in the art and refers to oligomeric compounds, primarily oligonucleotides, that can induce DNA synthesis by a template-dependent DNA polymerase, i.e., e.g., The 3 'end of the primer provides a free 3'-OH group, where additional nucleotides can be attached by a template-dependent DNA polymerase that establishes a 3'- to 5'-phosphodiester bond, whereby the deoxynucleoside It also refers to modified oligonucleotides in which triphosphate is used to release pyrophosphate.

  "Probe" also refers to a natural or modified oligonucleotide. As is known in the art, a probe serves for the purpose of detecting an analyte or amplificate. In the case of the method described above, the probe can be used to detect an amplification of the target nucleic acid. For this purpose, the probe typically has a label.

  A "label", often referred to as a "reporter group," is a group that generally distinguishes a nucleic acid, particularly an oligonucleotide or modified oligonucleotide, as well as any nucleic acid attached thereto, from the rest of the sample (label The nucleic acid to which is attached may also be referred to as a labeled nucleic acid binding compound, a labeled probe or simply a probe). Preferred labels of the present invention are fluorescent labels which are fluorescent dyes such as, for example, fluorescein dyes, rhodamine dyes, cyanine dyes and coumarin dyes. Preferred fluorescent dyes of the present invention are FAM, HEX, JA270, CAL635, Coumarin 343, Quasar705, Cyan 500, CY5.5, LC-Red 640, LC-Red 705.

  In the context of the present invention, any primer and / or probe may be chemically modified, ie, the primer and / or probe comprises a modified nucleotide or non-nucleotide compound. The probe or primer is then a modified oligonucleotide.

  A preferred method of nucleic acid amplification is the polymerase chain reaction (PCR) disclosed in U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159 and 4,965,188, among other references. PCR typically employs two or more oligonucleotide primers that bind to a selected nucleic acid template (eg, DNA or RNA). Primers useful for nucleic acid analysis include oligonucleotides that can act as initiation points for nucleic acid synthesis in the nucleic acid sequence of a target nucleic acid. Primers can be purified from restriction digests by conventional methods or made synthetically. Preferably, the primer is single-stranded for maximum efficiency in amplification, but the primer can be double-stranded. The double-stranded primer is first denatured, ie, treated to separate the strands. One method of denaturing double-stranded nucleic acids is by heating. A "thermostable polymerase" is a thermostable polymerase enzyme that catalyzes the formation of a primer extension product complementary to a template and is subjected to elevated temperatures for the time necessary to cause denaturation of the double-stranded template nucleic acid. It is an enzyme that does not irreversibly denature when performed. Generally, synthesis starts at the 3 'end of each primer and proceeds in the 5'-3' direction along the template strand. Thermostable polymerases include, for example, Thermus Flavas, T. ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillus stearothermophilus and Methanothermus fervidus. ). Nevertheless, polymerases that are not thermostable can also be used in the provided PCR assays when supplementing the enzyme.

  If the template nucleic acid is double-stranded, the two strands need to be separated before they can be used as templates in PCR. Strand separation can be achieved by any suitable denaturing method, such as physical, chemical or enzymatic means. One method of separating nucleic acid strands involves heating the nucleic acid until it is largely denatured (e.g., more than 50%, 60%, 70%, 80%, 90% or 95% denatured). Including. The heating conditions necessary for denaturation of the template nucleic acid depend, for example, on the buffer salt concentration and the length and nucleotide composition of the nucleic acid to be denatured, but typically for a time dependent on the characteristics of the reaction such as temperature and nucleic acid length, about 90 C. to about 105.degree. Denaturation is typically performed for about 5 seconds to 9 minutes. It is preferred to use a short denaturation step in order to expose the respective polymerases, for example Z05 DNA polymerase, to considerable elevated temperatures for prolonged periods without losing functional enzymes.

  In a preferred embodiment of the invention, the denaturation step is for up to 30 seconds, more preferably up to 20 seconds, more preferably up to 10 seconds, more preferably up to 5 seconds, most preferably about 5 seconds.

  If the double-stranded template nucleic acid is denatured by heating, the reaction mixture is cooled to a temperature that facilitates annealing of each primer with its target sequence on the target nucleic acid.

  The temperature for annealing is preferably from about 35C to about 70C, more preferably from about 45C to about 65C, more preferably from about 50C to about 60C, and more preferably from about 55C to about 58C. is there. Annealing times can be from about 10 seconds to about 1 minute (eg, from about 20 seconds to about 50 seconds, from about 30 seconds to about 40 seconds). In this context, it may be advantageous to use different annealing temperatures to increase the comprehensiveness of each assay. Briefly, this means that at relatively low annealing temperatures, variants of a particular sequence can also be amplified, since primers can also bind to targets with one mismatch. This may be desirable, for example, if a particular organism has a known or unknown genetic variant that should also be detected. On the other hand, relatively high annealing temperatures have the advantage of providing higher specificity, since they continuously reduce the likelihood that the primer will bind to a target sequence that does not exactly match the higher temperature. To reap the benefits of both phenomena, in some embodiments of the present invention, it is preferred that the method described above comprises annealing at different temperatures, preferably first at lower temperatures, and then at higher temperatures. . For example, if the first incubation is performed at 55 ° C. for about 5 cycles, an inexactly matching target sequence may be amplified (prior). This may be followed, for example, by about 45 cycles at 58 ° C., resulting in higher specificity throughout the main part of the experiment. In this way, the specificity remains relatively high without losing potentially important genetic variants.

  The reaction mixture is then adjusted to a temperature at which the activity of the polymerase is promoted or optimized, i.e., sufficient to cause extension from the annealed primer and produce a product complementary to the nucleic acid being analyzed. The temperature should be sufficient to synthesize an extension product from each primer annealed to the nucleic acid template, but not so high as to denature the extension product from the complementary template (e.g., Temperature generally ranges from about 40 ° C. to 80 ° C. (eg, about 50 ° C. to about 70 ° C .; about 60 ° C.) The extension time ranges from about 10 seconds to about 5 minutes, preferably from about 15 seconds to about 2 minutes, more preferably about 20 seconds to about 1 minute, more preferably about 25 seconds to about 35 seconds.The newly synthesized strand is a double-stranded molecule that can be used in the next step of the reaction. The steps of strand separation, annealing and extension can be repeated as often as necessary to produce the desired amount of amplification product corresponding to the target nucleic acid. The amount of primer, thermostable enzyme and nucleoside triphosphate cycle. The steps (ie, denaturation, annealing and extension) are preferably repeated at least once.For use in detection, the number of cycling steps depends, for example, on the nature of the sample.If the sample is a complex mixture of nucleic acids, More cycling steps are required to amplify a target sequence sufficient for detection, typically the cycling steps are repeated at least about 20 times, but 40, 60 or even 100 times. Is also good.

  Within the scope of the present invention, the PCR may be performed in the same step where the annealing and extension steps are the same (one-step PCR), or may be performed in separate steps as described above (two-step PCR). Performing annealing and extension together, under the same physicochemical conditions, using a suitable enzyme such as, for example, Z05 DNA polymerase, saves time for further steps in each cycle and further anneals and extends. This has the advantage of eliminating the need for further temperature adjustment during the process. Thus, one-step PCR reduces the overall complexity of each assay.

  In general, a shorter time is preferred for the entire amplification, as the time to result is reduced and allows for a faster diagnosis.

  Other preferred nucleic acid amplification methods used in the context of the present invention are ligase chain reaction (LCR; Wu DY and Wallace RB, Genomics 4 (1989) 560-69; and Barany F., Proc. Natl. Acad. Sci. USA 88 (1991) 189-193); Polymerase ligase chain reaction (Barany F., PCR Methods and Applic. 1 (1991) 5-16); Gap-LCR (WO 90/01069); Repair chain reaction (EP 0439182 A2) Natl.Acad.Sci. USA 86 (1989) 1173-1177; Guatelli JC, et al., Proc.Natl.Acad.Sci. USA 87 (1990) 1874-1878 WO 92/08808) and NASBA (US 5,130,238). In addition, there are strand displacement amplification (SDA), transcription-mediated amplification (TMA), and Qb amplification (for overview see, for example, Whelen AC and Persing DH, Annu. Rev. Microbiol. 50 (1996) 349-373; Abramson RD and Myers TW, see Curr Opin Biotechnol 4 (1993) 41-47).

