JP6754874B2 - Control nucleic acid for multiple parameters - Google Patents

Description

Field of Invention The present invention belongs to the field of in vitro diagnosis. In this art, the invention relates, in particular, to amplification of at least first and second target nucleic acids that may be present in at least one liquid sample using internal control nucleic acids for qualitative and / or quantitative purposes. ..

Background of the Invention In the field of molecular diagnosis, amplification of nucleic acids from a large 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), western Nile virus (WNV), or human immunodeficiency virus (HIV), hepatitis B virus (HBV) and / or type C. It is a routine screening of blood donations for the presence of hepatitis virus (HCV). In addition, the amplification technique is suitable for 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, among other things, ligase chain reaction, polymerase ligase chain reaction, Gap-LCR, repair chain reaction, 3SR, NASBA, chain substitution amplification (SDA), transcription mediation amplification (TMA) and Qβ amplification.

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 a reporter group or label.

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

For qualitative (performance control) and / or quantitative (determination of the amount of target nucleic acid using the control as a reference), it is possible to control each amplification using a control nucleic acid whose sequence is known. It is the most desirable or even essential in the field of clinical nucleic acid diagnosis. Control nucleic acids are usually designed in a particular manner, especially due to the diversity of diagnostic targets, including prokaryotic, eukaryotic and viral nucleic acids, and due to the diversity between different types of nucleic acids such as RNA and DNA. .. Briefly, these controls usually resemble target nucleic acids and serve as controls to mimic their performance during the process. This situation applies to both qualitative and quantitative assays. When multiple parameters are detected in a single or parallel experiment, different controls that resemble different target nucleic acids are usually used, such as Swanson et al. (Non-Patent Document 1). Stoecher et al. (Non-Patent Document 2) discloses a control nucleic acid in which a large number of virus-specific competitive controls are contained on the same DNA molecule.

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

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

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

That is, the gist of the present invention is
[1] A method for isolating and co-amplifying first and second target nucleic acids that may be present in one or more liquid samples.
The step of adding a quantitative standard nucleic acid to each of the liquid samples, where the sequence of the quantitative standard nucleic acid is derived from a source different from the origin of the liquid sample and is different from other nucleic acid sequences in the liquid sample. Unlike, derived from the plant genome and mixed, the quantitative standard nucleic acid has a concentration of 20 × LOD to 5000 × LOD.
b. One or more of the solid support material and the one or more liquid samples, with sufficient time and conditions for the nucleic acid containing the target nucleic acid and the quantitative standard nucleic acid to be immobilized on the solid support material. Process of putting together in a container
c. The step of isolating the solid support material from other materials present in the liquid sample at the separation station.
d. The step of purifying the nucleic acid at the separation station and washing the solid support material with the wash buffer at least once.
e. Amplification reagents comprising a purified target nucleic acid and a purified quantitative standard nucleic acid and at least one different set of primers for each of the target nucleic acids and the quantitative standard nucleic acid in at least two reaction vessels. Contact in at least two reaction vessels for amplification of at least the first and second target nucleic acids and quantitative standard nucleic acids
f. In the reaction vessel, the purified target nucleic acid, the purified quantitative standard nucleic acid, and the amplification reagent are used for a sufficient time for an amplification reaction indicating the presence or absence of the target nucleic acid to occur. Incubate under conditions to amplify the first target nucleic acid instead of the second target nucleic acid in the first reaction vessel and the second target nucleic acid instead of the first target nucleic acid in the second reaction vessel. Process to do
g. A step of detecting and measuring a signal produced by the amplification product of the target nucleic acid and proportional to the concentration of the target nucleic acid, and a step of detecting and measuring a signal produced by the quantitative standard nucleic acid.
h. The conditions for amplification and detection in steps e. To g. Include the step of automating the step of determining the amount of one or more of the target nucleic acids by using a quantitative standard nucleic acid as a reference. The same method for the first and second purified target nucleic acids and the quantitative standard nucleic acids.
[2] The presence of an amplification product of the quantitative standard nucleic acid indicates that amplification occurs in the reaction mixture even in the absence of amplification products for one or more of the target nucleic acids, [1]. the method of,
[3] The method according to [1] or [2], wherein the quantitative standard nucleic acid is an externalized nucleic acid.
[4] The method according to any one of [1] to [3], wherein the melting temperature of the quantitative standard nucleic acid is 50 ° C to 90 ° C.
[5] The method according to 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%.

The present invention may provide control nucleic acids for a number of parameters.

FIG. 1 is a schematic depiction of the sample preparation workflow used in aspects of the present invention. Arrows pointing down indicate the addition of components or reagents to each well of the deep well plates described above, and arrows pointing up indicate their respective removal. These operations are performed manually in steps 2, 3, 4, 21 and 22 and in the process heads of the equipment in steps 10, 14, 16, 18 and 24, steps 5, 6, 7, 11, 15 and In 19 it was done with the reagent head of the device. It should be understood that the volume used can be flexibly adjusted within the spirit of the invention, preferably up to at least about 30% of the disclosed values. In particular, in the case of step 2, the volume of the sample is preferably variable in order to take into account different types of liquid samples that may require some starting material to obtain appropriate results, as known to those skilled in the art. Is. Preferably, the range is from about 100 ul to about 850 ul. More preferably, the capacity 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 diagram 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 LightCycler 480 (Roche Diagnostics GmbH, Mannheim, DE) described in Example 1. The "signal" shown on the y-axis is a standardized fluorescent signal. The x-axis shows the number of each PCR cycle. The HIV and HBV increase curves are shown along with the corresponding internal control nucleic acid increase curves. The curve of each target nucleic acid is shown by the solid line, and the curve of the control nucleic acid is shown by the dotted line. Figure 2a is a qualitative HIV assay measured in the channel for detection of the target probe. Figure 2b is a qualitative HIV assay measured in the channel for detection of control probes. Figure 2c is a quantitative HIV assay measured in the channel for detection of the target probe. Figure 2d is a quantitative HIV assay measured in the channel for detection of control probes. Figure 2e is a quantitative HBV assay measured in the channel for detection of the target probe. Figure 2f is a quantitative HBV assay measured in the channel for detection of control probes. Figure 2g is a CT assay measured in the channel for detection of the target probe. FIG. 3 is a perspective view of the processing plate. FIG. 4 is a perspective view of the processing plate from the opposite angle. FIG. 5 is a top view of the processing plate. FIG. 6 is a cross-sectional view taken along the long side surface of the processing plate. FIG. 7 is a partial view of a cross-sectional view. FIG. 8 is a perspective view of the long side surface of the processing plate. 9a) to 9d) show different views of the second aspect of the magnetic separation station. FIGS. 10 (a) to 10 (c) show a magnetic separation station holding a processing plate having a first-type magnet at the highest Z position and a second-type magnet at the lowest Z position. The figure of the first aspect is shown. FIG. 11 is a schematic representation of an analyzer that includes various stations, modules or cells. FIG. 12 shows the analytical system of the present invention. FIG. 13 shows the linearity of the quantitative HBV assay in EDTA plasma according to the data of Example 2. FIG. 14 shows the linearity of the quantitative HBV assay in serum according to the data of Example 2. FIG. 15 shows the linearity of the quantitative HCV assay in EDTA plasma according to the data of Example 2. FIG. 16 shows the linearity of the quantitative HCV assay in serum according to the data of Example 2. FIG. 17 shows the linearity of the quantitative HIV assay in EDTA plasma according to the data of Example 2.

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

In a first aspect, the invention relates to a method of isolating and simultaneously amplifying at least the first and second target nucleic acids that may be present in one or more liquid samples.
a. Step of adding internal control nucleic acid to each of the liquid samples
b. Sufficient time for the nucleic acid containing the target nucleic acid and the internal control nucleic acid to be immobilized on the solid support material in one or more containers of the solid support material and the one or more liquid samples. And the process of matching together under conditions
c. The step of isolating the solid support material from other materials present in the liquid sample at the separation station.
d. The step of purifying the nucleic acid at the separation station and washing the solid support material with the wash buffer at least once.
e. At least two reaction vessels containing a purified target nucleic acid and a purified internal control nucleic acid and one or more amplification reagents containing at least one different set of primers for each of the target nucleic acids and the internal control nucleic acid. The step of contacting in, where at least the first reaction vessel comprises at least the first target nucleic acid, at least the second reaction vessel comprises at least the second target nucleic acid, and the second target nucleic acid is the first. Does not exist in the reaction vessel of
f. In the reaction vessel, the purified target nucleic acid, the purified internal control nucleic acid, and the one or more amplification reagents are sufficient for an amplification reaction indicating the presence or absence of the target nucleic acid to occur. Step of incubating for time and conditions
g. Includes an automated step of detecting and measuring a signal produced by the amplification product of the target nucleic acid and proportional to the concentration of the target nucleic acid, and a step of detecting and measuring the signal produced by the internal control nucleic acid. The conditions for amplification and detection in steps d to g are the same for at least the first and second purified target nucleic acids and the internal control nucleic acid, and the sequences of the internal control nucleic acids are at least the first and second purified nucleic acids. It is the same for the purified target nucleic acid of.

The present invention allows 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. As such, the present invention contributes to reducing the overall complexity of the corresponding experiments at various levels. For example, it is only necessary to design one internal control nucleic acid sequence and add it to each amplification mixture, saving time and money in designing and synthesizing or purchasing multiple control nucleic acid sequences. One or more assays can be streamlined, reducing the risk of handling errors. Also, the more different control nucleic acid sequences are used in one assay or parallel assays performed under the same conditions at the same time, the more complex the regulation of each condition can be. In addition, using one control suitable for multiple nucleic acids, the control can be dispensed from one source, for example, to different vessels containing the different target nucleic acids. Within the scope of the invention, one control nucleic acid sequence can also serve as a qualitative and quantitative control.

As a further advantage of the methods described above, the controls used in the present invention can be used to serve as controls for the amplification of various nucleic acids, so that in possible subsequent experiments of a particular biological sample for other nucleic acids. The test need not include a separate sample preparation procedure with the addition of different internal control nucleic acids. Therefore, once the internal control nucleic acid is added, other parameters can be tested in the same sample under the same conditions.

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

Competitive internal control nucleic acids have essentially the same primer binding site as the target and therefore compete with the target for the same primer. This principle results in a less sensitive assay as they allow good mimicry of each target nucleic acid due to their similar structure and can reduce amplification efficiency for one or more target nucleic acids.

The non-competitive internal control nucleic acid has a different primer binding site from the target and therefore binds to a different primer. Advantages of such a setting include, among other things, the fact that one amplification event of different nucleic acids in the reaction mixture can occur independently of each other without any competing effect. Therefore, there is no adverse effect on the detection limit of the assay that can occur in the case of competitive settings.

Finally, in amplification using the 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 comprises a different set of primers for each of the target nucleic acids and for the internal control nucleic acid makes the method quite 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 drawbacks of the competitive setting described above. In a non-competitive setting, the internal control nucleic acid has a sequence different from any target sequence so as not to compete with their primers and / or probes. Preferably, the sequence of the internal control nucleic acid is different from the other nucleic acid sequences in the liquid sample. As an example, if the liquid sample is of human origin, the internal control nucleic acid preferably does not have a sequence that is also endogenously present in humans. Therefore, sequence differences should be at least significant so that under stringent conditions the primers and / or probes do not bind to each one or more endogenous nucleic acids and the settings are not competitive. .. To avoid such interference, the sequences of the internal control nucleic acids used in the present invention are preferably from a source different from that of the liquid sample. Preferably, the sequence is derived from a naturally occurring genome, preferably from a plant genome, more preferably from a grape genome. In a highly preferred embodiment, naturally occurring genome-derived nucleic acids are mixed. As is known in the art, "scrambling" means introducing some degree of 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 that include the automation steps described above also show various additional advantages.

