JP6121591B2 - Qualitative and quantitative detection of microbial nucleic acids - Google Patents

Description

  The present invention relates to a novel method and use for qualitative and quantitative detection of microbial nucleic acids using at least a first control nucleic acid, or different concentrations of first and second control nucleic acids. The method is based on nucleic acid amplification, preferably polymerase chain reaction. Further provided are kits comprising components for performing the methods and uses described above.

  In the field of molecular diagnostics, the detection and quantification of microbial nucleic acids using nucleic acid amplification reactions plays an important role. Typed screening for the presence of donated hepatitis C virus (HCV), human immunodeficiency virus (HIV), and / or hepatitis B virus (HBV) is an example of a large-scale application of nucleic acid amplification and detection reactions is there. The latter includes a variety of different techniques, and the most commonly used technique is the polymerase chain reaction (PCR) introduced by Kary Mullis in 1984. Automated systems for PCR-based analysis often utilize real-time detection of product amplification during the PCR process. The key to such a method is the use of modified oligonucleotides with reporter groups or labels.

  Qualitative detection of microbial nucleic acids in a biological sample is important, for example, to recognize an individual's infection. Thereby, one important requirement for assays for the detection of microbial infections is to avoid false negative or false positive results, which inevitably relate to the treatment of each patient. This is because it is considered to lead to a serious result. Thus, particularly in PCR-based methods, a qualitative internal control nucleic acid is added to the detection mixture. The above controls are particularly important to confirm the validity of the test results: at least in the case of negative results for microbial nucleic acids, the qualitative internal control reaction must show a response within a given setting. That is, the qualitative internal control must be detected, otherwise the test itself is considered invalid. However, in a qualitative configuration, the qualitative internal control does not necessarily have to be detected in the case of a positive result. For qualitative testing, it is particularly important that the sensitivity of the reaction is guaranteed and therefore strictly controlled. As a result, the concentration of the qualitative internal control must be relatively low so that, for example, even in the presence of slight inhibition, the qualitative internal control is not detected and thus the test is ineffective.

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

  In the case of external calibration, a standard curve is generated in another reaction using known amounts of the same or comparable nucleic acid. Subsequently, the absolute amount of microbial nucleic acid is determined by comparing the results obtained with the sample to be analyzed with the standard function. However, external calibration has the disadvantage that potential extraction procedures, their diverse effectiveness, and potential, and often the presence of unpredictable factors that inhibit the amplification and / or detection reactions are not reflected in the control. Have

  This situation applies to any action associated with the sample. Therefore, it is possible that the microbial nucleic acid to be detected and quantified is actually present in the sample while the sample is judged negative due to unsuccessful extraction procedures or other factors based on the sample.

For these and other reasons, it is convenient to add an internal quantitative standard to the test reaction itself. The internal quantitative standard has at least two functions in quantitative tests:
i) It monitors the effectiveness of the reaction.

  ii) It serves as a reference in titer calculations, thus correcting for the effects of inhibition and controlling the preparation and amplification process to allow more accurate quantification. Thus, in contrast to qualitative internal control nucleic acids in qualitative tests that must be positive only in reactions where the target is negative, quantitative standard nucleic acids in quantitative tests have two functions: reaction control and reaction calibration. Therefore, it must be positive and effective in both reactions where the target is negative and reactions where the target is positive.

It must also be suitable to give a reliable reference value for the calculation of high nucleic acid concentrations. Therefore, the concentration of the internal quantitative standard nucleic acid needs to be relatively high.
The qualitative internal control nucleic acid and / or internal quantification standard nucleic acid can be competitive, non-competitive or partially competitive. A competitive qualitative internal control nucleic acid and / or internal quantification standard nucleic acid has essentially the same primer binding site as its target and thus competes as a target for the same primer. Among the advantages of a competitive configuration, for example, there are fewer different primer sets that must be introduced into the assay, thus reducing its cost and overall complexity. Furthermore, the functionality of the primer is monitored as an inhibitory effect that is specific to the target primer. Non-competitive qualitative internal control nucleic acids and / or internal quantitation standard nucleic acids have different primer binding sites than their targets and thus bind to different primers. Advantages of such a configuration include, inter alia, the fact that a single amplification event of different nucleic acids in the reaction mixture can occur independently of each other without any competitive effects. In PCR using partially competitive internal quantitative standard nucleic acids, at least one of its respective control and target nucleic acids competes for the same primer, but at least one other target nucleic acid binds to a different primer.

  Because the principles of quantitative and qualitative assays as described above present different requirements when compared to each other, and considering regulatory requirements in various countries, the general approach used in the art is Development of separate quantitative and qualitative assays for the same target nucleic acid (see, eg, Yang et al., J Agr Food Chem 2005, 53, 6222-6229).

  The present invention provides an alternative solution that exhibits several advantages.