The internal control nucleic acid used in the present invention is preferably with respect to its sequence as follows:
-A melting temperature of 55C to 90C, more preferably 65C to 85C, more preferably 70C to 80C, and most preferably about 75C.
-Up to 500 bases or base pairs, more preferably 50 to 300 bases or base pairs, more preferably 100 to 200 bases or base pairs, most preferably about 180 bases or base pairs in length
-Demonstrates a GC content of 30% to 70%, more preferably 40% to 60%, most preferably about 50%.

  In the context of the present invention, a “sequence” is the primary structure of a nucleic acid, that is, the specific sequence of the single nucleobase that each nucleic acid is composed of. The term "sequence" does not specify a particular type of nucleic acid, such as RNA or DNA, but should be understood to apply to both and other types of nucleic acids, such as, for example, PNA or others. When the nucleobases correspond to one another, especially in the case of uracil (present in RNA) and thymine (present in DNA), these bases are used between the RNA and DNA sequences as is well known in the relevant art. Can be considered equivalent.

  Clinically relevant nucleic acids are often, for example, DNA viruses, such as hepatitis B virus (HBV), cytomegalovirus (CMV) and others, or bacteria, such as Chlamydia tracomatis (CT), gonococcus (NG). ) And others. In such cases, it may be advantageous to use an internal control nucleic acid consisting of DNA to reflect the properties of the target nucleic acid.

  Therefore, a preferred aspect of the present invention is the above method, wherein said internal control nucleic acid is DNA.

  On the other hand, many nucleic acids relevant to clinical diagnosis are ribonucleic acids such as human immunodeficiency virus (HIV), hepatitis C virus (HCV), West Nile virus (WNV), human papilloma virus (HPV), Japanese encephalitis virus ( JEV), St. Louis encephalitis virus (SLEV) and others such as nucleic acids derived from RNA viruses. The present invention can be easily applied to such nucleic acids. In this case, it may be advantageous to use an internal control nucleic acid consisting of RNA to reflect the properties of the target nucleic acid. When both RNA and DNA are analyzed in the method described above, the internal control nucleic acid preferably mimics the most sensitive target of an assay involving multiple targets, and the RNA target usually needs to be more tightly controlled. Therefore, the internal control nucleic acid is preferably RNA.

  Accordingly, a preferred aspect of the present invention is the above method, wherein said internal control nucleic acid is RNA.

  Internal control nucleic acids made from RNA are preferably provided as armored particles, as RNA is more prone to degradation than DNA due to the effects of, for example, alkaline pH, ribonucleases, and the like. In particular, exterior particles such as exterior RNA are described in, for example, EP910643. Briefly, RNA that can be produced chemically, or preferably heterologously, eg, by a bacterium, eg, E. coli, is at least partially enveloped by a viral coat protein. The latter renders the RNA resistant to external influences, especially ribonucleases. It should be understood that the internal control DNA can also be provided as an exterior particle. Both armored RNA and armored DNA are useful as internal control nucleic acids in the context of the present invention. In a preferred embodiment, the RNA control nucleic acid is sheathed in E. coli by an MS2 coating protein. In a further preferred embodiment, the DNA control nucleic acid is packaged using phage lambda GT11.

  Therefore, a preferred aspect of the present invention is the above method, wherein said internal control nucleic acid is an exterior nucleic acid.

  Typically, in amplification system nucleic acid diagnostics, an RNA template is transcribed into DNA before amplification and detection.

  Therefore, a preferred aspect of the present invention, wherein the amplification reagent comprises a polymerase having a reverse transcriptase activity, wherein the method comprises, between step e and step f, in the reaction vessel, the purified nucleic acid, The above method, further comprising the step of incubating with said one or more amplification reagents for a time and under conditions suitable for transcription of RNA by said polymerase having reverse transcriptase activity.

  "Polymerase having reverse transcriptase activity" is a nucleic acid polymerase capable of synthesizing DNA based on an RNA template. This may also form double-stranded DNA once the RNA has been reverse transcribed into single-stranded cDN. In a preferred embodiment of the invention, the polymerase having reverse transcriptase activity is thermostable.

  In a preferred embodiment, the method according to the present invention comprises the step of combining a sample comprising an RNA template with an oligonucleotide primer sufficiently complementary to hybridize to said RNA template and preferably a thermostable DNA polymerase, at least a total of at least four. Incubating in the presence of a natural or modified deoxyribonucleoside triphosphate in a suitable buffer, including a metal ion buffer that buffers both pH and metal ion concentration in a preferred embodiment. This incubation allows the primer to hybridize to the RNA template and catalyze the DNA polymerase to polymerize the deoxyribonucleoside triphosphate to form a cDNA sequence complementary to the sequence of the RNA template. It is performed at a sufficient temperature.

  As used herein, the term “cDNA” refers to a complementary DNA molecule synthesized using a ribonucleic acid strand (RNA) as a template. The RNA can be another form of RNA, such as, for example, mRNA, tRNA, rRNA, or viral RNA. A cDNA can be single-stranded, double-stranded, or hydrogen-bonded to a complementary RNA molecule when in an RNA / cDNA hybrid.

  Primers suitable for annealing to an RNA template may also be suitable for amplification by PCR. For PCR, a second primer complementary to the reverse transcribed cDNA strand provides a starting site for synthesis of the extension product.

  In the amplification of an RNA molecule by a DNA polymerase, the first elongation reaction is reverse transcription using an RNA template, and a DNA chain is generated. A second extension reaction using a DNA template produces a double-stranded DNA molecule. Thus, the synthesis of a complementary DNA strand from an RNA template by a DNA polymerase provides a starting material for amplification.

  A thermostable DNA polymerase can be used in a combined one-enzyme reverse transcription / amplification reaction. The term "homogenous" in this context refers to a two-step single addition reaction for reverse transcription and amplification of an RNA target. Homologous means that after the reverse transcription (RT) step, there is no need to open the reaction vessel or otherwise adjust the reaction components before the amplification step. In a heterogeneous RT / PCR reaction, after reverse transcription and before amplification, one or more of the reaction components, such as amplification reagents, are adjusted, added or diluted, for example, so that the reaction vessel needs to be opened or at least The components need to be manipulated. While both homogeneous and non-homogeneous embodiments are included within the scope of the present invention, homogeneous formats are preferred for RT / PCR.

  Reverse transcription is an important step in RT / PCR. For example, it is known in the art that RNA templates exhibit a tendency to form secondary structures that can impair primer binding and / or elongation of the cDNA strand by the respective reverse transcriptase. Thus, for the efficiency of transcription, relatively high temperatures are advantageous for RT reactions. On the other hand, increasing the incubation temperature also means higher specificity, ie, the RT primer does not anneal to sequences that show a mismatch to the expected sequence or sequences. In particular, in the case of a large number of different target RNAs, for example, where there may be a substrain or subspecies of an unknown or rare organism in a liquid sample, the sequence with a single mismatch is transcribed and then amplified. , May also be desirable to detect.

  It is preferred to carry out the RT incubation at more than one different temperature in order to obtain both the advantages mentioned above, namely the reduction of secondary structure and the benefit of reverse transcription of mismatched templates.

  Therefore, a preferred aspect of the present invention is the above-mentioned, wherein the incubation of the polymerase having reverse transcriptase activity is performed at a different temperature of 30 ° C to 75 ° C, preferably 45 ° C to 70 ° C, more preferably 55 ° C to 65 ° C. Is the way.

  As a further important aspect of reverse transcription, a long RT step can damage DNA templates that may be present in a liquid sample. Thus, if the liquid sample contains both RNA and DNA species, the duration of the RT step is kept as short as possible, while at the same time sufficient amounts of cDNA for subsequent amplification and optional detection of the amplificate It is preferred to ensure the synthesis of

  Accordingly, a preferred aspect of the present invention is the above method, wherein the incubation time of the polymerase having reverse transcriptase activity is up to 30 minutes, 20 minutes, 15 minutes, 12.5 minutes, 10 minutes, 5 minutes or 1 minute.

In a further preferred aspect of the present invention, a polymerase having a reverse transcriptase activity and containing a mutation,
a) CS5 DNA polymerase
b) CS6 DNA polymerase
c) Thermotoga maritima DNA polymerase
d) Thermus aquaticus DNA polymerase
e) Thermus thermophilus DNA polymerase
f) Thermus flavus DNA polymerase
g) Thermus filiformis DNA polymerase
h) Thermus species (sp.) sps17 DNA polymerase
i) Thermus species (sp.) Z05 DNA polymerase
j) Thermotoganea polytana DNA polymerase
k) Thermosiphon africanus DNA polymerase
l) The method as described above selected from the group consisting of Thermus caldophilus DNA polymerase.

  Enzymes having mutations in the polymerase domain that increase reverse transcription efficiency for faster extension rates are particularly suitable for these requirements.