Prior art has made it difficult to limit the number of different target nucleic acids by the appropriate number of labels in a multiplex assay performed in a single reaction vessel. For example, in a real-time PCR assay, possible duplication of the fluorochrome spectrum has a significant impact on assay performance (risks of false positive results, low accuracy, etc.). Therefore, the respective fluorophores need to be carefully selected and separated sufficiently spectrally to ensure the desired performance of the diagnostic test. Typically, the number of different available fluorophores corresponds to a single digit number of fluorescent channels in the PCR device.

In contrast, in the method described above, amplification with at least internal control of the first and second target nucleic acids occurs in at least two different reaction vessels, and signals in different reaction vessels are detected independently of each other. Allows for simultaneous amplification of a larger number of different target nucleic acids. Moreover, it is within the scope of the invention that the multiplex reaction occurs in one or more multiple reaction vessels, thereby increasing the number of targets that can be amplified simultaneously under the same conditions. In such an embodiment, 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.

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

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

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

First target nucleic acids, especially 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. And the method described above in which the second target nucleic acid is from a different organism is advantageous and is therefore a preferred embodiment of the invention.

A more preferred aspect of the invention is the method described above in which the first and / or second target nucleic acid is a non-viral nucleic acid.

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

As mentioned earlier, the methods described above are useful for qualitative or quantitative control of the amplification of at least the first and at least second target nucleic acids.

Qualitative detection of nucleic acids in biological samples is important, for example, in the recognition of infections in individuals. Thereby, one important requirement for assays for the detection of microbial infections is to avoid such results, as false-negative or false-positive results almost inevitably have significant consequences for the treatment of each patient. It is to be. Therefore, a qualitative internal control nucleic acid is added to the detection mixture, especially in PCR-based methods. The control is particularly important for confirming the validity of the test results. In the case of a negative result, at least for each target nucleic acid, the qualitative internal control reaction must be reactive in a given setting, i.e., a qualitative internal control must be detected, otherwise tested. It is considered inactive. However, in a qualitative setting, the qualitative internal control does not necessarily have to be detected in the case of a positive result. For qualitative testing, it is especially important that the sensitivity of the reaction is guaranteed and therefore tightly controlled. As a result, the concentration of the qualitative internal control should be relatively low so that, for example, even in the case of slight inhibition, no qualitative internal control is detected, which invalidates the test.

Thus, a preferred embodiment of the invention described above shows that the presence of an amplification product of the internal control nucleic acid causes amplification in the reaction mixture even in the absence of the amplification product for one or more of the target nucleic acids. This is the method.

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

In the case of external calibration, standard curves are created in separate reactions using known amounts of the same or equivalent nucleic acid. The absolute amount of target nucleic acid is subsequently determined by comparing the standard function with the results obtained from the analyzed sample. However, external calibration has the disadvantage that possible extraction procedures, their altered efficiencies, and the presence of possible and often unpredictable agents that inhibit amplification and / or detection reactions are not reflected in the control.

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

For these and other reasons, the 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) Internal control nucleic acid monitors the validity of the reaction.
ii) The internal control nucleic acid acts as a reference in titer calculations, thus compensating for the effects of inhibition and controlling the preparation and amplification process to allow for more accurate quantification. Therefore, in contrast to the qualitative internal control nucleic acid in a qualitative test, which must be positive only in a target negative reaction, the quantitative control nucleic acid in a quantitative test has two functions: reaction control and reaction calibration. Therefore, the quantitative control nucleic acid must be positive and valid in both target-negative and target-positive reactions.

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

Therefore, the preferred aspects of the present invention are as follows:
h. The method described above further comprising determining the amount of one or more of the target nucleic acids.

The above-mentioned internally controlled method requires a considerably shorter operation time, and the test is considerably simpler than the real-time PCR method used in the prior art. The method offers great advantages, for example, in the field of clinical virology, as it allows for parallel amplification of several viruses in parallel experiments. The method is particularly useful for the management of post-transplant patients who require frequent virus monitoring. Thereby, the method facilitates economic diagnosis and contributes to the use of antiviral agents and reduction of viral complications and hospitalizations. This applies similarly to the field of clinical microbiology. In general, efficiency is increased with faster travel times and improved test flexibility. As a result, this can reduce the number of trials required of the patient to make the diagnosis and shorten the length of hospital stay (eg, patients in need of antimicrobial therapy if the diagnosis can be provided earlier). Receives the therapy faster and recovers faster). Patients also show lower morbidity, resulting in lower costs for adjuvant therapy (eg, intensive care for delayed diagnosis of sepsis). Providing negative results earlier may have important implications for overprescribing antibiotics. For example, clinicians are not forced to use empirical antibiotics if the test results obtained by the method according to the invention can rule out pathogens faster than standard real-time PCR methods. Alternatively, if empirical antibiotics are used, the duration of each treatment can be shortened.

One of ordinary skill in the art, with respect to designing a particular test based on the methods of the invention, has the following advantages:
· Reduced software complexity (lowering the risk of programming errors)
• Focus 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 carry out this protocol. The reliability of the system is significantly increased, as those skilled in the art performing the above internally contrasted methods provide the flexibility to perform a number of different assays in parallel as part of the same method.・ Benefit from cost reduction.

In the sense of the present invention, "purification", "isolation" or "extraction" of a nucleic acid relates to the following: Nucleic acids typically vary in variety before they can be analyzed in a diagnostic assay, eg, by amplification. It needs to be purified, isolated or extracted from a biological sample containing a complex mixture of ingredients. Often, for the first step, a process that allows the enrichment of nucleic acids is used. To release the contents of a cell or viral particle, the cell or viral particle can be treated with an enzyme or chemical to dissolve, degrade or denature the cell wall or viral particle. This process is commonly referred to as lysis. The resulting solution containing such a dissolved substance is referred to as a solution. A problem that often arises 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 can also be present outside the cell or can be spatially separated into different cell compartments prior to lysis. As lysis occurs, the component of interest 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 the treatment of humans or animals.

There are various means to tackle the above issues. It is common to use chaotropic agents such as guanidinium thiocyanate or anionic, cationic, zwitterionic or nonionic surfactants when the nucleic acid is intended to be released. It is also advantageous to use the aforementioned enzymes or proteases that rapidly degrade unwanted proteins. However, this can raise another problem that the substance or enzyme can interfere with the reagent or component in subsequent steps.

Enzymes that can be used favorably in such lysis or sample preparation processes described above are enzymes that cleave amide bonds in protein substrates and are classified as proteases or (compatiblely) peptidases (Walsh, 1979, Enzymatic Reaction). Mechanisms. See WH Freeman and Company, San Francisco, Chapter 3). Proteases used in the prior art include alkaline proteases (WO 98/04730) or acidic proteases (US 5,386,024). Proteases widely used for sample preparation in the isolation of nucleic acids in the prior art are derived from the Tritiracium album, which is active near neutral pH and belongs to a family of proteases known to those of skill in the art as subtilisin. Proteinase K (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 1 201 753), a potent protease that maintains its activity at both high alkalinity and high temperature, is particularly advantageous for use in the process of lysis or sample preparation described above.

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

Several methods for purification of nucleic acids:
--Sequence-dependent or biospecific methods, such as:
-Affinity chromatography-Hybridization to immobilized probes
--Sequence-independent or physicochemical methods, such as:
・ For example, liquid-liquid extraction using phenol-chloroform ・ For example, precipitation using pure alcohol ・ Extraction using filter paper ・ Extraction using micelle forming agent such as cetyltrimethylammonium bromide ・ Immobilized intercalation dye, For example, there are binding to an acrysin derivative, adsorption to silica gel or silica soil, and adsorption to magnetic glass particles (MGP) or organo-silane particles under chaotropic conditions.

Adsorption of nucleic acids to the glass surface is of particular interest for purification purposes, although other surfaces are possible. Many techniques have been recently proposed for isolating nucleic acids from the natural environment by using the binding behavior of nucleic acids to glass surfaces. When the unmodified nucleic acid is the target, the direct binding of the nucleic acid to a substance having a silica surface is preferable because the nucleic acid does not need to be modified and even a natural nucleic acid can be bound. These methods are described in detail in various documents. Vogelstein B. et al., Proc. Natl. Acad. USA 76 (1979) 615-9 states, for example, the binding of nucleic acids from agarose gels to ground flint glass in the presence of sodium iodide. Procedure is advocated. 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 isolation of single-stranded M13 phage DNA on a glass fiber filter by precipitation of phage particles with acetic acid and lysis of phage particles with perchlorate. The nucleic acid bound to the fiberglass filter is washed and then eluted with methanol-containing Tris / EDTA buffer. A similar procedure for the purification of DNA from λ phage is described in Jakobi R. et al., Anal. Biochem. 175 (1988) 196-201. The procedure requires selective binding of nucleic acids to the glass surface in a chaotropic salt solution and separation of nucleic acids from contaminants such as agarose, proteins or cell residues. To separate the glass particles from the contaminants, the particles can be centrifuged or the liquid can be extracted with a fiberglass filter. However, this is a limited step that prevents the technique from being used to process large volumes of samples. The use of magnetic particles to immobilize nucleic acids after precipitation by the addition of salts and ethanol is more advantageous, eg Alderton RP et al., S., Anal. Biochem. 201 (1992) 166-169 and PCT GB. It is described in 91/00212. In this technique, the nucleic acid aggregates with the magnetic particles. The agglomerates are separated from the original solvent by applying a magnetic field and performing a washing step. After one wash step, the nucleic acid is dissolved in Tris buffer. However, this approach has the disadvantage that precipitation is not selective for nucleic acids. Rather, various solid and dissolved substances also aggregate. As a result, this technique cannot be used to remove a significant amount of any inhibitor of a particular enzymatic reaction that may be present. Magnetic porous glass containing magnetic particles in a specific porous glass matrix and coated with a layer containing streptavidin is also commercially available. This product can be used to isolate a biological material, such as a protein or nucleic acid, when the biological material is modified to covalently bind to biotin in a complex preparation process. Certain magnetizable adsorbents have proved 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 described in WO 01/37291. In the context of the present invention, the method by 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 above-mentioned solid materials, such as magnetic glass particles, glass fibers, glass fiber filters, filter paper, etc., with respect to the immobilization of nucleic acids, the solid support material to which. Not limited.

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

In the context of the present invention, "fixation" means capturing an object, such as a nucleic acid, in a reversible or irreversible manner. In particular, "fixed on a solid support material" means that the object is bound to the solid support material in order to separate one or more objects from any ambient medium, and at a later time, It means that it can be recovered from the solid support material, for example by separation. In this context, "fixation" can include, for example, adsorption of nucleic acids to a glass surface or other suitable surface of a solid substance as described above. In addition, binding to a capture probe can specifically "fix" the nucleic acid, where base pairing binds to an essentially complementary nucleic acid bound to a solid support. In the latter case, such specific fixation results in predominant binding of the target nucleic acid.

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

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

The "first target nucleic acid" and the "second target nucleic acid" are different nucleic acids.