Yang et al., J Agr Food Chem 2005, 53, 6222-6229

The present invention relates to novel methods and uses for the simultaneous qualitative and quantitative detection of microbial nucleic acids. The method is based on nucleic acid amplification, preferably polymerase chain reaction. Briefly, a specific sequence portion of the microbial nucleic acid and at least a first control nucleic acid are amplified and detected using one or more specific primer pairs. Accordingly, one subject of the present invention is the following:
A method for the simultaneous detection and quantification of microbial nucleic acids in a biological sample, said method comprising:
a) isolating and purifying said microbial nucleic acid;
b) one or more primer pairs that specifically hybridize to at least a first control nucleic acid, to a separate sequence portion of said microbial nucleic acid, and to a separate sequence portion of said control nucleic acid, and one or more of these Providing a reaction mixture comprising a probe that specifically hybridizes to each of the sequences amplified by the primer pairs, wherein the microbial nucleic acid and the control nucleic acid hybridize to different probes;
c) adding the biological sample to the reaction mixture;
d) performing one or more cycle steps, wherein the cycle step comprises an amplification step, the amplification step comprising one or more amplification products derived from said microbial nucleic acid if present in said sample; And generating an amplification product derived from the control nucleic acid, wherein the cycling step includes a hybridization step, wherein the hybridization step converts the sequence amplified by the primer pair into the probe. Wherein the probes are labeled with a donor fluorescent moiety and a corresponding acceptor fluorescent moiety, each of the probes having a different fluorescent dye;
e) detecting and measuring a fluorescent signal produced by the amplification product, which is proportional to the concentration of the control nucleic acid and the microbial nucleic acid, wherein the presence of the amplification product of the control nucleic acid is amplified with respect to the microbial nucleic acid; It shows that amplification is occurring in the reaction mixture even in the absence of product.

FIG. 1a graphically shows an internal control growth curve for a quantitative real-time PCR reaction of a commercial COBAS AmpliPrep / COBAS TaqMan HIV-1 test. The PCR reaction included HIV-1 target concentrations ranging from 5 log 10 and target negative samples. If a high target concentration is present in the reaction (see “Target High Positive”), the fluorescence level reached by the internal quantification standard nucleic acid (QS) is determined by the PCR reaction without the HIV-1 target (“Target Negative”). Significantly lower than in the case of reference). The minimum fluorescence intensity level setting (“RFImin”) for the QS is low because the QS must be valid at all target concentrations in order to be able to calculate the titer with respect to the output of quantitative results. . The RFImin settings for the output of quantitative results in three commercial tests: COBAS AmpliPrep / COBAS TaqMan HBV, HCV and HIV-1 are shown in Table 1. FIG. 1b graphically shows an internal control growth curve for a real-time PCR reaction of a commercial COBAS AmpliPrep / COBAS TaqMan HIV-1 test. All PCR reactions included HIV-1 target negative samples. The fluorescence level of the internal control growth curve is bundled within a narrow range, and the minimum fluorescence intensity level setting (“RFImin”) for the qualitative internal control nucleic acid (IC) is high. In a target positive reaction, the IC may be below RFImin and may be ineffective; in the presence of the target, the results for the PCR reaction are still valid. The optimized RFImin settings for the output of qualitative results for the COBAS AmpliPrep / COBAS TaqMan HBV, HCV and HIV-1 tests are shown in Table 1. FIG. 2 shows the generation of armored RNA particles: expression vector for generation of armored RNA particles and electron microscopic observation of armored RNA particles. Armored RNA particles are assembled in E. coli. Following the introduction of the lac-operon, an mRNA containing the MS-2 bacteriophage MS-2 coat protein gene, control sequence, packaging signal and plasmid sequence upstream of the TrrnB terminator is transcribed. After the coat protein is translated, the composition of the particles spontaneously packs one copy of mRNA. FIG. 3 shows the normalized growth curve obtained for a real-time PCR reaction containing an HCV target, a first control nucleic acid and a second control nucleic acid. FIG. 4a shows simultaneous amplification of HCV-RNA and QS. The fluorescence intensity of both QS and HCV-RNA is lower than in the reaction that yields a standard signal (see Figure 4b). FIG. 4b shows simultaneous amplification of HCV-RNA and QS. Both reactions result in standard fluorescence intensity. For comparison, see FIG. 4a with a suppressed fluorescence curve; note also the different intensities in the two figures.

  The present invention exhibits numerous advantages over the prior art. In particular, the present invention allows the general design of qualitative and quantitative assays for any given microbial nucleic acid using such same reagents.

  In some embodiments, the first control nucleic acid described above can serve as both an internal quantitative standard nucleic acid and a qualitative internal control nucleic acid. Thus, it eliminates the need for each of two separate tests or test kits. The performance of the two separate tests burdens the patient with additional blood collections and can incur additional costs to the health care system if both the quantitative and qualitative tests are reimbursed. Furthermore, the time to results for both quantitative and qualitative tests is reduced when compared to sequential qualitative and quantitative tests.

  Preferably, said first control nucleic acid is evaluated according to different criteria for obtaining qualitative and quantitative results. In a preferred embodiment, the same set of raw data obtained during real-time PCR is analyzed against two different test parameter settings. The qualitative test parameter settings applied for valid qualitative control nucleic acids are more rigorous compared to the parameter settings applied for internal quantitation standards required for output of quantitative results. The settings for the internal quantitative standard nucleic acid in the quantitative reaction cannot be so strict that the presence of the target can affect the respective growth curve of the internal quantitative standard nucleic acid (see FIG. 1a).

  Accordingly, a preferred embodiment of the present invention is the method described above, wherein the first control nucleic acid is analyzed according to different criteria to serve as a quantitative standard nucleic acid or as a qualitative internal control nucleic acid. .

  In other embodiments, it may be advantageous to use the first and second control nucleic acids at different concentrations for the following reasons: Usually, the internal quantitative standard nucleic acid (s) in a quantitative test are It has a somewhat high concentration so that it can still be amplified and detected in a sample with a high concentration of target nucleic acid. Thus, sometimes it is not possible to use it as a control for the detection of low positive samples close to the limit of detection (LOD), especially if the amplification reaction is partially suppressed. In that case, the highly enriched control nucleic acid cannot serve as a measure of sufficient sensitivity for the respective assay.