  Therefore, a preferred aspect of the present invention is a polymerase wherein the polymerase having reverse transcriptase activity comprises a mutation that imparts an improved rate of nucleic acid elongation and / or improved reverse transcriptase activity compared to the respective wild-type polymerase. This is the method described above.

  In a more preferred embodiment, in the above method, the polymerase having reverse transcriptase activity is a polymerase containing a mutation that confers improved reverse transcriptase activity compared to the respective wild-type polymerase.

Polymerases with point mutations that make the polymerase particularly useful in the context of the present invention are disclosed in WO 2008/046612. In particular, preferred polymerases used in the context of the present invention have at least the following motifs in the polymerase domain:
A mutant DNA polymerase comprising TGRLSSX _b7 -X _b8 -PNLQN, wherein X _b7 is an amino acid selected from S or T, and X _b8 is selected from G, T, R, K or L An amino acid, wherein the polymerase comprises 3'-5 'exonuclease activity, has an improved nucleic acid extension rate and / or an improved reverse transcription efficiency compared to a wild-type DNA polymerase, In wild-type DNA polymerase, _Xb8 is an amino acid selected from D, E or N.

  One particularly preferred example is a mutant of a thermostable DNA polymerase from Thermus sp.Z05 (e.g. as described in US 5,455,170), wherein said variant has a mutation in the polymerase domain compared to the respective wild-type enzyme Z05. including. A mutant Z05 DNA polymerase in which the amino acid at position 580 is selected from the group consisting of G, T, R, K and L is particularly preferred for the method of the present invention.

  For reverse transcription using a thermostable polymerase, Mn is preferred as a divalent cation, typically as a salt, such as manganese chloride (MnCl), manganese acetate (Mn (OAc)) or manganese sulfate (MnSO). included. MnCl is included in reactions containing 50 mM Tricine buffer, e.g., MnCl is generally present at a concentration of 0.5-7.0 mM, 0.8-1.4 when 200 mM dGTP, dATP, dUTP and dCTP are used, respectively. mM is preferred, and 2.5-3.5 mM MnCl2 is most preferred. Furthermore, the use of Mg2 + as a divalent cation for reverse transcription is also preferred in the context of the present invention.

  Since it is within the scope of the present invention that the RNA target nucleic acid can be reverse transcribed into cDNA while preserving the DNA target nucleic acid, and both cDNA and DNA can be used for subsequent amplification, It is particularly useful for the simultaneous amplification of target nucleic acids from both organisms having an RNA genome or an organism having a DNA genome. This advantage significantly increases the spectrum of different organisms, especially pathogens, that can be analyzed under the same physical conditions.

  Therefore, a preferred aspect of the present invention is a method as described above, wherein the at least two target nucleic acids comprise RNA and DNA.

  As used herein, "organism" means any living single or multicellular form of life. In the context of the present invention, a virus is an organism.

  Due to particularly suitable temperature optima, enzymes such as Tth polymerase or preferably the mutated Z05 DNA polymerase described above are suitable for performing the steps of subsequent amplification of the target nucleic acid. Utilizing the same enzyme for both reverse transcription and amplification because the liquid sample does not need to be manipulated between the RT and amplification steps contributes to the simplicity of the method and facilitates its automation .

  Therefore, in a preferred embodiment, the same polymerase having reverse transcriptase activity is used for reverse transcription and amplification in step f in the manner described above. Preferably, the enzyme is a mutated Z05 DNA polymerase as described above.

  In a preferred embodiment, the step above 90 ° C. is up to 20 seconds, preferably up to 15 seconds, more preferably up to 20 seconds, in order not to expose the polymerase or other components of the reaction mixture used in the context of the invention to elevated temperatures for longer than necessary. Is up to 10 seconds, more preferably up to 5 seconds, and most preferably 5 seconds long. This also reduces the time to results and reduces the overall time required for the assay.

  In such a homogeneous setting, it may be quite advantageous to seal the reaction vessel before the start of RT and amplification, thereby reducing the risk of contamination. Sealing can be achieved, for example, by placing a preferably transparent foil, cap, or by adding oil to the reaction vessel to form a lipophilic phase as a sealing layer on top of the liquid.

  Accordingly, a preferred aspect of the present invention is the above method, further comprising the step of sealing at least two reaction vessels after step e.

  For ease of handling and ease of automation, it is preferred that at least two reaction vessels be brought together in an integrated row so that they can be operated together.

  Consequently, a preferred aspect of the present invention is the above-described method of matching at least two reaction vessels to the same integrated array.

  The integrated array can be, for example, vials or tubes reversibly or irreversibly attached to each other or arranged in a rack. Preferably, the integrated array is a multi-well plate.

  The target of the amplification step can be an RNA / DNA hybrid molecule. The target can be a single-stranded or double-stranded nucleic acid. The most widely used PCR techniques use double-stranded targets, but this is not essential. After the first amplification cycle of the single-stranded DNA target, the reaction mixture contains double-stranded DNA molecules consisting of the single-stranded target and the newly synthesized complementary strand. Similarly, after the first amplification cycle of the RNA / cDNA target, the reaction mixture contains double-stranded cDNA molecules. At this point, the successive cycles of amplification proceed as described above.

  Nucleic acid amplification is particularly effective not only in the case of PCR but also when carried out as a cycle reaction.Therefore, a preferred aspect of the present invention is the method described above, wherein the amplification reaction in step f comprises multiple cycle steps. is there.

  Suitable nucleic acid detection methods are known to those of skill in the art and are described in Sambrook J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989 and Ausubel F. et al .: Current Protocols. It is described in standard textbooks such as in Molecular Biology 1987, J. Wiley and Sons, NY. Before performing the nucleic acid detection step, there may be further purification steps such as, for example, a precipitation step. Detection methods include, but are not limited to binding or intercalating a specific dye, such as ethidium bromide, that intercalates into double-stranded DNA and then changes its fluorescence. Purified nucleic acids can also optionally be separated by electrophoresis after restriction digestion and subsequently visualized. Some probe system assays utilize oligonucleotide hybridization to a specific sequence and subsequent detection of the hybrid.

  In order to evaluate the results of the analysis, it is preferable to detect the amplified target nucleic acid during or after the amplification reaction. It is advantageous to use nucleic acid probes, especially for real-time detection.

  Accordingly, a preferred aspect of the present invention is the method as described above, wherein the cycling step comprises an amplification step and a hybridization step, wherein the hybridization step comprises hybridizing the amplified nucleic acid with the probe.

  It may be preferable to monitor the amplification reaction in real time, ie, to detect the target nucleic acid and / or amplification product during the amplification itself.

  Therefore, a preferred aspect of the invention is the above-described method of labeling a probe with a donor fluorescent moiety and a corresponding acceptor fluorescent moiety.

The method described above is preferably based on fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor fluorescent moiety. An exemplary donor fluorescent moiety is fluorescein, and exemplary corresponding acceptor fluorescent moieties include LC-Red 640, LC-Red 705, Cy5 and Cy5.5. Typically, detection involves excitation of the sample at a wavelength that is absorbed by the donor fluorescent moiety, and visualization and / or measurement of the wavelength emitted by the corresponding acceptor fluorescent moiety. In the method of the invention, detection is preferably followed by quantification of FRET. Preferably, the detection is performed after each cycle step. Most preferably, the detection is performed in real time. Commercial real-time PCR instrument (e.g., LightCycler ^TM or the TaqMan (R)) By using the detection of the PCR amplification and the amplified product, and dramatically reduce the cycle time, combining in a single closed cuvette be able to. Because detection and amplification occur simultaneously, real-time PCR avoids the need for manipulation of amplification products and reduces the risk of cross-contamination between amplification products. Real-time PCR greatly reduces turnaround time and makes it an attractive alternative to traditional PCR technology in clinical laboratories.

The following patent applications describe real-time PCR used in LightCycler ^™ technology: WO 97/46707, WO 97/46714 and WO 97/46712. The LightCycler ^™ instrument is a rapid thermal cycler combined with a microvolume fluorimeter using high quality optics. In this rapid thermal cycling technique, a thin glass cuvette is used as the reaction vessel. Heating and cooling of the reaction chamber is controlled by exchanging heated air with ambient air. Due to the low mass of air and the high surface area to volume ratio of the cuvette, very fast temperature exchange rates can be achieved in the thermal chamber.