A "fluid sample" is any liquid substance that can be subjected to a diagnostic assay targeting nucleic acids, preferably from a biological source. Also, preferably, the liquid sample is of human origin and is a body fluid. In a preferred embodiment of the invention, the liquid sample is human blood, urine, sputum, sweat, swab, pipetteable feces or spinal fluid. Most preferably, the liquid sample is human blood.

The term "reaction vessel" is not limited to a tube in which a reaction for analysis of a liquid sample, such as a reverse transcription reaction or a polymerase chain reaction, takes place, or a microwell, deepwell or other type of multiwell plate. Includes plate wells such as. The outer boundary or wall of such a container is chemically inert so as not to interfere with the analytical reaction occurring therein. Preferably, the above-mentioned nucleic acid isolation is also performed in a multi-well plate.

In this context, multi-well plates in analytical systems allow for parallel separation and analysis or storage of large numbers of samples. Multi-well plates can be optimized for maximum liquid uptake or maximum heat transfer. Preferred multi-well 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 the magnetic and / or heating devices.

The preferred multi-well plate, interchangeably referred to as a "treated plate" in the context of the present invention, is:
-Includes an upper surface containing a large number of containers with an opening at the top arranged in a row. The container includes a top part, a center part and a bottom part. The upper part is connected to the upper surface of the multiwell plate and contains two long sides and two short sides. The central part has 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 includes openings constructed and arranged to place a multi-well plate in contact with said magnetic and / or heating devices.

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

Preferably, the multi-well plate comprises a contiguous space arranged between adjacent rows of containers. The contiguous 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 ribs, which surround the opening of the container. Preferably, one short side of the upper part of the container comprises a recess, the recess comprising a curved surface extending from the rib to the inside of the container.

Further, in a preferred embodiment, the container comprises a round inner shape.

For fixation to a processing station or incubation station, the base preferably comprises a rim containing a recess. The latch clip on the station of the analyzer can engage the recess to secure the plate on the station.

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

A preferred treatment plate (101) in the context of the present invention is a one-component plate. Its upper surface (110) contains a large number of containers (103) (FIGS. 5, 6). Each container has an opening (108) at the top and is closed at the bottom edge (112). The top surface (110) is preferably elevated relative to the top surface (110) and includes ribs (104) surrounding the opening (108) of the container (103). This prevents contamination of the contents of the container (103) with droplets that may fall on the top surface (110) of the plate (101). Diagrams of preferred treatment plates are shown in Figures 3-8.

The footprint of the treated plate (101) preferably includes the length and width of the base corresponding to the ANSI SBS installation surface type. More preferably, the length is 127.76 mm +/- 0.25 mm and the width is 85.48 mm +/- 0.25 mm. Therefore, the plate (101) has two opposing short side walls (109) and two opposing long side walls (118). The processing plate (101) includes a morphological fixing element (106) for interacting with the controller (500, FIG. 12). The processing plate (101) can be quickly, safely and quickly grabbed, moved and placed while maintaining the correct orientation and position. Preferably, the morphofixing element (106) for grabbing is located within the upper central part, preferably within the upper central 1/3 of the processing plate (101). This has the advantage that the possible distortion of the treated plate (101) has only a slight effect on the morphological fixing element (106), and the handling of the plate (101) becomes more robust.

Preferably, the processing plate (101) comprises the hardware identifiers (102) and (115). The hardware identifiers (102) and (115) are unique 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 a recess (125) on the side wall of the consumable, the pattern of the ridge (119) and / or the recess (125) being specific. Type of consumables, preferably treatment 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 orientation to the appropriate stacker position on the analyzer. The sides of the treated plate (101) include guiding elements (116) and (117) (FIGS. 3 and 4). They prevent tilting of the treatment plate (101). The induction element (116, 117) allows the user to load the processing plate (101) onto the analyzer using the induction element (116, 117) as a stack, and then vertical the plate in the stacker in the apparatus without tilting the plate. Allows you to move to.

The central part (120) of the container (103) has a nearly rectangular cross section (FIGS. 6 and 7). They are separated by a common wall (113) along a nearly rectangular long side (118) (Fig. 3). The row of containers (103) thus formed has the advantage of having a large volume of preferably 4 ml at the cost of limited available space. Another advantage is that the manufacturing is very economical due to the essentially constant wall thickness. A further advantage is that the vessels (103) are reinforced with each other so that high shape stability can be obtained.

A continuous space (121) is located between the rows of containers (103) (FIGS. 6 and 7). The space (121) may accommodate a magnet (202, 203) or a heating device (128) (FIG. 11). These magnets (202, 203) and heating device (128) are preferably solid devices. Therefore, the magnetic particles (216) contained in the liquid (215) that can be retained in the container (103) will move the magnets (202, 203) proximal to the container (103) in the container (103). ) Can be separated from the liquid (215) by applying a magnetic field. Alternatively, the contents of the container (103) can be incubated at a controlled high temperature when the treatment plate (101) is placed on the heating device (128). Since the magnets (202, 203) or heating devices (128) can be solid, high energy densities can be achieved. The nearly rectangular shape of the central part (120) of the container (103) (FIG. 10) is also of the container by optimizing the contact surface between the container (103) and the magnet (202) or heating device (128). Optimized contact between the wall (109) and the flat-shaped magnet (202) or heating device (128) enhances energy transfer to the vessel (103).

In the area of the conical bottom (111) of the vessel, the space (121) is much clearer and can accommodate additional magnets (203). The combination of a large magnet (202) in the upper region of the container and a small magnet (203) in the conical region allows the separation of magnetic particles (216) in large or small amounts of liquid (215). Therefore, the small magnet (203) makes it easier to isolate the magnetic particles (216) during pipetting of the eluate. This makes it possible to pipette the eluate with minimal loss by reducing the dead capacity of the magnetic particle (216) pellets. In addition, the presence of magnetic particles (216) in the displaced eluate is minimized.

At the top of the vessel (103), one of the short sidewalls (109) of the vessel (103) includes a reagent inflow channel (105) extending to the surrounding ribs (104) (FIGS. 3, 4, 7). The reagent is pipetted into the reagent inflow channel (105) and flows from the channel (105) into the container (103). Therefore, contact between the pipette needle or tip (3, 4) and the liquid contained in the container is prevented. In addition, it prevents bounce caused by liquids that are distributed directly to another liquid (215) contained in the container (103), which can cause contamination of the pipette needle or tip (3, 4) or adjacent container (103). Is done. After a small amount of reagent, continuous pipetting of the maximum amount of another reagent into the reagent inflow channel (105) ensures that only a small amount of reagent is completely influx into the vessel (103). It becomes. Therefore, it is possible to pipette a small amount of reagent without compromising the accuracy of the tests performed.

Inside, at the bottom of the container (111, 112), the shape is conical (111) and the end has a spherical bottom (112) (FIGS. 6, 7). The internal shape (114) of the container, including the rectangular central part (120), is round. The combination of spherical bottom (112), round inner shape (114), conical part (111) and fine surface of vessel (103) facilitates effective separation and purification of the analyte in the treated plate (101). Causes favorable fluid engineering. The spherical bottom (112) allows essentially complete use of the separated eluate and reduced dead volume to reduce reagent carry-over or cross-contamination of the sample.

The base rim (129) of the processing plate (101) has a recess (107) for connection with the latch clip (124) or of the heating device (128) or analyzer (126) on the processing station (201). Includes (Figs. 5 and 9). The connection of the latch clip (124) and the recess (107) allows the installation and fixation of the processing plate (101) on the processing station (201). Due to the presence of the recess (107), a latching force approximately perpendicular to the base (129) acts on the processing plate (101). Therefore, only a small force applied sideways can occur. As a result, the occurrence of strain is reduced, so that the deformation of the processing plate (101) is reduced. The vertical latching force also negates any deformation of the processing plate (101), resulting in a more accurate placement of the spherical bottom (111) within the processing station (201). In general, the exact interface between the processing plate (101) and the processing station (201) or heating device (128) in the analyzer reduces dead capacity and further reduces the risk of cross-contamination of the sample.

A "separation station" is a device or component of an analytical system that allows the isolation of a solid support material from other substances present in a liquid sample. Such separation stations can include, but are not limited to, for example, centrifugation, racks with filter tubes, magnets or other suitable constructs. In a preferred embodiment of the invention, the separation station comprises 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, when a liquid sample and a solid support material are combined together in the wells of a multi-well plate, one or more magnets contained in the separation station come into contact with the liquid sample itself, for example by introducing the magnets into the wells. Alternatively, the one or more magnets may be brought closer to the outer wall of the well to attract the magnetic particles and then separate the magnetic particles from the surrounding liquid.

In a preferred embodiment, the separation station is a device comprising a multi-well plate comprising a container having an opening and a closed bottom on the top surface of the multi-well plate. The container comprises a top part, a center part and a bottom part, the top part being connected to the top surface of a multiwell plate, preferably containing two long sides and two short sides. The central part has a substantially rectangular cross section with two long sides and the containers are arranged in a row. Between the two adjacent rows, at least two Z positions, there is a continuous space for selective contact between at least one magnet mounted on the fixture and the side wall. 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 moving mechanism that vertically moves at least one of the fixtures, including at least one magnet, between at least the first and second positions of the multiwell plate container. Preferably, the at least two Z positions of the container include a side wall and bottom part of the container. The magnetic field of the at least one magnet preferably attracts the magnetic particles to the inner surface of the 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 comprising at least one magnet includes a frame. The container has the preferred characteristics as described above in the context of multi-well plates / treated plates. 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 to the contents of the container, such as exerting a magnetic field, or in physical contact with the container.

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

A second preferred embodiment comprising a spring that exerts pressure on a frame containing the magnet so that the magnet is pressed against the container of the multiwell plate is described below.

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

The separation station is a further method of isolating and purifying nucleic acids and is useful in the context of the present invention. The method comprises binding the nucleic acid to magnetic particles in a container of a multiwell plate. The container includes a top opening, a central part and a bottom part. If most of the liquid is located above the field where the conical part of the container replaces the central part with a rectangular shape, move the magnet from the second position to the first position, and said At position 1, by applying a magnetic field to the central part and, optionally, further magnetic field to the bottom part of the container, the bound material is then separated from the unbound material contained in the liquid. The magnetic particles can be optionally washed with a washing solution. If most of the liquid is located below the part where the conical part of the container replaces the central part having a rectangular shape, a small amount of liquid will be said 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, where the separation station is mulched to apply a magnetic field to a large amount of liquid containing the magnetic particles held in the container. It interacts with a multi-well plate container to apply a magnetic field to a first magnet constructed and arranged to interact with the well plate container, as well as a small amount of liquid containing magnetic particles held in the container. The first and second magnets may be moved to different Z positions, including a second magnet constructed and arranged in such a manner. 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 magnet (202) is constructed and arranged to fit the space (121) of the processing plate (101). Therefore, the magnet (202) applies a magnetic field to the liquid (215) in the container (103) to isolate the magnetic particles (216) inside the wall of the container. This allows the magnetic particles (216) inside the container (103) and any substance bound to it to be separated from the liquid (215) in the presence of a large amount of liquid (215). The magnet (202) has an elongated structure and is constructed and arranged to interact with the substantially rectangular central part (120) of the vessel. Therefore, the magnet (202) is used when the majority of the liquid (215) is located above the part where the conical part (111) of the container (103) replaces the rectangular central part (120). Will be done. As shown in FIG. 40, a preferred construction of the magnet (202) is a fixture (204,) comprising a magnet (202) that fits the space (121) between rows of containers (103) in the processing plate (101). 204a) is included. Another preferred embodiment of the magnet (202) comprises 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 into the space (121) of the processing plate (101). Each magnet (202, 203) is preferably constructed to interact with two vessels (103) in two adjacent rows. In a preferred embodiment, the treatment plate (101) has 6 rows of 8 containers (103). The separation station (201) capable of interacting with the preferred processing plate (101) has three fixtures (204, 204a) including a magnet (202) and four bases (205) including a magnet (203). .. Also included is an embodiment in which the separation station has four magnetic fixtures (204, 204a) including a magnet (202) and three magnetic bases (205) including a magnet (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) are interconnected by a base (217) and therefore move in concert. All magnets (203) are connected 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 types of magnetic plates (202, 203) to a total of four terminal positions.