Accordingly, preferred aspects of the present invention are:
A method for the simultaneous detection and quantification of microbial nucleic acids in a biological sample, said method comprising:
a) isolating and purifying said microbial nucleic acid;
b) one or more primer pairs that specifically hybridize to different concentrations of the first and second control nucleic acids, to separate sequence portions of the microbial nucleic acid, and to separate sequence portions of the control nucleic acid, and Provided is a reaction mixture comprising a probe that specifically hybridizes to each of the sequences amplified by one or more of these primer pairs, wherein said microbial nucleic acid and said first control nucleic acid and said second control The nucleic acid hybridizes to different probes;
c) adding the biological sample to the reaction mixture;
d) performing one or more cycle steps, wherein the cycle step comprises an amplification step, the amplification step comprising one or more amplification products derived from said microbial nucleic acid if present in said sample; And generating an amplification product derived from the first control nucleic acid and the second control nucleic acid, wherein the cycling step comprises a hybridization step, wherein the hybridization step comprises said primer pair Hybridizing the sequences amplified by the above probes, wherein the probes are labeled with a donor fluorescent moiety and a corresponding acceptor fluorescent moiety, each of the probes having a different fluorescent dye;
e) detecting and measuring fluorescent signals proportional to their concentration produced by the amplification product of the first control nucleic acid and the microbial nucleic acid and / or simultaneously by the amplification product of the second control nucleic acid. The generated fluorescent signal is detected, where the presence of the amplification product of the second control nucleic acid indicates that amplification is occurring in the reaction mixture even in the absence of the amplification product for the microbial nucleic acid.

  The use of different concentrations of the first and second control nucleic acids in the same control reagent according to the present invention overcomes the problems noted above. Nucleic acids with lower or lowest concentrations correspond to qualitative internal control nucleic acids for qualitative assays, ensuring the ability to determine whether a negative result is valid, i.e. a negative result is its internal control Invalid if no nucleic acid is detected.

  According to the present invention, those skilled in the art will develop quantitative and qualitative configurations by performing both in a single assay, thus reducing development costs, requiring less material and labor A synergy between reducing manufacturing costs, as well as reducing the time required to establish an assay. In addition, double patient testing, which burdens the patient with additional blood collection, is avoided, and the costs associated with the health care system are reduced if both quantitative and qualitative testing are reimbursed.

The qualitative internal control nucleic acid and / or internal quantification standard nucleic acid can be competitive, non-competitive or partially competitive.
“Competitive” means that in an amplification reaction involving primers, each control nucleic acid and target nucleic acid (s) have at least essentially the same primer binding site and thus compete for the same primers. Among the advantages of a competitive configuration, for example, there are fewer different primer sets that must be introduced into the assay, thus reducing its cost and overall complexity. Furthermore, the functionality of the primer is monitored as an inhibitory effect that is specific to the target primer.

  “Non-competitive” means that its respective control and target nucleic acid (s) have different primer binding sites and thus bind to different primers. Advantages of such a configuration include, inter alia, the fact that a single amplification event of different nucleic acids in the reaction mixture can occur independently of each other without any competitive effects.

  “Partially competitive” means that at least one of its respective control nucleic acid and its target nucleic acid competes for the same primer, but at least one other target nucleic acid binds to a different primer.

According to the method (s) of the present invention, the internal quantification standard nucleic acid is used as the first control nucleic acid to simultaneously prevent not only sample-specific inhibitory effects, but also amplification and detection reactions, possibly with respect to the quantification standard nucleic acid and the microbial nucleic acid. Sample non-specific inhibitory effects (inhibition independent of target region) are also leveled, resulting in more accurate titers. “Internal” means that the first control nucleic acid is amplified, detected and quantified in the same reaction mixture as the microbial nucleic acid, but not in a separate experiment.

Another preferred aspect of the invention is the method described above, wherein the first control nucleic acid is a quantitative standard nucleic acid and the second control nucleic acid is a qualitative internal control nucleic acid.
One skilled in the art can derive not only reliable qualitative information from the same experiment, but also quantitative information that is equally reliable in some cases. Accordingly, in another aspect, the invention relates to:
The method described above, further including:
In step e), the amount of the microbial nucleic acid in the biological sample is determined by comparing the signals generated by the microbial nucleic acid and the first control nucleic acid.

  In a preferred embodiment, the first control nucleic acid and the second control nucleic acid have essentially the same sequence. Preferably they have the same primer binding site but different probe binding sites.

  This is because the control nucleic acid for its qualitative and quantitative measurements is based on the same selected binding sequence, with the same convenience in terms of assay performance and in terms of its analyte sequence (s). Give the advantage of sharing unique properties.

Commonly used molecular biology and nucleic acid chemistry techniques are within the skill of the art and are described in the literature. For example, Sambrook J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, Gait, MJ (editor) 1984; Nucleic Acid Hybridization, Hames, BD, and Higgins, SJ (Editor) 1984; and series, Methods in Enzymology, Academic Press, Inc.

“Simultaneously” in the sense of the present invention means that two actions are carried out in the same reaction or reaction mixture, for example the detection and quantification of nucleic acids.
A “reaction mixture”, as used in the present invention, includes at least all components for promoting a biological or chemical reaction. It is a single volume without any separate compartments, i.e. all components present in said "reaction mixture" are in direct contact with each other.