  TaqMan® technology uses a single-stranded hybridization probe labeled with two fluorescent moieties. When the first fluorescent moiety is excited with light of the appropriate wavelength, the absorbed energy is transferred to the second fluorescent moiety according to FRET principles. The second fluorescent moiety is generally a quench molecule. Typical fluorescent dyes used in this format are, for example, FAM, HEX, CY5, JA270, Cyan and CY5.5, among others. During the annealing step of the PCR reaction, the labeled hybridization probe binds to the target nucleic acid (i.e., the amplification product) and during the subsequent extension phase another known to those of skill in the art, such as Taq or a preferred mutant Z05 polymerase. Degraded by the 5'-3 'exonuclease activity of the appropriate polymerase. As a result, the excited fluorescent moiety and the quenched moiety are spatially separated from each other. As a result, upon excitation of the first fluorescent moiety in the absence of the quencher, fluorescence emission from the first fluorescent moiety can be detected.

  In both detection formats described above, the intensity of the luminescent signal can be correlated with the number of original target nucleic acid molecules.

  As an alternative to FRET, the amplification product can be detected using a double-stranded DNA binding dye such as a fluorescent DNA binding dye (eg, SYBRGREEN I® or SYBRGOLD® (Molecular Probes)). Upon interaction with a double-stranded nucleic acid, such fluorescent DNA-binding dyes emit a fluorescent signal after excitation with light of an appropriate wavelength. Double-stranded DNA binding dyes such as nucleic acid intercalating dyes can also be used. When a double-stranded DNA binding dye is used, a melting curve analysis is usually performed to confirm the presence of the amplification product.

  For detection of the presence of an amplification product using the real-time PCR method of the present invention, a molecular beacon coupled to FRET may also be used. Molecular beacon technology uses a hybridization probe labeled with a first fluorescent moiety and a second fluorescent moiety. The second fluorescent moiety is generally a quencher, and a fluorescent label is typically located at each end of the probe. Molecular beacon technology uses probe oligonucleotides with sequences that allow secondary structure formation (eg, hairpins). As a result of the formation of secondary structures within the probe, both fluorescent moieties become spatially proximal when the probe is in solution. After hybridization to the amplification product, the secondary structure of the probe is broken and the fluorescent moieties are separated from one another such that emission of the first fluorescent moiety can be detected after excitation with light of the appropriate wavelength.

  Thus, a preferred method of the invention is that said probe comprises a nucleic acid sequence allowing secondary structure formation, said secondary structure formation resulting in a spatial proximity between said first and second fluorescent moieties. , FRET described above.

  Efficient FRET can only occur when the fluorescent moiety is in direct proximity and when the emission spectrum of the donor fluorescent moiety overlaps the absorption spectrum of the acceptor fluorescent moiety.

  Thus, in a preferred embodiment, the donor and acceptor fluorescent moieties are within 5 nucleotides of each other on the probe.

  In a further preferred embodiment, said acceptor fluorescent moiety is a quencher.

  As described above, in the TaqMan format, during the annealing step of the PCR reaction, the labeled hybridization probe binds to the target nucleic acid (i.e., the amplification product), and in the subsequent extension phase, such as Taq or a preferred mutant Z05 polymerase. Degraded by the 5'-3 'exonuclease activity of another suitable polymerase known to the art.

  Thus, in a preferred embodiment, in the above method, the amplification uses a polymerase enzyme having 5'-3 'exonuclease activity.

  It is further advantageous to carefully select the length of the amplicon resulting from the above method. Generally, relatively short amplicons increase the efficiency of the amplification reaction. Accordingly, a preferred aspect of the present invention is the above method, wherein the amplified fragment comprises up to 450 bases, preferably up to 300 bases, more preferably up to 200 bases, more preferably up to 150 bases.

  The internal control nucleic acid used in the present invention can serve as a reference for quantifying, ie, determining the amount of a target nucleic acid, and can serve as a "quantitative standard nucleic acid" used. For this purpose, one or more quantitative standard nucleic acids, together with the target nucleic acid, are subjected to all possible sample preparation steps. In addition, the quantitative standard nucleic acid is processed throughout the method in the same reaction mixture. This must result, directly or indirectly, in a signal that is detectable both in the presence and absence of the target nucleic acid. For this purpose, the concentration of the quantitative standard nucleic acid needs to be carefully optimized in each test, e.g., in order not to compromise the sensitivity, but to produce a detectable signal even at very high target concentrations . For the detection limit (LOD, see below) of each assay, the concentration range of the "quantitative standard nucleic acid" is preferably 20-5000x LOD, more preferably 20-1000x LOD, most preferably 20-5000x LOD. The final concentration of the quantitative standard nucleic acid in the reaction mixture depends on the quantitative measurement range achieved.

  "Limit of detection" or "LOD" means the lowest detectable amount or concentration of nucleic acid in a sample. A low "LOD" corresponds to a high sensitivity, and a high "LOD" corresponds to a low sensitivity. Usually, "LOD" is expressed in either "cp / ml" or IU / ml, especially if the nucleic acid is a viral nucleic acid. “Cp / ml” means “copy per milliliter”, and “copy” is the number (copy) of each nucleic acid. IU / ml stands for “international unit / ml” and refers to the WHO standard.

  A widely used method for calculating LOD is "probit analysis", which is a method for analyzing the relationship between stimulation (dose) and discontinuous (total or none) response. In a typical discontinuous response experiment, groups of animals are given different doses of the drug. Mortality at each dose level is recorded. These data can then be analyzed using probit analysis. The probit model assumes that the response percentage is related to the log dose as the cumulative normal distribution. That is, the log dose can be used as a variable to read mortality from a cumulative standard. Using the standard distribution, rather than the other probability distributions, affects the estimated response rates at the high and low end of the possible dose, but has little effect near the center.

  Probit analysis can be applied with a separate "hitrate". As known in the art, "hit ratio" is generally expressed as a percentage [%] and indicates the percentage of positive results at a particular concentration of an analyte. Thus, for example, if the LOD can be determined with a 95% hit rate, it means that the LOD is calculated for a setting where a 95% positive result is positive.

  In a preferred embodiment, the above method comprises 1-100 cp / ml or 0.5-50 IU / ml, more preferably 1-75 cp / ml or 0.5-30 IU / ml, more preferably 1-25 cp / ml or 1-20 IU / ml Provide LOD.

For some examples of possible target nucleic acids from a particular virus, the above method preferably provides the following LOD:
HIV: up to 60 cp / ml, more preferably up to 50 cp / ml, more preferably up to 40 cp / ml, more preferably up to 30 cp / ml, more preferably up to 20 cp / ml, more preferably up to 15 cp / ml. Up to 10 IU / ml, more preferably up to 7.5 IU / ml, more preferably up to 5 IU / mlHCV: up to 10 IU / ml, more preferably up to 7.5 IU / ml, more preferably up to 5 IU / mlWNVI: 20 cp / ml WNVII: up to 20 cp / ml, more preferably up to 15 cp / ml, more preferably up to 10 cp / ml, more preferably up to 5 cp / ml. JEV: up to 100 cp / ml, more preferably up to 75 cp / ml, more preferably up to 50 cp / ml, more preferably up to 30 cp / mlSLEV: up to 100 cp / ml, more preferably up to 75 cp / ml, more preferably 50 cp / ml / ml, more preferably up to 25 cp / ml, more preferably up to 10 cp / ml.

An example of how to calculate a quantitative result in TaqMan format based on an internal control nucleic acid that serves as a quantitative standard nucleic acid is described below: from input data of instrument corrected fluorescence values from all PCR runs Calculate the titer. A set of samples containing a target nucleic acid and an internal control nucleic acid that functions as a quantitative standard nucleic acid is subjected to PCR in a thermal cycler using a specific temperature profile. Illuminate the sample with filtered light at the selected temperature and time in the PCR profile and collect filtered fluorescence data for the target nucleic acid and internal control nucleic acid for each sample. After the completion of the PCR run, the fluorescence readings are processed to generate a set of dye concentration data for the internal control nucleic acid and a set of dye concentration data for the target nucleic acid. Each set of dye density data is processed in the same manner. After several plausibility checks, an elbow value (CT) is calculated for the internal control nucleic acid and the target nucleic acid. The point at which the fluorescence of the target nucleic acid or internal control nucleic acid crosses a predetermined threshold (fluorescence concentration) is defined as the elbow value. Determination of the titer is based on the fact that the target nucleic acid and the internal control nucleic acid are amplified with the same efficiency and that, at the calculated elbow value, equal amounts of amplicon copies of the target nucleic acid and the internal control nucleic acid are amplified and detected. Based on assumptions. So (CTQS-CT target) is linear to log (target concentration / QS concentration). In this context, QS indicates an internal control nucleic acid that serves as a quantitative standard nucleic acid. The titer T is then calculated, for example, by the following equation:
T '= 10 (a (CTQS-CT target) 2 + b (CTQS-CT target) + c)
Can be calculated using a polynomial calibration equation such as

  Since the polynomial constant and the concentration of the quantitative standard nucleic acid are known, only the variables in the equation differ (CTQS-CT target).