In FIGS. 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 the separation position. In this figure, the magnet (202) is placed at the lowest position. When these are in this position, the magnets are not involved in the separation.

In a preferred embodiment shown in FIG. 10, the base (217) of the magnet (202) is connected to the positioning wheel (206). The base (217) includes a bottom end (207) that flexibly contacts the connecting element (208) by the moving element (209). The moving elements are constructed and arranged to move the connecting element (208) from one side to the other along the rail (212). The moving element (209) is fixed to the connecting element (208) with a pin (220). The connecting element (208) is fixed to the positioning wheel (206) with a screw (210). The connecting element (208) is also connected to the axis (211). The connecting element (208) is preferably a rectangular plate. The positioning wheel (206) moves eccentrically around the axis (211), so that the screw (210) moves and moves from a point above the eccentric axis to a point below the eccentric axis. The bottom end (207) of the base (204) to which the element (209) and magnet (202) are attached is moved from the highest position to the lowest position. The base (218) rests on the bottom part (219) and at its lower end is connected to the moving element (214), preferably the wheel, by a pin (213) and interacts with the positioning wheel (206). If the positioning wheel (206) rotates around the axis (211), the wheel (214) moves along the positioning wheel (206). If the wheel (214) is located where the distance between the positioning wheel (206) and the shaft (211) is short, then the magnet (203) is at its lowest position. If the wheel (214) is located where the distance between the positioning wheel (206) and the shaft (211) is maximum, then the magnet (203) is at its highest position. Therefore, in a preferred embodiment of the first aspect of the separation station, the position of the magnet (203) is controlled by the shape of the positioning wheel (206). When the moving element (209) moves along the central round upper or lower part (212a) of the rail (212), the small magnet (203) moves up and down. If 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. This positioning wheel can be rotated by any motor (224).

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

As used herein, the term "pin" relates to any fixing element, including screws or pins.

In a second preferred embodiment, the separation station (230) is at least one fixture containing at least one magnet (232), preferably a large number of magnets as many as the number of containers (103) in the row (123). Includes (231). Preferably, the separation station (230) comprises a large number of fixtures (231) as many as the number of rows (123) of the multiwell plates (101) described above. More preferably, six fixtures (231) are placed on the separation station (230). At least one magnet (232) is placed 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 placed on one side towards a container that interacts with the magnet.

The fixture (231) rests on the base (233). Preferably, the loading is flexible. The base (233) includes a spring (234) resting on it. The number of springs (234) is at least one per fixture (231) mounted on the base (233). The base further includes a round groove (236) that limits the movement of the spring and, as a result, the movement of the fixture (231), including the magnet (232). Preferably, any one of the springs (234) is constructed and arranged to interact with the fixture (231). More preferably, the spring (234) is a yoke spring. The interaction controls the horizontal movement of the fixture (231). In addition, the separation station (230) includes a frame (235). The base (233) having the fixture (231) is connected to the frame (235) by the moving mechanism described above for the magnet (232) of the first aspect.

Preferably, the base (233) and 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 brings the magnet (232) mounted on the fixture (231) into contact with the container (103). The Z position is selected depending on the volume of liquid (215) in the container (103). In large quantities, the magnet (232) contacts the container (103) at the central position (120) where the container (103) is approximately rectangular. For 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 the conical part (111) of the container (103). Contact with.

A spring is attached to the base (233) of any one frame (231) (Fig. 9a, 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 containers are treated the same. To 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) and magnetic particles (215) contained in the multi-well plate (101) described above are contained. Especially suitable for separation of 216).

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

Cleaning in the methods of the invention requires somewhat strong contact between the solid support material and the nucleic acids immobilized therein and the cleaning buffer. This can be achieved in a variety of ways, including shaking the wash buffer and solid support material in or with one or more of each container. Another advantageous method is to aspirate and dispense the suspension containing the wash buffer and solid support material at least once. This method is preferably performed using a pipette, wherein the pipette preferably comprises a disposable pipette tip that aspirates and redistributes the 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, still more preferably about 1 ml. The pipette used in the context of the present invention can be a pipetting needle.

Therefore, a preferred aspect of the present invention is the method described above, wherein the cleaning in step d comprises suctioning and distributing a cleaning buffer containing a solid support material.

For subsequent 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 method described above, wherein the method further comprises the step of washing the solid support material and then eluting the purified nucleic acid from the solid support material with an elution buffer in step d.

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

Preferably, since elution is carried out at a high temperature, a preferred embodiment of the present invention is the method described above in which step d is carried out at a temperature of 70 ° C. to 90 ° C., more preferably 80 ° C.

An "amplifying reagent" in the context of the present invention is a chemical or biochemical component that allows amplification of nucleic acids. 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 is 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.

A "nucleotide" is a nucleoside that further comprises a phosphate group covalently attached to the sugar moiety of the nucleoside. For these nucleosides, including pentoflanosyl sugars, the phosphate group can be attached to either the 2'-, 3'- or 5'-hydroxyl moiety of the sugar. Nucleotides are monomeric units of "oligonucleotides" that are more commonly referred to as "oligomer compounds" or "polynucleotides" and more commonly referred to as "polymer compounds". Another common expression mentioned above is deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

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

"Oligonucleotides" and "modified oligonucleotides" (or "oligonucleotide analogs") are subgroups of oligomeric compounds. In the context of the present invention, the term "oligonucleotide" refers to a component formed of a plurality of nucleotides as its monomer unit. A phosphate group generally refers to an oligonucleotide that forms a backbone between nucleosides. The usual binding or backbone of RNA and DNA is a 3'-5'phosphodiester bond. Oligonucleotides and modified oligonucleotides useful in the present invention (see below) are primarily described in the art and can be synthesized as known to those of skill in the art. Methods for preparing oligomeric compounds with specific sequences are known in the art and include, for example, cloning and restriction of appropriate sequences as well as direct chemical synthesis. Examples of the chemical synthesis method include the phosphodiester 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. Phosphodiester method disclosed, Beaucage et al., Tetrahedron Letters 22 (1981) Phosphoramidite method disclosed in 1859, Garegg et al., Chem. Scr. 25 (1985) H- disclosed in 280-282. Phosphonate methods and solid support methods disclosed in US 4,458,066 can be mentioned.

In the methods described above, oligonucleotides can be chemically modified, i.e. primers and / or probes include modified nucleotides or non-nucleotide compounds. The probe or primer is then a modified oligonucleotide.

"Modified nucleotides" (or "nucleotide analogs") differ from natural nucleotides due to some modifications, but are still bases, pentofranosyl sugars, phosphate moieties, base-like moieties, pentoflanos. It consists of sylsaccharide-like moieties and phosphate-like moieties or combinations thereof. For example, if the label can be attached to the base portion of the nucleotide, a modified nucleotide is obtained. If the natural base in the nucleotide can also be replaced, for example with 7-deazapurine, a modified nucleotide is obtained as well.

"Modified oligonucleotides" (or "oligonucleotide analogs") that belong to another particular 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 like an oligonucleotide and can be used interchangeably in the context of the present invention. From a synthetic point of view, modified oligonucleotides (or oligonucleotide analogs) can be made, for example, by chemical modification of the oligonucleotide by appropriate modification of the phosphate backbone, ribose units or nucleotide bases (Uhlmann and Peyman, Chemical Reviews 90 (Uhlmann and Peyman, Chemical Reviews 90). 1990) 543; Verma S., and Eckstein F., Annu. Rev. Biochem. 67 (1998) 99-134). Illustrative modifications include phosphorothioate, phosphorodithioate, methylphosphonate, phosphortriester or phosphoramidate nucleoside linkages in place of phosphodiester nucleoside linkages; deaza- or azapurines in place of natural purines and pyrimidine bases. And-Pyrimidine, a pyrimidine base with a substituent at the 5- or 6-position; a purine base with a substituent at the 2, 6 or 8-position or modified to the 7-position as 7-deazapurin; alkyl-, alkenyl-, alkynyl-or Aryl moieties such as methyl, ethyl, propyl, butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl groups and other lower alkyl groups or bases with aryl groups such as phenyl, benzyl, naphthyl; eg, part 2 Sugars having a substituent at the'position'; or carbocyclic or acyclic sugar analogs. Other modifications in line with the spirit of the invention are known to those of skill in the art. It is best described that such modified oligonucleotides (or oligonucleotide analogs) are functionally interchangeable with natural oligonucleotides but structurally different. More specifically, exemplary modifications are disclosed in Verma S., and Eckstein F., Annu. Rev. Biochem. 67 (1998) 99-134 or WO 02/12263. In addition, modifications can be made to link the nucleoside units via a group that is an alternative to the phosphate bond between nucleosides or the sugar phosphate bond. Such bindings include those disclosed in Verma S., and Eckstein F., Annu. Rev. Biochem. 67 (1998) 99-134. When a nucleoside unit is bound using a method other than a phosphate bond, such a structure is also described as "oligonucleoside".

"Nucleic acid" and "target nucleic acid" are polymer compounds of nucleotides known to those of skill in the art. A "target nucleic acid" is used herein to indicate a nucleic acid in a sample that should be analyzed, i.e., its presence in the sample, its absence and / or its amount in the sample.

The term "primer", as used herein, as known to those skilled in the art, refers to oligomeric compounds, primarily oligonucleotides, which can induce DNA synthesis by template-dependent DNA polymerase, eg, for example. The 3'end of the primer provides a free 3'-OH group to which additional nucleotides can be attached by a template-dependent DNA polymerase that establishes a 3'- to 5'-phosphodiester bond, thereby deoxynucleoside. It also refers to a modified oligonucleotide in which triphosphate is used to release pyrophosphate.

"Probe" also refers to a natural or modified oligonucleotide. As is known in the art, probes act for the purpose of detecting analytes or amplifications. 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 nucleic acids, especially oligonucleotides or modified oligonucleotides, and any nucleic acid attached to them from the rest of the sample (labeling). Nucleic acids bound to are also referred to as labeled nucleic acid binding compounds, labeled probes or simply probes). The preferred label of the present invention is a fluorescent label which is a fluorescent dye such as a fluorosane dye, a rhodamine dye, a cyanine dye and a coumarin dye. 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 can be chemically modified, i.e. 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 US Pat. Nos. 4,683,202, 4,683,195, 4,800,159 and 4,965,188, among other references. PCR typically uses 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 starting points for nucleic acid synthesis in the nucleic acid sequence of the target nucleic acid. Primers can be purified from the restricted digest by conventional methods or can be made synthetically. Preferably, the primer is single-stranded for maximum efficiency of amplification, but the primer can be double-stranded. Double-stranded primers are first denatured, i.e. treated to separate the strands. One of the methods for denaturing double-stranded nucleic acids is heating. A "heat-stable polymerase" is a heat-stable polymerase enzyme that catalyzes the formation of primer extension products complementary to the template and is exposed to high temperatures for the time required to cause denaturation of the double-stranded template nucleic acid. It is an enzyme that does not irreversibly denature when it is used. In general, synthesis begins at the 3'end of each primer and proceeds in the 5'-3' direction along the template strand. Thermostable polymerases include, for example, Tharma flavus, T. ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillus stearothermophilus and Methanothermus fervidus. ) Was isolated. Nevertheless, non-thermostable polymerases can also be used in the PCR assays provided when supplementing the enzyme.