  A “biological sample” can be any sample of natural origin. Preferably, the “biological sample” is derived from a human and is a body fluid. In a preferred embodiment of the invention, the “biological sample” is blood.

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

  A “nucleotide” is a “nucleoside” that further includes a phosphate group covalently linked to the sugar moiety of the nucleoside. For “nucleosides” that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2 ′, 3 ′ or 5 ′ hydroxyl moiety of the sugar. “Nucleotide” is an “oligonucleotide”, more commonly referred to herein as an “oligomer compound” or “polynucleotide”, and more commonly a “monomer” of what is referred to as a “polymer compound”. Unit ”. Another common expression for the above is deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

According to the present invention, an “oligomer compound” is a compound consisting of “monomer units”, which is either “nucleotides” only, or “non-natural compounds” (see below), more specifically “modified nucleotides” ( Or “nucleotide analogue”) or “non-nucleotide compound” alone, or a combination thereof. “Oligonucleotides” and “modified oligonucleotides” (or “oligonucleotide analogs”) are a subpopulation of “oligomer compounds” in the context of the present invention.

  In the context of this invention, the term “oligonucleotide” refers to a “polynucleotide” formed from a plurality of “nucleotides” as “monomer units”, ie, “oligonucleotide” refers to “monomer units”. Belongs to a specific subpopulation of “oligomer compounds” or “polymer compounds” of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). The phosphate group is generally referred to as forming the internucleoside backbone of the “oligonucleotide”. The normal linkage or backbone of RNA and DNA is a 3 'to 5' phosphodiester linkage.

“Oligonucleotides” and “modified oligonucleotides” (see below) according to the present invention can be synthesized as described primarily in the art and known to those skilled in the art. Methods for preparing oligomeric compounds of a specific sequence are known in the art and include, for example, cloning and restriction of appropriate sequences, and direct chemical synthesis. Chemical synthesis methods include, for example, the phosphotriester method described by Narang SA et al., Methods in Enzymology 68 (1979) 90-98, Brown EL, et al.,
Methods in Enzymology 68 (1979) 109-151, the phosphodiester method disclosed in Beaucage et al., Tetrahedron Letters 22 (1981) 1859, the phosphoramidite method, Garegg et al., Chem. Scr. 25 ( 1985) 280-282 and the H-phosphonate method disclosed in US Pat. No. 4,458,066 may be included.

  With respect to the methods described above, the nucleic acid can be present in double-stranded or single-stranded form, so that the double-stranded nucleic acid is denatured by heating (ie, heat-denatured) before the method is performed. ), That is, a single strand.

  In another embodiment, the primer and / or its probe may be chemically modified, i.e., the primer and / or its probe comprises a modified nucleotide or non-nucleotide compound. The probe or its primer is therefore a modified oligonucleotide.

  “Modified nucleotides” (or “nucleotide analogs”) differ from natural “nucleotides” by some modifications, but still include bases, pentofuranosyl sugars, phosphate moieties, base-like, pentofuranosyl sugar-like and phosphate-like It consists of a part or a combination thereof. For example, a “label” can be attached to the base moiety of a “nucleotide”, resulting in a “modified nucleotide”. Natural bases in “nucleotides” can also be replaced by, for example, 7-deazapurine, which also results in “modified nucleotides”. The terms “modified nucleotide” or “nucleotide analog” are used interchangeably in this application. “Modified nucleosides” (or “nucleoside analogs”) differ from natural “nucleosides” by several modifications in the manner as outlined above for “modified nucleotides” (or “nucleotide analogs”).

“Non-nucleotide compounds” are different from natural “nucleotides”, but in the sense of this invention—as well as “nucleotides” —can be “monomer units” of “oligomer compounds”. Thus, “non-nucleotide compounds” must be able to form “oligomeric compounds” with “nucleotides”. In addition, “non-nucleotide compounds” can contain base-like, pentofuranosyl sugar-like or phosphate-like moieties, but not all of them are present simultaneously in the “non-nucleotide compound”.

“Modified oligonucleotides” (or “oligonucleotide analogs”) belong to another specific subpopulation of “oligomer compounds”, one or more “nucleotides”, one or more “non-nucleotide compounds” or “modified nucleotides”. "As a" monomer unit ". Thus, the term “modified oligonucleotide” (or “oligonucleotide analog”) refers to a structure that functions in a manner substantially similar to “oligonucleotide” and is used interchangeably throughout this application. From a synthetic point of view, “modified oligonucleotides” (or “oligonucleotide analogs”) can be made by chemical modification of “oligonucleotides”, eg by appropriate modification of the phosphate backbone, ribose units or nucleotide bases (Uhlmann and Peyman, Chemical Reviews 90 (1990) 543; Verma S., and Eckstein F., Annu. Rev. Biochem. 67 (1998) 99-134). Typical modifications include phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester or phosphoramidate internucleoside linkages instead of phosphodiester internucleoside linkages; deaza- or azaa instead of natural purine and pyrimidine bases. -Purine and -pyrimidine pyrimidine bases having substituents in the 5 or 6 position; purine bases having substituents changed in the 2, 6 or 8 position or 7 position (as 7-deazapurines); alkyl-, alkenyl- Alkynyl or aryl moieties such as lower alkyl groups such as methyl, ethyl, propyl, butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, or aryl such as phenyl, benzyl, naphthyl Contained or carbocyclic or acyclic sugar analogs; for example saccharides having their 2 'position a substituent; base having a. Other modifications consistent with the spirit of this invention are known to those skilled in the art. Such “modified oligonucleotides” (or “oligonucleotide analogues”) are structurally compatible with natural “oligonucleotides” (or synthetic “oligonucleotides” along the natural line) but structurally Are best described as different. More particularly, typical 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 in which nucleoside units are linked through groups that substitute for internucleoside phosphate or sugar phosphate linkages. Such linkages include those disclosed in Verma S., and Eckstein F., Annu. Rev. Biochem. 67 (1998) 99-134. Such structures have also been described as “oligonucleosides” when other than phosphate linkages are used to link the nucleoside units.