  Furthermore, in the sense of the present invention, an internal control nucleic acid can function as a "qualitative internal control nucleic acid". “Qualitative internal control nucleic acids” are particularly useful for validating test results of qualitative detection assays. Even in the case of a negative result, a qualitative internal control must be detected, otherwise the test itself is deemed not working. However, in a qualitative setting, a qualitative internal control need not necessarily be detected in case of a positive result. As a result, its concentration must be relatively low. Qualitative internal controls need to be carefully adapted for each assay and sensitivity. Preferably, the concentration range of the qualitative internal nucleic acid, ie the second control nucleic acid, comprises the range from 1 copy per reaction to 1000 copies per reaction. With respect to the limit of detection (LOD) of each assay, the concentration is preferably between the LOD of the assay and 25 times the LOD, more preferably between LOD and 10x LOD. More preferably, this is between 2x to 10x LOD. More preferably, it is between 5x and 10x LOD. Most preferably, this is a 5x or 10x LOD.

  As used in the present invention, the internal control nucleic acid is not limited to a particular sequence. It may be advantageous to add different internal control nucleic acids to the liquid sample, but it is advantageous to use only one of them for amplification, for example by adding only primers for one of said internal control nucleic acids. It can be advantageous. In such embodiments, the internal control nucleic acid that is amplified in a particular experiment can be selected by one of skill in the art, thus increasing the flexibility of the analysis performed. In a particularly advantageous embodiment, said different internal control nucleic acids may be comprised by a single nucleic acid construct, such as a plasmid or a different suitable nucleic acid molecule.

  Thus, a preferred aspect of the present invention is the above method, wherein more than one internal control nucleic acid is added in step a, but only one of said internal control nucleic acids is amplified in step f.

  The above results may be degraded and may include false positives, for example, in the case of cross-contamination with nucleic acids from sources other than liquid samples. In particular, the amplification of the previous experiment contributes to such undesirable effects. Certain methods for minimizing the effects of cross-contamination of nucleic acid amplification are described in US Pat. No. 5,035,996. The method involves the introduction of unusual nucleotide bases, such as dUTP, into the amplification product, and the enzymatic treatment and / or physicochemical treatment of the carryover product to prevent the product DNA from functioning as a template for subsequent amplification. Including exposure to treatment. Enzymes for such treatment are known in the art. For example, uracil-DNA glycosylase, also known as uracil-N-glycosylase or UNG, removes uracil residues from PCR products containing uracil. Enzymatic treatment results in the degradation of the carry-over PCR product and serves to "sterilize" the amplification reaction.

Therefore, a preferred aspect of the present invention is that between step d and step e,
Treating the liquid sample with an enzyme under conditions in which products from amplification of cross-contaminated nucleic acids from other samples are enzymatically degraded;
-The above method, further comprising the step of inactivating the enzyme.

  Preferably, the enzyme is uracil-N-glycosylase.

  In the method of the present invention, it is preferable that all the steps are automated. "Automated" means that the method steps are suitable to be performed by a device or machine that can operate with little or no external control or human influence. Only the preparation steps of the method may need to be performed manually, e.g., the storage container needs to be filled and placed in a predetermined location, the selection of the sample needs to be made by a human, known to those skilled in the art. Further steps, such as operation of a control computer, need to be performed by a human. The device or machine can add liquids, mix samples, or perform the incubation step at a particular temperature, for example, automatically. Typically, such machines or devices are robotically controlled by a computer executing a program in which one set of steps and one set of commands are specified.

A further aspect of the invention is an analysis system (440) for isolating and simultaneously amplifying at least two target nucleic acids that may be present in a liquid sample, said analysis system comprising the following modules:
A separation station (230) comprising a solid support material, said separation station being constructed and arranged to separate and purify a target nucleic acid contained in a liquid sample;
An amplification station (405) comprising at least two reaction vessels, said reaction vessels comprising an amplification reagent, at least a first purified target nucleic acid in at least a first reaction vessel and at least a second in a second reaction vessel. Two purified target nucleic acids, an internal control nucleic acid, and a polymerase having reverse transcriptase activity, wherein the second target nucleic acid is absent in the first reaction vessel; Further comprising a mutation that imparts an improved nucleic acid elongation rate and / or improved reverse transcriptase activity compared to a wild-type polymerase,
including.

  An "analysis system" is an array of components, such as devices, that interact with each other for the ultimate purpose of analyzing a given sample.

  The advantages of the analysis system are the same as those described above for the method of the invention.

  The analysis system (440, FIG. 11) of the present invention is a system (440) including a module (401) for isolating and / or purifying an analyte. Further, the system (440) further includes a module (403) for analyzing the analyte to obtain a detectable signal. The detectable signal can be detected in the same module (401, 402, 403), or alternatively can be detected in separate modules. The term “module” as used herein relates to any spatially defined location in the analyzer (400). The two modules (401, 403) can be separated by a wall or in an open relationship. Any one module (401, 402, 403) may be controlled autonomously, or control of the module (401, 402, 403) may be shared with other modules. Preferably, all modules are controlled centrally. Transfer between modules (401, 402, 403) can be manual, but is preferably automated. Accordingly, many different embodiments of the automated analyzer (400) are encompassed by the present invention.

  "Separation stations" are described above.

  An "amplification station" includes a temperature controlled incubator for incubating the contents of at least two reaction vessels. It further includes various reaction vessels such as tubes or plates in which reactions for analysis of a sample, such as PCR, take place. The outer border or wall of such a container is chemically inert so as not to interfere with the amplification reaction taking place therein. For ease of handling and ease of automation, it is preferred that at least two reaction vessels can be combined in an integrated array and operated together.

  Consequently, a preferred aspect of the present invention is an analytical system as described above, wherein at least two reaction vessels are combined in an integrated array.

  The integrated array can be, for example, vials or tubes attached reversibly or irreversibly to one another or arranged in a rack. Preferably, the integrated array is a multi-well plate.

  Preferably, the multi-well plate is held in a holding station. In a more preferred embodiment, one maneuver transfers a multi-well vessel from a holding station to an airlock (460), and a second maneuver transfers the multiwell plate from the airlock to the amplification station; Both pilots interact with the multi-well plate by form-locking interactions.

  In a preferred embodiment, the analysis system is fully automated.

  In one embodiment, at least two reaction vessels combined in an integrated array are transferred between stations of the system.

  In a second embodiment, a purified target nucleic acid is transferred from the separation station to the amplification station. Preferably, the liquid containing the purified nucleic acid is transferred by a pipettor comprising a pipette with a pipette tip attached.

  In a third embodiment, the purified nucleic acids are transferred from the separation station to a reaction vessel in an integrated sequence held in a holding station. Preferably, the reaction vessels in an integrated array are then transferred from the holding station to the amplification station.

  The analysis system of the present invention preferably further includes a pipetting unit. The pipetting unit comprises at least one pipette, preferably a number of pipettes. In a preferred embodiment, the multiple pipettes are combined in one or more integrated arrays, in which the pipettes can preferably be operated individually. The pipette used in the context of the present invention is preferably a pipette comprising a pipette tip as described above. In another preferred embodiment, the pipette is a pipetting needle.

  Alternatively, a reaction vessel or an array of reaction vessels used for sample preparation in a separation station and containing a liquid containing purified target nucleic acid can be transferred from the separation station to an amplification station.

  To this end, the analysis system of the invention preferably further comprises a transfer unit, said transfer unit preferably comprising a robotic device, said device preferably comprising a pilot.

For the reasons mentioned above in the context of the method of the invention, the following are further preferred aspects of the invention:
-The analysis system (440) above, wherein at least one reaction vessel comprises an RNA target nucleic acid and a DNA target nucleic acid.
• The analysis system (440) above, wherein at least one reaction vessel contains an RNA target nucleic acid and at least one other reaction vessel contains a DNA target nucleic acid.

Preferably, the above analysis system (440) comprises:
A detection module (403) for detecting a signal induced by the analyte
・ Sealer (410)
Storage modules for reagents and / or disposables (1008)
-It further comprises one or more elements selected from the group consisting of control units for controlling the components of the system.