If the template nucleic acid is double-stranded, the two strands must be separated before they can be used as a template in PCR. Chain separation can be achieved by any suitable denaturing method, such as physical, chemical or enzymatic means. One method of separating nucleic acid strands is to heat the nucleic acid until it is largely denatured (eg, denatured above 50%, 60%, 70%, 80%, 90% or 95%). Including. The heating conditions required for denaturation of the template nucleic acid depend on, for example, buffer salt concentration and the length and nucleotide composition of the nucleic acid to be denatured, but typically time depending on the characteristics of the reaction such as temperature and nucleic acid length, about 90. It is in the range of ° C to about 105 ° C. Denaturation typically takes about 5 seconds to 9 minutes. It is preferable to use a short denaturation step to prevent loss of functional enzymes by exposing each polymerase, such as Z05 DNA polymerase, to a fairly high temperature for extended periods of time.

In a preferred embodiment of the invention, the modification step is up to 30 seconds, more preferably up to 20 seconds, even more preferably up to 10 seconds, even 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 about 35 ° C to about 70 ° C, more preferably about 45 ° C to about 65 ° C, still more preferably about 50 ° C to about 60 ° C, still more preferably about 55 ° C to about 58 ° C. is there. The annealing time can be from about 10 seconds to about 1 minute (eg, about 20 seconds to about 50 seconds, 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, primers can also bind to targets with one mismatch, so variants of a particular sequence can also be amplified. 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, a relatively high annealing temperature has the advantage of providing higher specificity, as it continuously reduces the likelihood that the primers will bind to target sequences that do not exactly match at higher temperatures. In order to enjoy the benefits of both phenomena, in some embodiments of the invention, the methods described above preferably include 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, inaccurate target sequences can be amplified (first). This can be followed, for example, at 58 ° C. for about 45 cycles, resulting in higher specificity throughout the main part of the experiment. This method does not lose potentially important genetic variants and maintains relatively high specificity.

The reaction mixture is then adjusted to a temperature at which the activity of the polymerase is promoted or optimized, i.e. enough 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 the extension product from each primer annealed to the nucleic acid template, but not high enough to denature the extension product from the complementary template (eg, of extension). The temperature is generally in the range of about 40 ° C to 80 ° C (eg, about 50 ° C to about 70 ° C; about 60 ° C). The elongation time is about 10 seconds to about 5 minutes, preferably about 15 seconds to. It can be 2 minutes, more preferably about 20 seconds to about 1 minute, even more preferably about 25 seconds to about 35 seconds. The newly synthesized strand contains double-stranded molecules that can be used in the next step of the reaction. The steps of chain 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 limiting factor of the reaction is present during the reaction. The amount of primer, thermostabilizing enzyme and nucleoside triphosphate. The cycle step (ie, denaturation, annealing and elongation) is preferably repeated at least once. For use in detection, the number of cycle steps is eg. Depends on the nature of the sample. If the sample is a complex mixture of nucleic acids, more cycle steps are required to amplify the target sequence sufficient for detection. Generally, the cycle steps are at least about about. It is repeated 20 times, but may be repeated 40, 60 or even 100 times.

Within the scope of the present invention, PCR can be performed in the same step of annealing and extension (one-step PCR), or in separate steps as described above (two-step PCR). Performing annealing and elongation together under the same physicochemical conditions with a suitable enzyme, such as Z05 DNA polymerase, saves time for further steps in each cycle, and further annealing and elongation. It has the advantage of eliminating the need for further temperature control during. Therefore, one-step PCR reduces the overall complexity of each assay.

In general, shorter times are preferred for the entire amplification, as it reduces the time to result and allows for faster diagnosis.

Other preferred nucleic acid amplification methods used in the context of the present invention are the 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) ), 3SR (Kwoh DY et al., Proc. Natl. Acad. Sci. USA 86 (1989) 1173-1177; Guatelli JC, et al., Proc. Natl. Acad. Sci. USA 87 (1990) 1874-1878 Includes WO 92/08808) and NASBA (US 5,130,238). In addition, there are strand substitution amplification (SDA), transcription mediation amplification (TMA), and Qb amplification (for overview, eg Whelen AC and Persing DH, Annu. Rev. Microbiol. 50 (1996) 349-373; Abramson RD and Myers. See TW, Curr Opin Biotechnol 4 (1993) 41-47).

The internal control nucleic acid used in the present invention preferably relates to the following:
--55 ° C to 90 ° C, more preferably 65 ° C to 85 ° C, more preferably 70 ° C to 80 ° C, most preferably about 75 ° C melting temperature
--Up to 500 bases or base pairs, more preferably 50-300 bases or base pairs, more preferably 100-200 bases or base pairs, most preferably about 180 bases or base pairs in length.
-Exhibits properties of 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 major structure of a nucleic acid, i.e. a specific sequence of a single nucleobase that constitutes each nucleic acid. It should be understood that the term "sequence" does not specify a particular type of nucleic acid, such as RNA or DNA, but applies to both and other types of nucleic acids, such as PNA or others. When the nucleobases correspond to each other, especially in the case of uracil (present in RNA) and thymine (present in DNA), these bases are located 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), gonococci (NG). ) And others, etc. 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 method described above in which the internal control nucleic acid is DNA.

On the other hand, many nucleic acids related to clinical diagnosis are ribonucleic acid, such as human immunodeficiency virus (HIV), hepatitis C virus (HCV), West Nile virus (WNV), human papillomavirus (HPV), and Japanese encephalitis virus (Japanese encephalitis virus). JEV), St. Louis encephalitis virus (SLEV) and others, such as RNA-derived nucleic acids. The present invention can be readily 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 manner described above, the internal control nucleic acid preferably mimics the most sensitive target of the assay involving a large number of targets, and the RNA target usually needs to be more closely controlled. Therefore, the internal control nucleic acid is preferably RNA.

Therefore, a preferred aspect of the present invention is the method described above in which the internal control nucleic acid is RNA.

Since RNA is more prone to degradation than DNA due to the effects of, for example, alkaline pH, ribonuclease, etc., internal control nucleic acids made from RNA are preferably provided as armored particles. In particular, exterior particles such as exterior RNA are described, for example, in EP910643. Briefly, chemically, or preferably RNA that can be heterologously produced by, for example, a bacterium, such as E. coli, is at least partially encapsulated in a virus-coated protein. The latter confer resistance to RNA against external effects, especially ribonucleases. It must be understood that internal control DNA can also be provided as exterior particles. Both outer RNA and outer 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 covered with MS2-coated protein in E. coli. In a more preferred embodiment, the DNA control nucleic acid is externalized using λ phage GT11.

Therefore, a preferred aspect of the present invention is the method described above in which the internal control nucleic acid is an outer nucleic acid.

Typically, in amplification system nucleic acid diagnosis, RNA templates are transcribed into DNA prior to amplification and detection.

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

A "polymerase having reverse transcriptase activity" is a nucleic acid polymerase capable of synthesizing DNA based on an RNA template. It can also form double-stranded DNA once RNA has been reverse transcribed into a 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 invention comprises a sample containing an RNA template with an oligonucleotide primer that is sufficiently complementary to hybridize to the RNA template, and preferably a thermostable DNA polymerase, at least a total of four types. In the presence of natural or modified deoxyribonucleoside triphosphate, the step of incubating in a suitable buffer comprising a metal ion buffer that buffers both pH and metal ion concentration in a preferred embodiment is included. This incubation catalyzes the primer hybridizing to the RNA template and the DNA polymerase to polymerize the deoxyribonucleoside triphosphate to form a cDNA sequence complementary to the sequence of the RNA template. It is carried out at a sufficient temperature.

As used herein, the term "cDNA" refers to complementary DNA molecules synthesized using ribonucleic acid strands (RNAs) as templates. RNA can be another form of RNA, such as mRNA, tRNA, rRNA, or viral RNA. The cDNA can be single-stranded, double-stranded, or hydrogen-bonded to complementary RNA molecules when present in an RNA / cDNA hybrid.

Primers suitable for annealing to RNA templates 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 extension product synthesis.

In the amplification of RNA molecules by DNA polymerase, the first extension reaction is reverse transcription using an RNA template, which produces DNA strands. A second extension reaction using a DNA template produces a double-stranded DNA molecule. Therefore, the synthesis of complementary DNA strands from RNA templates by DNA polymerase provides an initiator for amplification.

A thermostable DNA polymerase can be used in the combined one-enzyme reverse transcription / amplification reaction. The term "homogeneous" in this context refers to a two-step single addition reaction for reverse transcription and amplification of RNA targets. Homogeneity means that it is not necessary to open the reaction vessel after the reverse transcription (RT) step or otherwise adjust the reaction components before the amplification step. In a non-homogeneous RT / PCR reaction, one or more of the reaction components, such as amplification reagents, are prepared, added or diluted, for example, after reverse transcription and prior to amplification, which requires opening the reaction vessel or at least. You need to manipulate that component. Both homogeneous and non-homogeneous aspects are within the scope of the invention, but homogeneous forms are preferred for RT / PCR.

Reverse transcription is an important step in RT / PCR. For example, in the art, RNA templates are known to exhibit a tendency to form secondary structures that can impair primer binding and / or extension of cDNA strands by their respective reverse transcriptases. Therefore, relatively high temperatures are advantageous for RT reactions for transcription efficiency. On the other hand, an increase in incubation temperature also means higher specificity, i.e., RT primers do not anneal to sequences that show a mismatch for one or more expected sequences. In particular, in the case of a large number of different target RNAs, for example if unknown or rare substrains or variants can be present in the liquid sample, the sequence with a single mismatch is transcribed and then amplified. , It may also be desirable to detect.

It is preferred to perform RT incubation at more than one different temperature in order to benefit from both of the above, namely reduction of secondary structure and reverse transcription of the template with mismatch.

Therefore, a preferred aspect of the present invention is that the incubation of the polymerase having reverse transcriptase activity is carried out at different temperatures of 30 ° C. to 75 ° C., preferably 45 ° C. to 70 ° C., more preferably 55 ° C. to 65 ° C. The method.

As a more important aspect of reverse transcription, a long RT step can damage the DNA template that may be present in the liquid sample. Therefore, if the liquid sample contains both RNA and DNA species, the duration of the RT process should be kept as short as possible, while at the same time sufficient amount of cDNA for subsequent amplification and any detection of the amplification. It is preferable to ensure the synthesis of.

Therefore, a preferred aspect of the present invention is the method described above, 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.

A more preferred aspect of the present invention is that a polymerase having reverse transcriptase activity and containing mutations
a) CS5 DNA polymerase
b) CS6 DNA polymerase
c) Thermotogamalitima DNA polymerase
d) Tharma Aquaticus DNA Polymerase
e) Tharma Thermophilus DNA Polymerase
f) Tharma flavus DNA polymerase
g) Thermus filiformis DNA polymerase
h) Tharma species (sp.) Sps17 DNA polymerase
i) Tharma species (sp.) Z05 DNA polymerase
j) Thermotoganea polytana DNA polymerase
k) Thermoscipio Africanus DNA polymerase
l) The method described above selected from the group consisting of Thermus caldophilus DNA polymerase.