  “Nucleic acid” and “target nucleic acid” or “microbial nucleic acid” are polymeric compounds of “nucleotides” as known to those skilled in the art. “Target nucleic acid” or “microbial nucleic acid” as used herein means “nucleic acid” in a sample to be analyzed, ie, “nucleic acid” whose presence, absence and / or amount in a sample is to be determined. Used as follows. Therefore, in this case, the nucleic acid is the target, and therefore can also be expressed as “target nucleic acid”. According to the present invention, since the target nucleic acid is derived from a microorganism, the target nucleic acid is also referred to as “microbial nucleic acid”. For example, if it must be determined whether blood contains HCV, the “target nucleic acid” or “microbial nucleic acid” is an HCV nucleic acid.

“Microorganism” means any virus, bacterium, archaean, fungus or any unicellular eukaryote.
“Microbial” means derived from or belonging to “microorganism”.

“Detecting” means determining the presence or absence of a particular object, eg, a target or signal.
“Quantifying” means determining the amount of a particular subject, eg, a target or signal.

“Measuring” means determining at least the relative value of a particular object, eg, target or signal.
In the sense of a particular embodiment of the invention using a first and second control nucleic acid, the first control nucleic acid serves as an “internal quantification standard nucleic acid”. An “internal quantitative standard nucleic acid” is a “nucleic acid” and is therefore a polymer compound of “nucleotides” as known to those skilled in the art. In the case of an “internally quantified standard nucleic acid”, the nucleic acid is simultaneously “as a control and quantifying for the effectiveness of the reaction”, ie a criterion for determining the amount of its “target nucleic acid” or “microbial nucleic acid”. Is suitable for use as such. For this purpose, the “internal quantification standard nucleic acid” goes through all possible sample preparation steps together with its “target nucleic acid” or “microbial nucleic acid”. Furthermore, it is processed in the same reaction mixture throughout the process. The “internal quantification standard nucleic acid” must produce a detectable signal, either directly or indirectly, both in the presence or absence of the target nucleic acid. For this purpose, the concentration of the “internally quantified standard nucleic acid” has been carefully optimized in each test so as not to impair sensitivity but to produce a detectable signal, for example even at very high target concentrations. There must be. Preferably, the concentration range for that “internally quantified standard nucleic acid”, ie the first control nucleic acid, will comprise a range of 100 copies per reaction to 100,000 copies per reaction. Possible concentrations of the “internal quantification standard nucleic acid” may be, for example, for HIV: 1000 copies / reaction, for HCV: 7500 copies / reaction, but these concentrations are as known to those skilled in the art. It may be adapted to a specific assay. In terms of the limit of detection (LOD) of the respective assay, the concentration range for the “internally quantified standard nucleic acid” is preferably 20-5000 × LOD, more preferably 20-1000 × LOD, most preferably 20-500 × LOD. It is. The final concentration of that “internal quantitative standard nucleic acid” in the reaction mixture depends on the quantitative measurement range to be achieved. The “internal quantification standard nucleic acid” can be, for example, DNA, RNA or PNA, armored DNA or armored RNA, and modified forms thereof.

  Furthermore, in the sense of the present invention, the second control nucleic acid serves as a “qualitative internal control nucleic acid”. A “qualitative internal control nucleic acid” is particularly important to confirm the validity of a test result of a qualitative detection assay: at least in the case of a negative result, the qualitative internal control nucleic acid must be detected Otherwise, the test itself may not be functioning. However, in a qualitative configuration, in the case of a positive result, the qualitative internal control nucleic acid does not necessarily have to be detected. For qualitative testing, it is important that the sensitivity of the reaction is guaranteed and therefore strictly controlled. As a result, the concentration of the qualitative internal control nucleic acid must be relatively low so that the test is considered ineffective even in the presence of slight inhibition. It must be carefully adapted to each assay and its sensitivity. Preferably, the concentration range for that “qualitative internal nucleic acid”, ie the second control nucleic acid, comprises a range of 1 copy per reaction to 1000 copies per reaction. With respect to its respective assay limit of detection (LOD), its concentration is preferably between the LOD of the assay and 25 times its LOD. More preferably, it is 2x to 20x LOD, or 2x to 15x LOD, or 2x to 10x LOD. Even more preferably, it is 3 × -7 × LOD.

“Detection limit” or “LOD” means the lowest detectable amount or concentration of nucleic acid in a sample having a predetermined hit rate. Low “LOD” corresponds to high sensitivity, and high “LOD” corresponds to low sensitivity. The “LOD” is usually expressed either by the unit “cp / ml” or as IU / ml, especially if the nucleic acid is a viral nucleic acid. “Cp / ml” means “number of copies per milliliter”, where “copy” is a copy of each nucleic acid. IU / ml stands for “international units / ml” and refers to the WHO standard. Depending on the target, LOD values in clinical molecular diagnostic assays are typically less than 1000 cp / ml. Preferably, the LOD in an assay performed in the context of the present invention is between 1 and 500 cp / ml.