  The “detection module” (403) may be, for example, an optical detection unit for detecting the result or effect of the amplification procedure. The optical detection unit includes a light source, e.g., a xenon lamp, a mirror, a lens, an optical filter, an optical fiber for guiding and filtering light, one or more reference channels, or optics such as a CCD camera or a different camera. May be included.

  The "sealer" (410) is constructed and arranged to seal any vessel used in connection with the analysis system of the present invention. Such a sealer may, for example, seal the tube with a suitable cap or seal the multi-well plate with foil or other suitable sealing material.

  The “storage module” (1008) stores reagents necessary to cause a chemical or biological reaction important for the analysis of a liquid sample. This may also include additional components useful in the methods of the invention, such as disposables such as pipette tips or vessels used as reaction vessels in separation and / or amplification stations.

  Preferably, the analysis system of the present invention further includes a control unit for controlling the system components.

  Such a `` control unit '' (1006) ensures that the different components of the analysis system operate and interact with precise and precise timing, e.g., moving and manipulating components such as pipettes in a coordinated manner. Software may be included. The control unit may also include a processor running a real-time operating system (RTOS), which is a multitasking operating system intended for real-time applications. In other words, the system processor may manage real-time constraints, ie, deadlines from events to system responses regardless of system load. This controls in real time that different units in the system will operate and respond accurately according to predetermined instructions.

In a preferred embodiment, the present invention provides
a.A first receptacle (1001) in a linear array containing a liquid sample (1010), a processing plate (101) containing a receptacle (103) in an nxm array for holding a liquid sample (1011), at least in the linear array. A first pipetting device (700) comprising two pipetting units (702) and a first position comprising a tip rack (70) comprising pipette tips (3, 4) in an ax (nxm) array, said Pipetting unit (702) is connected to pipette tips (3, 4);
b. Holders (201, 128) for the processing plate (101), holders (330) for multi-well plates, holders (470) for the tip rack (70) and a second pipetting device ( A second position comprising a pipetting unit (702) in an nxm array for coupling a pipette tip (3, 4), the second position comprising a pipetting tip (3, 4). An analysis system (440) for processing (FIG. 12). As used herein, the term "holder" relates to any arrangement capable of receiving a rack or processing plate.

  The advantages of the analysis system (440) of the present invention are as described above for the method of the present invention.

  Preferably, the position of said pipetting unit (702) of the first pipetting device (700) is variable. Preferred embodiments of the first pipetting device (700) are described later herein.

  In one embodiment, the tip rack (70) includes pipette tips (3, 4) in an ax (nxm) array. Preferably, the first type (4) and the second type (3) of pipette tips are contained in a tip rack (70). In this embodiment, the first type of pipette tips (4) are arranged in an nxm array and the second type of pipette tips (3) are arranged in an nxm array. In this context, "n" indicates the number of rows, m indicates the number of columns, where n is preferably 6, and m is preferably 8. More preferably, the first type of pipette tip (4) has a different volume than the second type of pipette tip (3), most preferably the volume of the first type of pipette tip (4) Is greater than 500 ul and the volume of the second type of pipette tip (3) is less than 500 ul. In this embodiment, a = 2. However, embodiments of the present invention with more than two types of pipette tips, and thus a> 2, are also included in the present invention.

  In one aspect, the analysis system (440) of the present invention includes a control unit (1006) for specifying sample types and individual tests for individual locations on the processing plate (101). Preferably, said location is a separate cell (401, 402).

  In one aspect of the invention, the system further comprises a transfer system (480) for transferring the processing plate (101) and the rack (70) between first (402) and second (401) positions. )including. A preferred embodiment of the transfer system (480) is a conveyor belt or, more preferably, one or more pilots.

  Furthermore, preferably the pipette unit of the second pipetting device (35) is connected to a pipette tip (3, 4) used in a first position (402).

  A preferred embodiment of the system (440) of the present invention further comprises a third station (403) comprising a temperature controlled incubator for incubating said analyte and the reagents necessary to obtain a detectable signal. In addition. Further preferred aspects of this system are described later herein.

  A more suitable control for assigning samples and tests to the nxm array is a first processor (1004) included in the first location (402) and a second processor included in the second location (401). (1005), wherein the first processor has instructions in the control unit to assign sample types and individual tests to specific locations in the nxm array of vessels (103) of the processing plate (101). Transported by (1006), the second processor is instructed by the control unit (1006) to assign sample types and individual tests to specific locations in the nxm array of containers (103) of processing plates. Transported by

  Preferably, the system further includes a first processor located at the first location and a second processor located at the second location.

  More preferably, said first processor (1004) controls said first pipetting device (700) and said second processor (1005) controls said second pipetting device (35).

  All other preferred embodiments and specific descriptions of embodiments of the analytical system of the present invention have been set forth for the method of the present invention.

Examples The following examples describe embodiments in which the invention may be operated. It is obvious to those skilled in the art that these examples are not limiting and can be modified without departing from the spirit of the invention.

Example 1:
This example describes a method for isolating and co-amplifying at least a first and a second target nucleic acid using a single general internal control nucleic acid.

  Briefly, in the embodiment shown, real-time PCR is performed simultaneously and under the same conditions on a panel of several different targets, including bacteria (Chlamydia trachomatis, CT), and DNA viruses (HBV) and RNA viruses (HIV). Done in All samples were processed and analyzed in each of the same experiments, ie, the same deep well plate (for sample preparation) or multi-well plate (for amplification and detection).

  The following samples were prepared and subsequently analyzed:

  Appropriate standards or other types of targets are available to those skilled in the art.

  The equipment listed in the table below was used according to the respective manufacturer's instructions:

  For sample preparation, the following reagents were used as diluents:

  The following dilutions were previously prepared and stored overnight (-60 to -90 ° C for plasma dilutions, 2 to 8 ° C for PreservCyt dilutions):

  Each respective sample (500 ul) and each respective sample dilution (350 ul) were manually pipetted into a deep well plate and each sample was added to three different wells for triplicate analysis. To each well containing HIV or HBV samples, 50 ul of internal control nucleic acid was added manually. For qualitative HIV assays, RNA was added to serve as a qualitative control (100 armored particles / sample). For the quantitative HIV assay, RNA was added to serve as a quantitation standard (500 armored particles / sample). For quantitative HBV assays, DNA serving as a quantitation standard was added (1E4 copies / sample). The sequence of the control nucleic acid was identical in all cases and was selected from the group of SEQ ID NOs: 45-48.

  Each control nucleic acid was stored in the following buffers:

  Sample preparation was performed on a Hamilton Star (Hamilton, Bonaduz, CH) using the following reagents according to the schematic workflow shown in FIG.

  After the final step, each master mix (Mmx) containing the amplification reagent was added to each well by the process head of the Hamilton Star apparatus, and the liquid containing the isolated nucleic acid was mixed with Mmx to obtain each of the obtained nucleic acids. The mixture was transferred to the corresponding well of the microwell plate where amplification was performed.

  The following master mix (each consisting of two reagents R1 and R2) was used:

  For amplification and detection, the microwell plate was sealed with an automatic plate sealer (see above) and the plate was transferred to a LightCycler 480 (see above).

  The following PCR profile was used:

  The pre-PCR program involves initial denaturation and incubation at 55, 60 and 65 ° C. for reverse transcription of the RNA template. Incubating at three different temperatures will result in the transcription of even slightly mismatched target sequences (such as genetic variants of organisms) at lower temperatures, while at higher temperatures the formation of RNA secondary structures will be suppressed, The advantageous effect that more effective transfer is provided is combined.

  The PCR cycle is split into two measurements, and both measurements have a one-step setting applied (annealing and extension combined). The first 5 cycles at 55 ° C allow for increased inclusivity by pre-amplifying slightly mismatched target sequences, and a 45 ° C second annealing measurement with an annealing / extension temperature of 58 ° C. The use of increases the specificity.

  This profile was used for all samples contained in the microwell plate described above to achieve amplification and detection on all samples as shown in FIG. This indicates that the sample preparation before amplification was also performed successfully.

  The results for the qualitative and quantitative HIV internal control and the quantitative HBV internal control are shown separately in FIG. 2 for clarity. It can be seen that the control was also successfully amplified in all cases. The amount of HIV target and HBV in a quantitative setting was calculated by comparison with an internal control nucleic acid that served as a quantification standard.

Example 2:
The general amplification process described above was performed on a variety of different target nucleic acids under identical conditions, but in separate experiments. Isolation of each nucleic acid was performed as described in Example 1.