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

Therefore, a preferred aspect of the present invention is a polymerase containing a mutation in which a polymerase having reverse transcriptase activity imparts an increase in nucleic acid elongation rate and / or an increase in reverse transcriptase activity as compared to each wild-type polymerase. This is the method described above.

In a more preferred embodiment, in the methods described above, the polymerase having reverse transcriptase activity is a polymerase comprising a mutation that imparts improved reverse transcriptase activity as compared to the respective wild-type polymerase.

Polymerases with point mutations that make them 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 containing TGRLSSX _b7 -X _b8 -PNLQN, in which X _b7 is an amino acid selected from S or T and X _b8 is selected from G, T, R, K or L. Amino acids, wherein the polymerase contains 3'-5'exonuclease activity, has improved nucleic acid elongation rates and / or improved reverse transcription efficiency compared to wild-type DNA polymerase, said. In wild-type DNA polymerase, X _b8 is an amino acid selected from D, E or N.

One particularly preferred example is a variant of the thermostable DNA polymerase from Thermus species Z05 (eg, described in US 5,455,170), the variant being mutated in the polymerase domain as 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 invention.

For reverse transcription using a thermostable polymerase, Mn2 + is preferred as a divalent cation, typically as a salt such as manganese chloride (MnCl2), manganese acetate (Mn (OAc) 2) or manganese sulfate (MnSO4). included. MnCl2 is included in reactions involving a 50 mM tricin buffer, for example MnCl2 is generally present at concentrations of 0.5-7.0 mM, 0.8-1.4 when 200 mM dGTP, dATP, dUTP and dCTP are used, respectively. mM is preferable, and MnCl2 of 2.5 to 3.5 mM is most preferable. 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 the cDNA and DNA can be used for subsequent amplification, the above-mentioned method with internal control was taken. , Is particularly useful for the simultaneous amplification of target nucleic acids from both organisms with an RNA genome or organisms with 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 the method described above in which at least two target nucleic acids contain RNA and DNA.

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

Enzymes such as Tth polymerase or preferably the mutant Z05 DNA polymerase described above are suitable for carrying out the step of amplification after the target nucleic acid, especially for suitable temperature optimum conditions. 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 ease of implementation 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 the mutant Z05 DNA polymerase described above.

In a preferred embodiment, steps above 90 ° C. are up to 20 seconds, preferably up to 15 seconds, more preferably, so that the polymerase or other components of the reaction mixture used in the context of the present invention are not exposed to high temperatures for longer than necessary. Is up to 10 seconds, more preferably up to 5 seconds, most preferably 5 seconds long. This also reduces the time to results and reduces the total time required for the assay.

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

Therefore, a preferred aspect of the invention is the method described above, which further comprises the step of sealing at least two reaction vessels after step e.

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

As a result, a preferred aspect of the invention is the method described above in which at least two reaction vessels are aligned into the same integrated sequence.

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

The target of the amplification step can be an RNA / DNA hybrid molecule. The target can be a single or double strand nucleic acid. The most widely used PCR technique uses double-stranded targets, but this is not essential. After the first amplification cycle of the single-stranded DNA target, the reaction mixture comprises a double-stranded DNA molecule 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 continuous cycle of amplification proceeds as described above.

Nucleic acid amplification is very effective not only in the case of PCR but also when it is carried out as a cycle reaction. Therefore, a preferable aspect of the present invention is the above-mentioned method in which the amplification reaction in step f consists of a multiple cycle step. is there.

Suitable nucleic acid detection methods are known to those of skill in the art and are known to those of skill in the art, Sambrook J. et al., Molecular Cloning: A Laboratory Manual, ColdSpring HarborLaboratory Press, Cold Spring Harbor, New York, 1989 and Ausubel F. et al .: Current Protocols. It is found 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 a precipitation step. Detection methods include, but are not limited to, binding or intercalation of specific dyes such as ethidium bromide that intercalate into double-stranded DNA and then alter its fluorescence. The purified nucleic acid can also be optionally separated by electrophoresis after restriction digestion and then visualized. There are also probe system assays that 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 targeted nucleic acid amplified during or after the amplification reaction. It is advantageous to use nucleic acid probes, especially for real-time detection.

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

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

Therefore, a preferred aspect of the present invention is the method described above in which the probe is labeled 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 fluorescence moiety and the acceptor fluorescence moiety. A typical donor fluorescent moiety is fluorescein and typical corresponding acceptor fluorescent moieties include LC-Red 640, LC-Red 705, Cy5 and Cy5.5. Detection typically involves excitation of the sample at a wavelength that is absorbed by the donor fluorescence moiety, and visualization and / or measurement of the wavelength that is emitted by the corresponding acceptor fluorescence moiety. In the methods 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 done in real time. By using a commercially available real-time PCR device (eg, LightCycler ^TM or TaqMan®), PCR amplification and detection of amplification products are combined in a single closed cuvette with dramatically reduced cycle time. be able to. Since 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 travel time and is an attractive alternative to traditional PCR techniques in clinical laboratories.

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

TaqMan® technology uses a single-stranded hybridization probe labeled with two fluorescent moieties. When the first fluorescent portion is excited by light of the appropriate wavelength, the absorbed energy is transferred to the second fluorescent portion according to the FRET principle. 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 (ie, amplification product) and during the subsequent elongation phase another known to those skilled in the art, such as Taq or the preferred mutant Z05 polymerase. Degraded by the 5'-3'exonuclease activity of the appropriate polymerase. As a result, the excited fluorescent and quenching moieties are spatially separated from each other. As a result, when the first fluorescent portion is excited in the absence of the quencher, fluorescence emission from the first fluorescent portion can be detected.

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

As an alternative to FRET, amplification products can be detected using double-stranded DNA-binding dyes such as fluorescent DNA-binding dyes (eg, SYBR GREEN I® or SYBR GOLD® (Molecular Probes)). Upon interaction with a double-stranded nucleic acid, such a fluorescent DNA-binding dye emits a fluorescent signal after being excited by light of an appropriate wavelength. Double-stranded DNA-binding dyes such as nucleic acid intercalate dyes can also be used. When double-stranded DNA-binding dyes are used, melting curve analysis is usually performed to confirm the presence of amplification products.

Molecular beacons bound to FRET can also be used to detect the presence of amplification products using the real-time PCR method of the present invention. Molecular beacon technology uses hybridization probes labeled with a first fluorescence moiety and a second fluorescence moiety. The second fluorescent moiety is generally a quencher and a fluorescent label is typically placed at each end of the probe. Molecular beacon technology uses probe oligonucleotides with sequences that allow secondary structure formation (eg, hairpins). When the probe is in solution, both fluorescent moieties are spatially proximal as a result of the formation of secondary structure within the probe. After hybridization to the amplification product, the secondary structure of the probe is disrupted and the fluorescent moieties are separated from each other so that emission of the first fluorescent moiety can be detected after excitation with light of the appropriate wavelength.

Therefore, the preferred method of the invention comprises a nucleic acid sequence in which the probe allows for secondary structure formation, which results in spatial proximity between the first and second fluorescent moieties. , The above method using FRET.

Efficient FRET can only occur if the fluorescence moiety is directly proximal and if the emission spectrum of the donor fluorescence moiety overlaps the absorption spectrum of the acceptor fluorescence moiety.

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

In a more preferred embodiment, the acceptor fluorescent portion is a quencher.

As mentioned above, in the TaqMan format, during the annealing step of the PCR reaction, the labeled hybridization probe binds to the target nucleic acid (ie, amplification product), and during the subsequent elongation phase, such as Taq or the preferred mutant Z05 polymerase. It is degraded by the 5'-3'exonuclease activity of another suitable polymerase known to the vendor.

Therefore, in a preferred embodiment, in the methods described above, amplification uses a polymerase enzyme with 5'-3'exonuclease activity.

It is even more advantageous to carefully select the length of the amplicon that results from the method described above. In general, relatively short amplicons increase the efficiency of the amplification reaction. Therefore, a preferred aspect of the present invention is the method described above, wherein the amplified fragment comprises up to 450 bases, preferably up to 300 bases, more preferably up to 200 bases, even more preferably up to 150 bases.

The internal control nucleic acid used in the present invention can serve as a quantification, a reference for determining the amount of target nucleic acid, and as a "quantitative standard nucleic acid" used. To this end, one or more quantitative standard nucleic acids, along 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 within the same reaction mixture. This must generate a detectable signal, either directly or indirectly, in the presence or 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, for example to produce a detectable signal even at very high target concentrations, although it does not compromise sensitivity. .. For the detection limit of each assay (LOD, see below), the concentration range of the "quantitative standard nucleic acid" is preferably 20-5000x LOD, more preferably 20-100x 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.

"Detection limit" or "LOD" means the minimum detectable amount or concentration of nucleic acid in a sample. A low "LOD" corresponds to high sensitivity, and a high "LOD" corresponds to low sensitivity. "LOD" is usually expressed in units of "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 of each nucleic acid (copy). 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 of analyzing the relationship between stimulus (dose) and discontinuous (all or no) responses. 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 associated with a log-dose as a cumulative normal distribution. That is, the logarithmic dose can be used as a variable for reading mortality from the cumulative standard. Using the standard distribution rather than the other probability distributions affects the estimated response rate at the high and low end of possible doses, but has little effect near the center.

Probit analysis can be applied with a separate "hit rate". As is known in the art, the "hit rate" is generally expressed as a percentage [%] and indicates the percentage of positive results at a particular concentration of the 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% sure result is positive.

In a preferred embodiment, the method described above is 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. LOD is provided.

For some examples of possible target nucleic acids from a particular virus, the methods described above preferably provide 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-HBV: Up to 10IU / ml, more preferably up to 7.5IU / ml, more preferably up to 5IU / ml-HCV: up to 10IU / ml, more preferably up to 7.5IU / ml, more preferably up to 5IU / ml-WNVI: 20cp / Up to ml, more preferably up to 15 cp / ml, more preferably up to 10 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 100cp / ml, more preferably up to 75cp / ml, more preferably up to 50cp / ml, more preferably up to 30cp / ml · SLEV: up to 100cp / ml, more preferably up to 75cp / ml, more preferably up to 50cp Up to / ml, more preferably up to 25 cp / ml, more preferably up to 10 cp / ml.

An example of how to calculate quantitative results in the TaqMan format based on an internal control nucleic acid that acts as a quantitative standard nucleic acid is described below: from instrument-corrected fluorescence input data from all PCR runs. Calculate the titer. A set of samples containing a target nucleic acid and an internal control nucleic acid that acts as a quantitative standard nucleic acid is subjected to PCR in a thermal cycler using a specific temperature profile. Samples are irradiated with filtered light at selected temperatures and times in the PCR profile, and filtered fluorescence data is collected for each sample for target and internal control nucleic acids. After completion of the PCR run, fluorescence readings are processed to yield 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 concentration data is processed in the same way. After several plausibility checks, elbow values (CT) are calculated for the internal control nucleic acid and the target nucleic acid. The point at which the fluorescence of the target nucleic acid or the internal control nucleic acid intersects a predetermined threshold (fluorescence concentration) is defined as an elbow value. The titer determination is that the target nucleic acid and the internal control nucleic acid are amplified with the same efficiency and that the calculated elbow value amplifies and detects equal amounts of amplicon copy of the target nucleic acid and the internal control nucleic acid. Based on assumptions. Therefore, (CTQS-CT target) is linear with respect to log (target concentration / QS concentration). In this context, QS refers to an internal control nucleic acid that acts as a quantitative standard nucleic acid. Then the titer T is, for example, the following equation:
T'= 10 (a (CTQS-CT target) 2 + b (CTQS-CT target) + c)
Can be calculated using a multinomial calibration formula such as.