  A widely used method for calculating LOC is “probit analysis”. It is a method of analyzing the relationship between stimulus (dose) and quantum (total or none) responses. In a typical quantum response experiment, groups of animals are given different doses of drug. Record the percent mortality at each dose level. These data can then be analyzed using probit analysis. The probit model assumes that the percent response is related to the log of dose as a cumulative normal distribution. That is, the logarithm of dose can be used as a variable to read percent mortality from its cumulative normal. Using the normal distribution rather than other probability distributions affects the expected response rate at the high and low ends of the possible dose, but has little effect near the middle.

  “Probit analysis” can be applied at different “hit rates”. As is known in the art, “hit rate” is generally expressed as a percentage [%] and indicates the percentage of positive results at a particular concentration of analyte. Thus, for example, LOD can be measured at 95% hit rate, which means that LOD is calculated for a setting where 95% of valid results are positive.

  The term “primer” is used herein as known to those skilled in the art and refers to “oligomer compound”, primarily “oligonucleotide”, but “prime” DNA synthesis by a template-dependent DNA polymerase. ) "Can be" modified oligonucleotide ", i.e., for example, the 3 'end of the oligonucleotide provides a free 3'-OH group to which further" nucleotide "is coupled by a template-dependent DNA polymerase, Also refers to those in which a phosphodiester bond from to 5 'is established, whereby deoxynucleoside triphosphates are used, thereby releasing the pyrophosphate.

  The term “probe” is also an oligonucleotide in the context of the present invention, but has the following specific function: it hybridizes to other nucleic acids in the reaction mixture, preferably to allow their detection. Accordingly, preferably those probes bind specifically to a particular nucleic acid. The probe preferably has at least one label. The probes can be labeled, for example with different dyes, so that each probe can be detected and measured independently of each other.

A “label”, often referred to as a “reporter group”, is a group that generally makes it possible to distinguish nucleic acids, in particular “oligomer compounds” or “modified oligonucleotides” and any nucleic acids bound to them from the rest of the sample (“label” The nucleic acid to which “is bound” can also be referred to as a labeled nucleic acid binding compound, a labeled probe, or simply a probe). A preferred label according to the present invention is a fluorescent label, which is a “fluorescent dye” such as, for example, a fluorescein dye, a rhodamine dye, a cyanine dye, and a coumarin dye. Preferred “fluorescent dyes” according to the invention are FAM, HEX, CY5, JA270, Cyan, CY5.5, LC-Red.
640, LC-Red 705.

  With respect to the methods described above, the nucleic acid can be present in double-stranded or single-stranded form, so that the double-stranded nucleic acid is denatured by heating (ie, heat-denatured) before the method is performed. ), That is, a single strand.

In another preferred embodiment, the primer and / or probe may be chemically modified, i.e., the primer and / or probe comprises a modified nucleotide or non-nucleotide compound. The probe or its primer is therefore a modified oligonucleotide.

  A “detectable signal” is a signal that is “generated” by a compound such as “microbial nucleic acid”, “internal control nucleic acid” or “qualitative standard nucleic acid” and that makes the compound distinguishable from the rest of the sample. It is. According to the present invention, the “detectable signal” can be quantified or analyzed in a qualitative manner. A “detectable signal” can be, for example, radioactive or optical, such as a luminescent signal. A preferred “detectable signal” according to the present invention is a fluorescent signal emitted by a “fluorescent dye”.

  “Inhibition” or “suppression” of a PCR reaction refers to a PCR reaction that is less efficient compared to the standard reaction observed in the majority of samples. The “inhibiting” or “suppressing” effect may be due to a reduced fluorescence level of the growth curve for the control and / or target, at a delayed CT value, at a change in the slope of the growth curve, It can be seen either in the change in characteristics. Its “inhibition” or “suppression” effect may be due to altered sample preparation effectiveness, sample-related effects, the presence of potentially and often unpredictable factors that inhibit the amplification and / or detection reactions, and other This can result in a reason. Thus, microbial nucleic acids to be detected and quantified may actually be present in the sample while the sample is judged negative due to unsuccessful extraction procedures or other sample-based factors .

  “Generating” means providing directly or indirectly. Thus, in the context of “detectable signal”, “generating” means “directly bringing on” or “inducing” or “inducing”, for example in the case of a fluorescent dye that emits a fluorescent signal. Means “indirectly”; eg, “detectable signal” by a “label” such as “fluorescent dye” or by a nucleic acid probe having a “label” such as “fluorescent dye” "Generate" "microbial nucleic acid".

  As known to those skilled in the art, the terms “specific” or “specifically hybridize” in the context of primers and probes refer to conditions under which a “specific” primer or probe for a separate nucleic acid is stringent. In the sense that it binds to the nucleic acid. Preferably, the primers and probes used in the method according to the invention are at least 80% identical to a part of the sequence of the microbial nucleic acid and / or the first and second control nucleic acids.