  Each common internal control nucleic acid was selected from SEQ ID NOs: 45-49 and was armored RNA for RNA targets and lambda packaged DNA for DNA targets. 300 particles were added per sample for qualitative RNA assays, 3000 for quantitative RNA assays and 500 for total DNA assays.

  The following PCR profiles were used for all targets:

In detail, the following experiments were performed:
1. Qualitative multiplex analysis of HBV, HCV and HIV
a. Master mix

Analytical sensitivity / LOD
For each detected virus (HIV-1 group M, HIV-1 group O, HIV-2, HBV and HCV), several concentrations / levels at and around the expected LOD for EDTA plasma. One virus and one panel per concentration were tested in at least 20 valid replicates per concentration. LOD was determined by probit analysis (see Tables 1-5).

HIV

WHO standard titers for HIV-1 group M were converted to IU / mL.

Therefore, IU / mL HIV-1 group M LOD is:
LOD (95% hit rate) by probit analysis: 6.77 IU / mL
95% confidence interval for LOD by probit analysis: 4.75 to 15.4 IU / mL

  The titer of the primary standard for HIV-1 Group O was rearranged against the CBER HIV-1 Group O panel; the calculation factor is 0.586.

Therefore, HIV-1 group O LOD
LOD (95% hit rate) by probit analysis: 8.8cp / mL
95% confidence interval for LOD by probit analysis: 6.4 to 18.5 cp / mL

  The primary standard titer for HIV-2 was rearranged against the CBER HIV-2 panel; the calculation factor is 26.7.

Therefore, HIV-2 LOD is
LOD (95% hit rate) by probit analysis: 34.44cp / mL
95% confidence interval for LOD by probit analysis: 21.89-83.04cp / mL

HBV

HCV

2. WNV Qualitative Analysis Master Mix

Analytical sensitivity / LOD
Independent panels were prepared for the viruses (WNV, SLEV and JEV) as serial dilutions of each standard containing several concentrations / levels at and around the expected LOD. One virus and one panel per concentration were tested with at least 20 valid replicates per concentration. LOD was determined by probit analysis.

3. Master mix for quantitative analysis of HBV

Analytical sensitivity / LOD
Four dilution panels with HBV secondary standards (indicating genotype A), two in HBV-negative serum for sample input volumes of 200 μL and 500 μL, and two for HBV negative and 200 μL sample input volumes Negative EDTA plasma was prepared. Each panel contained seven concentration levels at and around the expected LOD. One panel per matrix was tested in ≧ 21 replicates per concentration level. At least 20 replicates had to be valid. LOD was determined by probit analysis at 95% hit rate and ≧ 95% hit rate analysis.

LOD Overview:
EDTA plasma: Probit analysis at 95% hit rate yielded a 8.2 IU / mL LOD for a 200 μL sample input volume and 2.3 IU / mL for a 500 μL sample input volume for EDTA plasma.

  The 95% confidence interval ranges for these concentrations were 4.8-26.0 IU / mL for a 200 μL sample input volume and 1.6-4.2 IU / mL for a 500 μL sample input volume.

Serum: Probit analysis at 95% hit rate resulted in LOD of 9.02 IU / mL for 200 μL sample input volume and 4.1 IU / mL for 500 μL sample input volume for serum.

  The 95% confidence interval ranges for these concentrations were 6.2-19.0 IU / mL for a 200 μL sample input volume and 2.4-10.0 IU / mL for a 500 μL sample input volume.

Linearity
One EDTA plasma panel and one serum panel were prepared using HBV genotype A (provided by RMD Research Pleasanton, linearized plasmid, pHBV-PC_ADW2). Each panel was analyzed at 12 concentration levels for determination of the expected dynamic range of the assay (4-2E + 09 IU / mL). All concentration levels / panel members (PM) were tested in 21 replicates.

The test was performed with a sample input volume of 500 μL. Concentration levels were selected as follows: one level below the expected lower limit of quantification (LLOQ), one at the expected LLOQ, one above the expected LLOQ, and several concentrations at intermediate levels. Some concentrations at the expected upper limit of quantification (ULOQ), and one above the expected ULOQ.
PM12-2.0E + 09IU / mL-above expected ULOQ
PM11-1.0E + 09IU / mL-Expected ULOQ
PM10-1.0E + 08IU / mL-less than expected ULOQ
PM9-1.0E + 07IU / mL-intermediate concentration level
PM8-1.0E + 06IU / mL-intermediate concentration level
PM7-1.0E + 05IU / mL-intermediate concentration level
PM6-1.0E + 04IU / mL-intermediate concentration level
PM5-1.0E + 03IU / mL-intermediate concentration level
PM6a-2.0E + 02IU / mL-intermediate concentration level (PM6 diluted to 2.0E + 02IU / mL, used for serum panel titration)
PM4-1.0E + 02IU / mL-intermediate concentration levels (also used for plasma panel titration)
PM3-5.0E + 01IU / mL-above expected LLOQ
PM2-1.0E + 01IU / mL-Expected LLOQ
PM1-4.0E + 00IU / mL Less than expected LLOQ

  For all valid samples in the linearity panel, the observed HBV DNA titers were converted to log10 titers and the average log10 titer per concentration level was calculated.

  FIG. 13 shows a graphical representation of this result.

  FIG. 14 shows a graphical representation of this result.

Linearity overview:
The linear range, defined as the concentration range where the log10 deviation of the mean log10 observed titer is within ± 0.3 of the log10 nominal titer, is from 3.5E + 00 IU / mL to 1.7E + 09IU / mL for EDTA plasma and 3.3E + for serum. It was determined to be between 00 IU / mL and 1.7E + 09 IU / mL. The lower limit of quantification was found to be 4.0E + 00 IU / mL for EDTA plasma and serum.

4. Master mix for quantitative analysis of HCV

Analytical sensitivity / LOD
Dilution panels with Roche HCV secondary standards in HCV negative EDTA plasma and serum were prepared using 200 μL and 500 μL sample input volumes. Each concentration level was tested in 21 replicates. At least ≧ 20 replicates need to be completed. LOD was determined by probit analysis at 95% hit rate and> 95% hit rate analysis.

LOD Overview:
1. Probit analysis at 95% hit rate resulted in LOD of 17.4 IU / mL for 200 μL sample processing input volume and 9.0 IU / mL for 500 μL sample processing input volume for EDTA plasma. The 95% confidence intervals for these concentrations are 12.1 to 34.3 IU / mL for a 200 μL sample processing input volume and 5.5 to 25.4 IU / mL for a 500 μL sample processing input volume.

2. The values of the probit analysis at 95% hit rate are 20.2 IU / mL for 200 μL sample processing input volume and 8.2 IU / mL for 500 μL sample processing input volume for serum. The 95% confidence intervals for these concentrations are 14.0-39.3 IU / mL for a 200 μL sample processing input volume and 5.8-15.0 IU / mL for a 500 μL sample processing input volume.

Linearity
One preparation of an EDTA plasma panel and one preparation of a serum panel of HCV aRNA traceable to HCV WHO standards were analyzed. Linear panels were prepared by serial dilution and analyzed at 10 different concentrations. The test was performed with a 500 μL sample processing input volume. Concentrations were selected as follows: one level below the lower limit of expected quantification (LLoQ), one at LLoQ, one above LLOQ, several concentrations at intermediate levels, and an upper limit of expected quantification Several concentrations at (ULoQ) and one at or above ULoQ. For all concentrations, 21 replicates were tested.

PM1-2.0E + 08IU / mL-above expected ULoQ
PM2-1.0E + 08IU / mL-Expected ULoQ
PM3-1.0E + 07IU / mL-less than expected ULoQ
PM4-1.0E + 06IU / mL-intermediate concentration level
PM5 1.0E + 05IU / mL-intermediate concentration level
PM6-1.0E + 04IU / mL-intermediate concentration level for titer designation
PM7-1.0E + 03IU / mL-intermediate concentration level
PM8-1.0E + 02IU / mL-above expected LLoQ
PM9-1.0E + 01IU / mL-Expected LLoQ
PM10-8.0E + 00IU / mL-less than expected LLoQ

  FIG. 15 shows a graphical representation of this result.

  FIG. 16 shows a graph display of this result.

Linearity overview:
The linear range defined as the concentration range where the log10 deviation of the average log10 observed titer is within ± 0.3 of the log10 nominal titer is 4.87E + 00IU / mL to 1.22E + 08IU / mL for EDTA plasma and 3.90E + 00IU for serum. / mL to 9.92E + 07IU / mL.