Since the constants of the polynomial 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, the internal control nucleic acid can function as a "qualitative internal control nucleic acid". "Qualitative internal control nucleic acids" are particularly useful for validating the 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 considered inactive. However, in a qualitative setting, if the result is positive, the qualitative internal control does not necessarily have to be detected. As a result, its concentration should be relatively low. Definite internal controls need to be carefully adapted to their respective assays and sensitivities. Preferably, the concentration range of the qualitative internal nucleic acid, i.e. the second control nucleic acid, comprises the range of 1 copy per reaction to 1000 copies per reaction. For the detection limit (LOD) of each assay, its concentration is preferably between the LOD and 25 times the LOD of the assay, more preferably between the LOD and 10x LOD. More preferably, this is between 2x and 10x LOD. More preferably, this is a 5x to 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 possible to use only one of them for amplification, for example by adding only primers for one of the internal control nucleic acids. Can be advantageous. In such an embodiment, the internal control nucleic acid amplified in a particular experiment can be selected by one of ordinary skill in the art, thus increasing the flexibility of the analysis performed. In a particularly advantageous embodiment, the different internal control nucleic acids may be included by a single nucleic acid construct, eg, a plasmid or a different suitable nucleic acid molecule.

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

The above results may be of poor quality 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 amplifications of the previous experiments contribute to such undesired 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, as well as enzymatic treatment and / or physicochemical treatment of the carryover product to prevent the product DNA from serving as a template for subsequent amplification. Includes exposure to treatment. Enzymes for such treatments 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 contamination PCR product and functions to "sterilize" its amplification reaction.

Therefore, a preferred aspect of the present invention is between step d and step e.
-The step of treating a liquid sample with an enzyme under the condition that the product derived from the amplification of cross-contaminated nucleic acid derived from another sample is enzymatically decomposed;
-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 steps are automated. "Automation" means that the steps of the method are suitable for being carried out by a device or machine that can operate with little or no external control or human influence. Only the preparation step of the method may need to be done manually, for example the storage container needs to be filled and placed in place, the sample selection needs to be done by humans and is known to those skilled in the art. Further steps, such as the operation of the control computer, need to be performed by humans. The device or machine may, for example, automatically add liquids, mix samples, or perform an incubation step at a particular temperature. Typically, such machines or devices are robotically controlled by a computer that executes a program that identifies one process group and command group.

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

An "analytical 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 analytical system are the same as those described above for the methods of the invention.

The analytical system of the present invention (440, FIG. 11) is a system (440) that includes a module (401) for isolating and / or purifying the analyte. In addition, the system (440) further includes a module (403) for analyzing the analyte to obtain a detectable signal. Detectable signals can be detected in the same module (401, 402, 403) or, in alternative, in separate modules. The term "module" as used herein refers to any spatially defined location within 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 the other module. Preferably, all modules are centrally controlled. Transfers between modules (401, 402, 403) can be manual, but preferably automated. Therefore, many different aspects of the automated analyzer (400) are included in the present invention.

The "separation station" is described above.

The "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 on which the reaction for analysis of the sample such as PCR takes place. The outer boundary or wall of such a container is chemically inert so as not to interfere with the amplification reaction occurring therein. For ease of handling and for ease of automation, it is preferred that at least two reaction vessels can be combined in an integrated array and operated together.

As a result, a preferred aspect of the present invention is the analytical system described above in which at least two reaction vessels are combined in an integrated sequence.

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

Preferably, the multi-well plate is held in a holding station. In a more preferred embodiment, one maneuver transfers the multiwell vessel from the holding station to the airlock (460), and a second maneuver transfers the multiwell plate from the airlock to the amplification station. Both controls interact with the multiwell plate through morphologically fixed interactions.

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

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

In the second aspect, the purified target nucleic acid is transferred from the separation station to the amplification station. Preferably, a pipette containing a pipette with a pipette tip is used to transfer the liquid containing the purified nucleic acid.

In a third embodiment, the purified nucleic acid is transferred from the separation station to a reaction vessel in an integrated sequence held in a retention station. Preferably, the reaction vessel in the integrated sequence is then transferred from the retention station to the amplification station.

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

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

For this purpose, the analytical system of the present invention preferably further comprises a transfer unit, the transfer unit preferably comprising a robotic device, the device preferably including a maneuver.

For the reasons mentioned above in the context of the methods of the invention, the following are more preferred aspects of the invention:
-The above-mentioned analytical system (440) in which at least one reaction vessel contains RNA target nucleic acid and DNA target nucleic acid.
The above-mentioned analytical system (440), wherein at least one reaction vessel contains RNA target nucleic acid and at least one other reaction vessel contains DNA target nucleic acid.

Preferably, the analytical system (440) described above is:
-Detection module (403) for detecting signals induced by the analyte
・ Sealer (410)
Storage module for reagents and / or disposables (1008)
• Further includes one or more elements selected from the group consisting of control units for controlling the components of the system.

The "detection module" (403) can be, for example, an optical detection unit for detecting the result or effect of an amplification procedure. Optical detection units include light sources such as xenon lamps, mirrors, lenses, optical filters, optical fibers for guiding and filtering light, one or more reference channels, or optics such as CCD cameras or different cameras. Can include.

The "sealer" (410) is constructed and arranged to seal any container used in connection with the analytical system of the present invention. Such sealers can, for example, seal the tube with a suitable cap, or seal the multiwell plate with foil or other suitable sealing material.

The "Preservation Module" (1008) stores the reagents needed to trigger chemical or biological reactions that are important for the analysis of liquid samples. This may also include additional constructs useful for the methods of the invention, such as disposable items such as pipette tips or containers used as reaction vessels in separation and / or amplification stations.

Preferably, the analytical 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 analytical system operate and interact at precise and precise timings, eg, move and manipulate components such as pipettes in a coordinated manner. May include software for The control unit may also include a processor that operates a real-time operating system (RTOS), which is a multitasking operating system intended for real-time application. In other words, the system processor can manage real-time constraints, that is, event-to-system response deadlines regardless of system load. It controls in real time that different units in the system operate and respond accurately according to predetermined instructions.

In a preferred embodiment, the present invention
First receptacle (1001) in linear array containing liquid sample (1010), processing plate (101) containing nxm array receptacle (103) to hold liquid sample (1011), at least in linear array A first pipetting device (700) containing two pipetting units (702), and a first position containing a tip rack (70) containing pipette tips (3, 4) in an ax (nxm) array, said The pipetting unit (702) is connected to the pipette tips (3, 4);
b. Holders (201, 128) for the processing plate (101), holders (330) for the multi-well plate, holders (470) for the chip rack (70) and a second pipetting device ( A second location comprising 35), said second pipetting device (35), comprising a pipetting unit (702) in an nxm sequence for connecting pipette tips (3, 4). Regarding the analysis system (440) for processing (Fig. 12). As used herein, the term "holder" refers to any arrangement that can receive a rack or processing plate.

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

Preferably, the position of the pipetting unit (702) of the first pipetting device (700) is variable. A preferred embodiment of the first pipetting device (700) will be described later herein.

In one aspect, the tip rack (70) comprises pipette tips (3, 4) in the ax (nxm) sequence. Preferably, the first mold (4) and the second mold (3) of the pipette tip are included in the tip rack (70). In this embodiment, the first type pipette tip (4) is arranged in the nxm sequence and the second type pipette tip (3) is arranged in the nxm sequence. In this context, "n" indicates the number of rows and m indicates the number of columns, where n is preferably 6 and m is preferably 8. More preferably, the first type pipette tip (4) has a different volume than the second type pipette tip (3), and most preferably the volume of the first type pipette tip (4). Is larger than 500ul and the volume of the second type pipette tip (3) is smaller than 500ul. In this embodiment, a = 2. However, more than two types of pipette tips, and thus a> 2 aspects of the invention, are also included in the invention.

In one aspect, the analytical system (440) of the present invention includes a control unit (1006) for designating a sample type and individual test for each position of the processing plate (101). Preferably, the positions are separate cells (401, 402).

In one aspect of the invention, the system further transfers the processing plate (101) and the rack (70) between positions 1 (402) and 2 (401), a transfer system (480). )including. A preferred embodiment of the transfer system (480) is a conveyor belt, or more preferably one or more controls.

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

A preferred embodiment of the system (440) of the present invention further comprises a third station (403) comprising the analyte and a temperature controlled incubator for incubating the reagents required to obtain a detectable signal. Including further. A more preferred embodiment of this system will be described later herein.

More appropriate controls for assigning samples and tests to nxm sequences are the first processor (1004) contained in the first position (402) and the second processor contained in the second position (401). Achieved by (1005), the first processor is instructed by the control unit to assign sample types and individual tests to specific positions in the nxm array of containers (103) of processing plates (101). Transferred by (1006), the second processor is instructed by the control unit (1006) to assign sample types and individual tests to specific positions in the nxm sequence of the processing plate container (103). Transported by.

Preferably, the system further includes a first processor located in the first position and a second processor located in the second position.

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

All other preferred embodiments and specific description of aspects of the analytical system of the present invention describe the methods of the present invention.

Examples The following examples describe aspects in which the present invention can be applied. It will be apparent to those skilled in the art that these examples are not limited and can be modified without departing from the spirit of the present invention.

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

Briefly, in the embodiments shown, real-time PCR is performed simultaneously and under the same conditions against a panel of bacteria (Chlamydia trachomatis, CT) and several different targets including DNA virus (HBV) and RNA virus (HIV). It is done in. All samples were processed and analyzed in the same experiment, respectively, in the same deep well plate (for sample preparation) or multi-well plate (for amplification and detection).

The following samples were prepared and subsequently analyzed:

Suitable standards or other types of targets are available to those of skill in the art.

The devices listed in the table below were used according to their respective manufacturer's instructions:

The following reagents were used as diluents for sample preparation:

The following dilutions were prepared in advance and stored overnight (-60-90 ° C for plasma dilutions, 2-8 ° 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 3 different wells for triple analysis. 50 ul of internal control nucleic acid was manually added to each well containing the HIV or HBV sample. RNA acting as a qualitative control was added for the qualitative HIV assay (100 outer particles / sample). RNA acting as a quantitative standard was added for the quantitative HIV assay (500 exterior particles / sample). DNA acting as a quantitative standard was added for the quantitative HBV assay (1E4 copy / sample). The control nucleic acid sequence was identical in all cases and was selected from the group SEQ ID NO: 45-48.

Each control nucleic acid was stored in the following buffer:

Sample preparation was performed on Hamilton Star (Hamilton, Bonaduz, CH) using the following reagents according to the schematic workflow shown in Figure 1.

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 and Mmx were mixed to obtain each. The mixture was transferred to the corresponding well of the amplified microwell plate.

The following master mix (consisting of two reagents R1 and R2 respectively) 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 includes initial denaturation and incubation at 55, 60 and 65 ° C. for reverse transcription of the RNA template. Incubating at three different temperatures also transcribes slightly mismatched target sequences (such as genetic variants of the organism) at lower temperatures, while at higher temperatures the formation of RNA secondary structure is suppressed. Combine the beneficial effects of resulting in more effective transcription.