“Polymerase chain reaction” (PCR) is disclosed in US Pat. Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188, among other references, and is the most preferred nucleic acid for use in the method according to the present invention. Amplification technique. PCR typically uses two or more oligonucleotide primers that bind to a selected nucleic acid template (eg, DNA or RNA). Primers useful in the present invention include oligonucleotides that can act as a starting point for nucleic acid synthesis within the nucleic acid sequence of a microbial or quantitative standard nucleic acid. The primer can be purified from restriction digests by commonly used methods, or it can be produced synthetically. The primer is preferably single stranded for maximum efficiency in amplification, but the primer can be double stranded. First, the double-stranded primer is denatured, that is, treated to separate the strands. One method of denaturing double stranded nucleic acids is by heating. A “thermostable polymerase” is a polymerase enzyme that is thermostable, ie it catalyzes the formation of a primer extension product complementary to the template and the time required for the denaturation of the double-stranded template nucleic acid to be achieved. It is an enzyme that does not irreversibly denature when exposed to high temperatures. In general, the synthesis is initiated at the 3 ′ end of each primer and proceeds in the 5 ′ to 3 ′ direction along the template strand. Thermostable polymerases are described in Thermus flavus, T. et al. T. ruber, T. T. thermophilus, T. et al. Aquaticus, T. aquaticus T. lacteus, T. It has been isolated from T. rubens, Bacillus stearothermophilus, and Methanothermus fervidus. Nevertheless, polymerases that are not thermostable can also be used in PCR assays if the enzyme is supplemented. If the template nucleic acid is double stranded, it is necessary to separate the two strands before it can be used as a template in PCR. Strand separation can be accomplished by any suitable denaturing method including physical, chemical or enzymatic means. One method of separating the nucleic acid strands is to have the nucleic acid denatured to a large extent (eg, more than 50%, 60%, 70%, 80%, 90% or 95% denatured). With heating. The heating conditions necessary to denature the template nucleic acid will depend on, for example, the buffer salt concentration and the length and nucleotide composition of the nucleic acid to be denatured, but typically the reaction conditions such as temperature and nucleic acid length. Depending on the characteristics, the time ranges from about 90 ° C. to about 105 ° C. Denaturation is typically performed for about 3 seconds to 4 minutes (eg, 5 seconds to 2 minutes 30 seconds, or 10 seconds to 1.5 minutes). When the double-stranded template nucleic acid is denatured by heat, the reaction mixture is cooled to a temperature that promotes the annealing of each primer to its target sequence on the microbial nucleic acid and / or internal standard nucleic acid. The annealing temperature is usually from about 35 ° C to about 75 ° C (eg, from about 40 ° C to about 70 ° C; from about 45 ° C to about 66 ° C). The time for annealing can be from about 5 seconds to about 1 minute (eg, from about 10 seconds to about 50 seconds; from about 15 seconds to about 40 seconds). The reaction mixture is then allowed to elongate from the annealed primer to produce a product complementary to the microbial nucleic acid and / or internal standard nucleic acid at a temperature at which the polymerase activity is promoted or optimized. Adjust to a sufficient temperature. The temperature should be sufficient for the extension product to be synthesized from each primer annealed to the nucleic acid template, but not so high that the extension product is denatured from its complementary template (e.g., its The temperature for stretching is generally in the range from about 35 ° to 80 ° C. (eg, from about 40 ° C. to about 75 ° C .; from about 45 ° C. to about 72 ° C.). The extension time can be from about 5 seconds to about 5 minutes (eg, from about 10 seconds to about 3 minutes; from about 15 seconds to about 2 minutes; from about 20 seconds to about 1 minute). The newly synthesized chain forms a double-stranded molecule that can be used in the next reaction step. The strand separation, annealing, and extension steps can be repeated as many times as necessary to produce the desired amount of amplification product corresponding to the microbial nucleic acid and / or internal standard nucleic acid. The limiting factors in the reaction are the amount of primer, thermostable enzyme, and nucleoside triphosphate present in the reaction. The cycling steps (ie denaturation, annealing, and extension) are preferably repeated at least once. For use in detection, the number of cycle steps will depend, for example, on the nature of the sample. If the sample is a complex mixture of nucleic acids, more cycling steps will be required to amplify sufficient target sequence for detection. Generally, the cycle process is repeated at least about 20 times, but may be repeated as many times as 40, 60, or even 100 times.

For nucleic acid amplification reactions other than PCR, 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; WO 92/08808
), And NASBA (US Pat. No. 5,130,238). In addition, strand displacement amplification (S
DA), transcription-mediated amplification (TMA), and Q-beta-amplification (for review, see, eg, Whelen AC and Persing DH, Annu. Rev. Microbiol. 50 (1996) 349-373; Abramson
RD and Myers TW, Curr Opin Biotechnol 4 (1993) 41-47).

Suitable nucleic acid detection methods are known to those skilled in the art, and Sambrook J. et al., Molecular Cloning:
Described in standard textbooks such as A Laboratory Manual, Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY) 1989, and Ausubel F. et al .: Current Protocols in Molecular Biology 1987, J. Wiley and Sons (New York) Has been. Prior to performing the nucleic acid detection step, there may be a further purification step, for example a precipitation step. Such detection methods may include, but are not limited to, binding or intercalation of specific dyes, such as ethidium bromide that intercalates into double stranded DNA and then changes its fluorescence. Not. The purified nucleic acid may optionally be digested by restriction, separated by electrophoresis, and then visualized. There are also probe-based assays that utilize oligonucleotide hybridization to a specific sequence followed by detection of the hybrid. It is also possible to sequence the nucleic acid after further steps known to those skilled in the art. A preferred template-dependent nucleic acid polymerase is Z05 DNA polymerase and variants thereof. Other template-dependent nucleic acid polymerases useful in the present invention are Taq polymerase and Tth polymerase. Still other nucleic acid polymerases useful in the method according to the invention are known to those skilled in the art.