5. Master mix for quantitative analysis of HIV

Analytical sensitivity / LOD
Dilution panels with HIV-1M secondary standards in HIV-1 negative EDTA plasma were prepared for sample input volumes of 200 μL and 500 μL. Each concentration level was tested in 21 replicates. At least ≧ 20 replicates must be valid. LOD was determined by probit analysis and 95% hit rate analysis at 95% hit rate.

LOD overview
1. Probit analysis with a 95% hit rate resulted in a LOD of 41.8 cp / mL for a 200 μL input volume and 18.9 cp / mL for a 500 μL input volume.

2. The 95% confidence interval ranges for these concentrations were 30.9-74.9 cp / mL for a 200 μL input volume and 14.9-29.4 cp / mL for a 500 μL input volume.

Linearity The samples used for the linearity / dynamic range / accuracy tests consisted of HIV-1 cell culture supernatant material, a dilution panel of HIV-1 group M subtype B.

  A linear panel was prepared by serial dilution. The panel was analyzed at 10 concentration levels.

  Concentrations were selected as follows: one level below the lower limit of expected quantification (LLoQ), one at LLoQ, one above LLoQ, several concentrations at intermediate levels, and an upper limit of expected quantification Several concentrations in (ULoQ) and one above ULoQ. For all concentrations, 21 replicates were tested. The linearity test was performed with a 500 μL input volume:

PM1-2.0E + 07cp / mL-above expected ULoQ
PM2-1.0E + 07cp / mL-Expected ULoQ
PM3-1.0E + 06cp / mL-less than expected ULoQ
PM4-1.0E + 05cp / mL-intermediate concentration level
PM5 3.0E + 04cp / mL-intermediate concentration level for titer designation
PM6-1.0E + 04cp / mL-intermediate concentration level
PM7-1.0E + 03cp / mL-intermediate concentration level
PM8-1.0E + 02cp / mL-intermediate concentration level
PM9-5.0E + 01cp / mL-above expected LLoQ
PM10-2.0E + 01cp / mL-Expected LLoQ
PM11-1.5E + 01cp / mL-less than expected LLoQ

  FIG. 17 shows a graphical representation of this result.

Linearity Summary The linear range defined as the concentration range where the log10 deviation of the average log10 observed titer was within ± 0.3 of the log10 nominal titer was determined to be 1.5E + 01cp / mL to 2.0E + 07cp / mL.

Embodiments of the present invention include the following.
[1] A method for isolating and co-amplifying at least first and second target nucleic acids that may be present in one or more liquid samples, wherein the method comprises:
a. adding an internal control nucleic acid to each of the liquid samples
b. separating the solid support material and the one or more liquid samples for one or more times and under conditions and for a time and under conditions sufficient for the nucleic acid containing the target nucleic acid and the internal control nucleic acid to be immobilized on the solid support material The process of joining together in a container
c. isolating the solid support material from other materials present in the liquid sample at the separation station.
d. purifying the nucleic acid at the separation station and washing the solid support material one or more times with a washing buffer;
e. at least two reaction vessels comprising a purified target nucleic acid and a purified internal control nucleic acid, and one or more amplification reagents comprising at least one different set of primers for each of the target nucleic acids and the internal control nucleic acid. Contacting, wherein at least a first reaction vessel includes at least the first target nucleic acid, at least a second reaction vessel includes at least the second target nucleic acid, and a second target nucleic acid is the first target vessel. Not present in the reaction vessel
f. in the reaction vessel, the purified target nucleic acid and the purified internal control nucleic acid and the one or more amplification reagents are sufficient to cause an amplification reaction indicative of the presence or absence of the target nucleic acid to occur. Incubating under appropriate time and conditions
g. detecting and measuring a signal generated by the amplification product of the target nucleic acid and proportional to the concentration of the target nucleic acid; andautomating a step of detecting and measuring a signal generated by the internal control nucleic acid, The conditions for amplification and detection in steps d to g are the same for the at least first and second purified target nucleic acids and the internal control nucleic acid, and the sequence of the internal control nucleic acid is the at least first and second The method is the same for the two purified target nucleic acids.
[2] The method according to [1], wherein the presence of the amplification product of the internal control nucleic acid indicates that amplification occurs in the reaction mixture even in the absence of the amplification product of one or more of the target nucleic acids. Method.
[3] Following:
h. The method of any of the preceding, further comprising determining one or more amounts of said target nucleic acid.
[4] The amplification reagent comprises a polymerase having reverse transcriptase activity, and the method comprises the steps of: e) and (f), wherein the purified nucleic acid and the one or more amplification reagents are contained in the reaction vessel. The method according to any of the preceding claims, further comprising the step of incubating for a time and under conditions suitable for transcription of RNA by the polymerase having reverse transcriptase activity to occur.
[5] The method according to any one of the above, wherein the internal control nucleic acid is DNA.
[6] The method according to any one of the above, wherein the internal control nucleic acid is RNA.
[7] The method according to any one of the above, wherein the internal control nucleic acid is a sheathed nucleic acid.
[8] The method according to any one of the above, wherein the sequence of the internal control nucleic acid is different from the sequence of another nucleic acid present in the liquid sample.
[9] The method according to any one of the above, wherein the sequence of the internal control nucleic acid is derived from a naturally occurring genome.
[10] The method according to any one of the above, wherein the sequence of the internal control nucleic acid is mixed.
[11] The method according to any one of the above, wherein the melting temperature of the internal control nucleic acid is 50 ° C to 90 ° C.
[12] The method according to any one of the above, wherein the internal control nucleic acid has a length of up to 500 bases.
[13] The method according to any one of the above, wherein the sequence of the internal control nucleic acid has a GC content of 30% to 70%.
[14] The method according to any one of the above, wherein the internal control nucleic acid has a concentration of LOD to 20 × LOD.
[15] The method according to any one of the above, wherein the internal control nucleic acid has a concentration of 20 × LOD to 5000 × LOD.
[16] The method according to any of the preceding claims, wherein in step a more than one internal control nucleic acid is added, but only one of said internal control nucleic acids is amplified in step f.

Claims (6)

A method for isolating and co-amplifying first and second target nucleic acids that may be present in one or more liquid samples, said method comprising:
a. adding a quantitative standard nucleic acid to each of the liquid samples, wherein the sequence of the quantitative standard nucleic acid is derived from a source different from the source of the liquid sample and differs from other nucleic acid sequences in the liquid sample. Is different, is derived from the plant genome, is mixed, and the quantitative standard nucleic acid has a concentration of 20 × LOD to 5000 × LOD;
b. the solid support material and the one or more liquid samples for one or more times and under conditions sufficient for the nucleic acid containing the target nucleic acid and the quantitative standard nucleic acid to be immobilized on the solid support material; Of combining together in a container
c. isolating the solid support material from other materials present in the liquid sample at the separation station.
d. purifying the nucleic acid at the separation station and washing the solid support material one or more times with a washing buffer;
e. an amplification reagent comprising a purified target nucleic acid and a purified quantitative standard nucleic acid and at least one different set of primers for each of said target nucleic acids and said quantitative standard nucleic acid in at least two reaction vessels Contacting in at least two reaction vessels for the amplification of at least the first and second target nucleic acids and the quantitative standard nucleic acid of
f. in the reaction vessel, the purified target nucleic acid and the purified quantitative standard nucleic acid, and the amplification reagent, for a time sufficient for an amplification reaction to be performed indicating the presence or absence of the target nucleic acid, and Incubate under conditions, amplify the first target nucleic acid instead of the second target nucleic acid in the first reaction vessel, and amplify the second target nucleic acid instead of the first target nucleic acid in the second reaction vessel Process
g. detecting and measuring a signal generated by the amplification product of the target nucleic acid and proportional to the concentration of the target nucleic acid, and detecting and measuring a signal generated by the quantitative standard nucleic acid;
h. comprises an automated step of determining one or more amounts of the target nucleic acid by using a quantitative standard nucleic acid as a reference, wherein the conditions for amplification and detection in steps e.-g. The same is true for the first and second purified target nucleic acids and said quantitative standard nucleic acid.   2. The method of claim 1, wherein the presence of the quantitative standard nucleic acid amplification product indicates that amplification occurs in the reaction mixture even in the absence of the amplification product for one or more of the target nucleic acids.   3. The method according to claim 1, wherein the quantitative standard nucleic acid is a sheathed nucleic acid.   The method according to any one of claims 1 to 3, wherein the melting temperature of the quantitative standard nucleic acid is 50C to 90C.   The method according to any of claims 1 to 4, wherein the quantitative standard nucleic acid has a length of up to 500 bases. The method according to any one of claims 1 to 5, wherein the sequence of the quantitative standard nucleic acid has a GC content of 30% to 70%.

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