The PCR cycle is divided into two measurements, both of which apply a one-step setting (annealing and elongation combined). The first 5 cycles at 55 ° C allow for increased inclusivity by pre-amplifying the slightly mismatched target sequence, and the 45 cycles of the second measurement allow for an annealing / elongation temperature of 58 ° C. The use of is an increase in specificity.

Using this profile for all samples contained in the microwell plate described above, amplification and detection was achieved in all samples as shown in FIG. This indicates that the sample preparation before amplification was also successfully performed.

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

Example 2:
The general amplification process described above was performed on a variety of different target nucleic acids under the same conditions but in different 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 outer RNA for RNA targets and λ-packaged DNA for DNA targets. 300 particles per sample were added for the qualitative RNA assay, 3000 for the quantitative RNA assay and 500 for the total DNA assay.

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 around the expected LOD for EDTA plasma. One virus and one panel per concentration were tested in at least 20 reasonable repeat experiments per concentration. LOD was determined by probit analysis (see Tables 1-5).

HIV

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

Therefore, the HIV-1 group M LOD of IU / mL 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 primary standard titers for HIV-1 group O have been rearranged relative to the CBER HIV-1 group O panel; the computational factor is 0.586.

Therefore, the 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 titers for HIV-2 have been rearranged relative to the CBER HIV-2 panel; the computational 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 to 83.04 cp / mL

HBV

HCV

2. WNV Qualitative Analysis Master Mix

Analytical sensitivity / LOD
For viruses (WNV, SLEV and JEV), independent panels were prepared as serial dilutions of each standard containing several concentrations / levels in and around the expected LOD. One virus and one panel per concentration were tested by at least 20 reasonable repeat experiments per concentration. The LOD was determined by probit analysis.

3. Quantitative analysis of HBV Master mix

Analytical sensitivity / LOD
Four dilution panels with HBV secondary standards (showing genotype A), two for HBV-negative serum for 200 μL and 500 μL sample input volumes, and two for HBV for 200 μL and 500 μL sample input volumes. Negative EDTA was prepared in plasma. Each panel contained 7 concentration levels in and around the expected LOD. One panel per matrix was tested in ≥21 repeat experiments per concentration level. At least 20 repetitive experiments needed to be valid. The LOD was determined by a probit analysis with a 95% hit rate and a ≥95% hit rate analysis.

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

The 95% confidence interval range for these concentrations ranged from 4.8 to 26.0 IU / mL for a 200 μL sample input volume and 1.6 to 4.2 IU / mL for a 500 μL sample input volume.

Serum: Probit analysis at 95% hit rate yielded 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 range for these concentrations ranged from 6.2 to 19.0 IU / mL for a 200 μL sample input volume and 2.4 to 10.0 IU / mL for a 500 μL sample input volume.

Straightness
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 to determine the expected dynamic range of the assay (4-2E + 09IU / mL). All concentration levels / panel members (PM) were tested in 21 repeat experiments.

This 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 the intermediate level. , Some concentrations at the upper limit of expected quantification (ULOQ), and one above the expected ULOQ.
PM12 --2.0E + 09IU / mL --above the 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 to titer serum panels)
PM4-1.0E + 02IU / mL-Intermediate concentration level (also used to titer plasma panels)
PM3 --5.0E + 01IU / mL --above the expected LLOQ
PM2-1.0E + 01IU / mL-Expected LLOQ
PM1-4.0E + 00IU / mL Less than expected LLOQ

For all reasonable 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.

The graph display of this result is shown in FIG.

The graph display of this result is shown in FIG.

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

4. Quantitative analysis of HCV Master mix

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 repeat experiments. At least ≥20 repetitive experiments need to be done. LOD was determined by probit analysis with 95% hit rate and ≧ 95% hit rate analysis.

LOD overview:
1. Probit analysis at 95% hit rate yielded an LOD of 17.4 IU / mL for a 200 μL sample processing input volume and 9.0 IU / mL for a 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. Probit analysis values at 95% hit rate are 20.2 IU / mL for a 200 μL sample processing input volume and 8.2 IU / mL for a 500 μL sample processing input volume for serum. The 95% confidence intervals for these concentrations are 14.0 to 39.3 IU / mL for a 200 μL sample processing input volume and 5.8 to 15.0 IU / mL for a 500 μL sample processing input volume.

Straightness
One preparation of an EDTA plasma panel of HCV aRNA and one preparation of a serum panel that were traceable to the HCV WHO standard 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: 1 level below the lower limit of expected quantification (LLoQ), 1 at LLoQ, 1 above LLOQ, some concentrations at intermediate levels, upper limit of expected quantification (ULoQ) at several concentrations and one at ULoQ or above. Twenty-one repetitive experiments were tested for all concentrations.

PM1 --2.0E + 08IU / mL --above the 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 specification
PM7 --1.0E + 03IU / mL --Intermediate concentration level
PM8 --1.0E + 02IU / mL --above the expected LLoQ
PM9 --1.0E + 01IU / mL --Expected LLoQ
PM10 --8.0E + 00IU / mL --less than expected LLoQ

The graph display of this result is shown in FIG.

The graph display of this result is shown in FIG.

Outline of linearity:
The linear range defined as the concentration range in which the log10 deviation of the mean 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. It was determined to be / mL ~ 9.92E + 07IU / mL.

5. HIV Quantitative Analysis Master Mix

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 repeat experiments. At least ≥20 repetitive experiments need to be valid. LOD was determined by probit analysis with 95% hit rate and ≧ 95% hit rate analysis.

LOD overview
1. Probit analysis at 95% hit rate yielded 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 range for these concentrations ranged from 30.9 to 74.9 cp / mL for a 200 μL input volume and 14.9 to 29.4 cp / mL for a 500 μL input volume.

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

Linear panels were prepared by serial dilution. This panel was analyzed at 10 concentration levels.

Concentrations were selected as follows: 1 level below the lower limit of expected quantification (LLoQ), 1 at LLoQ, 1 above LLoQ, some concentrations at intermediate levels, upper limit of expected quantification (ULoQ) at several concentrations and one above ULoQ. Twenty-one repetitive experiments were tested for all concentrations. This linearity test was performed at a 500 μL input volume:

PM1 --2.0E + 07cp / mL --above the 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 specification
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 the expected LLoQ
PM10 --2.0E + 01cp / mL --Expected LLoQ
PM11 --1.5E + 01cp / mL --less than expected LLoQ

The graph display of this result is shown in FIG.

Outline of linearity The linear range defined as the concentration range in which the log10 deviation of the average log10 observed titer is within ± 0.3 of the log10 nominal titer was determined to be 1.5E + 01cp / mL to 2.0E + 07cp / mL.

The following aspects of the present invention can be mentioned.
[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.
a. Step of adding internal control nucleic acid to each of the liquid samples
b. Solid support material and one or more of the above liquid samples, with sufficient time and conditions 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 matching together in a container
c. The step of isolating the solid support material from other materials present in the liquid sample at the separation station.
d. The step of purifying the nucleic acid at the separation station and washing the solid support material with the wash buffer at least once.
e. At least two reaction vessels containing a purified target nucleic acid and a purified internal control nucleic acid and one or more amplification reagents containing at least one different set of primers for each of the target nucleic acids and the internal control nucleic acid. The step of contacting in, where at least the first reaction vessel comprises at least the first target nucleic acid, at least the second reaction vessel comprises at least the second target nucleic acid, and the second target nucleic acid is the first. Does not exist in the reaction vessel of
f. In the reaction vessel, the purified target nucleic acid, the purified internal control nucleic acid, and the one or more amplification reagents are sufficient for an amplification reaction indicating the presence or absence of the target nucleic acid to occur. Incubation step under various time and conditions
g. Includes an automated step of detecting and measuring a signal produced by the amplification product of the target nucleic acid and proportional to the concentration of the target nucleic acid, and a step of detecting and measuring the signal produced by the internal control nucleic acid. The conditions for amplification and detection in steps d-g are the same for the at least the first and second purified target nucleic acids and the internal control nucleic acid, and the sequences of the internal control nucleic acids are at least the first and first. The same method for 2 purified target nucleic acids.
[2] The description of [1], wherein the presence of an amplification product of the internal control nucleic acid indicates that amplification occurs in the reaction mixture even in the absence of amplification products for one or more of the target nucleic acids. Method.
[3] Below:
h. The method according to any of the above, further comprising determining the amount of one or more of the target nucleic acids.
[4] The amplification reagent comprises a polymerase having reverse transcriptase activity, and the method comprises the purified nucleic acid and the one or more amplification reagents in the reaction vessel during steps e and f. The method according to any of the above, further comprising the step of incubating the nucleic acid with a time and conditions suitable for transcription of RNA by the polymerase having reverse transcriptase activity.
[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 an outerized 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 other nucleic acids present in the liquid sample.
[9] The method according to any 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 sequences of the internal control nucleic acids are 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 of the above, wherein the internal control nucleic acid has a length of up to 500 bases.
[13] The method according to any 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 of the above, wherein the internal control nucleic acid has a concentration of LOD to 20xLOD.
[15] The method according to any of the above, wherein the internal control nucleic acid has a concentration of 20xLOD to 5000xLOD.
[16] The method according to any of the above, wherein more than one internal control nucleic acid is added in step a, but only one of the 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.
The step of adding a quantitative standard nucleic acid to each of the liquid samples, where the sequence of the quantitative standard nucleic acid is derived from a source different from the origin of the liquid sample and is different from other nucleic acid sequences in the liquid sample. is different, is derived from a plant genome, that have been mixed (scrambling),
b. One or more of the solid support material and the one or more liquid samples, with sufficient time and conditions for the nucleic acid containing the target nucleic acid and the quantitative standard nucleic acid to be immobilized on the solid support material. Process of putting together in a container
c. The step of isolating the solid support material from other materials present in the liquid sample at the separation station.
d. The that pollock fine nucleic acid in the separation station step
e. purified and quantitative standard nucleic acid the target nucleic acid and is purified, the amplification reagent comprising at least one different set of primers for each, and the quantitative standard nucleic acid of the target nucleic acid, two reaction vessels even without least Process of contacting inside
f. In the reaction vessel, the purified target nucleic acid, the purified quantitative standard nucleic acid, and the amplification reagent are used for a sufficient time for an amplification reaction indicating the presence or absence of the target nucleic acid to occur. Incubate under conditions to amplify the first target nucleic acid instead of the second target nucleic acid in the first reaction vessel and the second target nucleic acid instead of the first target nucleic acid in the second reaction vessel. Process to do
g. A step of detecting and measuring a signal produced by the amplification product of the target nucleic acid and proportional to the concentration of the target nucleic acid, and a step of detecting and measuring a signal produced by the quantitative standard nucleic acid.
h. The conditions for amplification and detection in steps e. To g. Include the step of automating the step of determining the amount of one or more of the target nucleic acids by using a quantitative standard nucleic acid as a reference. The same method for the first and second purified target nucleic acids and the quantitative standard nucleic acids. The method of claim 1, wherein the presence of an amplification product of the quantitative standard nucleic acid indicates that amplification occurs in the reaction mixture even in the absence of amplification products for one or more of the target nucleic acids. The method according to claim 1 or 2, wherein the quantitative standard nucleic acid is an outerized nucleic acid. The method according to any one of claims 1 to 3, wherein the melting temperature of the quantitative standard nucleic acid is 50 ° C to 90 ° C. The method according to any one 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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