  In the context of the present invention, each of the microbial nucleic acid and its control nucleic acid binds to different probes with different labels in order to make them distinguishable from each other during detection. Thus, the different probes preferably have different fluorescent dyes and emit different wavelengths of fluorescence. These different fluorescent signals can conveniently be detected independently of each other in different channels of the fluorescence detector as used in most devices for performing real-time PCR.

  Again preferably, the results for the microbial nucleic acid, the first control nucleic acid and the second control nucleic acid are visualized with different masks on one display or on different displays.

  In the sense of the present invention, “purification”, “isolation” or “extraction” of nucleic acids relates to the following: before nucleic acids can be analyzed in one of the above mentioned assays, It must be purified, isolated or extracted from a biological sample containing a complex mixture. Often, for the first step, a process is used that allows the nucleic acid to be concentrated. In order to release the contents of the cells or virus particles, they can be treated with enzymes or chemicals to dissolve, degrade or denature the cell walls or virus particles. This process is commonly referred to as dissolution. The resulting solution containing such dissolved material is called a lysate. A problem often encountered during lysis is that other enzymes that degrade the component of interest, such as deoxyribonucleases or ribonucleases that degrade nucleic acids, come into contact with the component of interest during the lysis procedure. These degrading enzymes may also be present extracellularly or may have been spatially separated into different cell compartments prior to lysis. As lysis occurs, the components of interest become exposed to the degrading enzyme. Other components released during this process, for example, belong to the lipopolysaccharide family, are toxic to cells and cause problems for products intended for use in human or animal therapy. It may be an endotoxin obtained.

There are various ways to address the problems mentioned above. When it is intended to liberate nucleic acids, it is common to use chaotropic agents such as guanidine thiocyanate, or anionic, cationic, zwitterionic or nonionic surfactants. It is also convenient to use the previously described enzymes or proteases that rapidly degrade unwanted proteins. However, this can cause another problem that the substances or enzymes can interfere with reagents or components in later steps.

Enzymes that can be conveniently used in such lysis or sample preparation processes referred to above are those that cleave amide bonds in protein substrates and are classified as proteases or (interchangeably) peptidases ( Walsh, 1979, Enzymatic Reaction Mechanisms. See WH Freeman and Company, San Francisco, Chapter 3. Proteases used in the prior art include alkaline proteases (WO 98/04730) or acidic proteases (US Pat. No. 5,386,024). Proteases that have been widely used for sample preparation in the prior art for nucleic acid isolation in the prior art are active near neutral pH and belong to a family of proteases known to those skilled in the art as subtilisins, the Trityrachium album. (See, for example, Sambrook J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
The enzyme esperase, a strong protease that retains its activity at both high alkalinity and high temperature, is particularly advantageous for use in the lysis or sample preparation processes referred to above (EP 1 201 753).

  In the sample preparation step after the dissolution step, the target component is further concentrated. Where the non-proteinaceous components of interest are, for example, nucleic acids, they are usually extracted from the complex lysis mixture before they are used in probe-based assays.

There are several methods for the purification of nucleic acids:
A sequence-dependent or biospecific method, for example as follows:
Affinity chromatographyHybridization to immobilized capture oligonucleotides--sequence-independent or physicochemical methods, for example:
Liquid-liquid extraction with, for example, phenol-chloroform, precipitation with, for example, pure ethanol, extraction with filter paper, extraction with a micelle-forming agent such as cetyl-trimethyl-ammonium-bromide, fixed intercalating dyes, eg acridine derivatives Adsorption on silica gel or diatomaceous earth Adsorption on magnetic glass particles (MGP) or organosilane particles under chaotropic conditions.

Although other surfaces are possible, the adsorption of nucleic acids to the glass surface is of particular interest for purification purposes. Many procedures have recently been proposed for isolating nucleic acids from their natural environment using the behavior of nucleic acids binding to glass surfaces. When an unmodified nucleic acid is the target, the nucleic acid does not need to be modified, and even natural nucleic acids can bind, among other reasons, so that direct binding of the nucleic acid to a material having a silica surface is possible. preferable. These processes are described in detail in various literature. For example, Vogelstein B. et al., Proc. Natl. Acad. USA 76 (1979) 615-9
Proposed a procedure for binding nucleic acids from an agarose gel to ground flint glass in the presence of sodium iodide. Purification of plasmid DNA from bacteria on glass powder in the presence of sodium perchlorate is described in Marko MA et al., Anal. Biochem. 121 (1982) 382-387. DE-A 37 34 442 describes the isolation of single-stranded M13 phage DNA on glass fiber filters by precipitation of phage particles with acetic acid and lysis of the phage particles by perchlorate. The nucleic acid bound to the glass fiber filter is washed and then eluted with methanol containing Tris / EDTA buffer. A similar procedure for purifying DNA from lambda phage is described in Jakobi R. et al., Anal. Biochem. 175 (1988) 196-201. The procedure involves the selective binding of nucleic acids to the glass surface in chaotropic salt solutions and the separation of nucleic acids from contaminants such as agarose, proteins or cell residues. To separate the glass particles from contaminants, the particles can be processed either by centrifuging or by drawing the fluid through a glass fiber filter. However, this is a limiting step that prevents the procedure from being used to process large quantities of samples. The use of magnetic particles to immobilize nucleic acid after precipitation by adding salt and ethanol is more convenient, eg Alderton
RP et al., S., Anal. Biochem. 201 (1992) 166-169 and PCT GB 91/00212. In this procedure, nucleic acids are aggregated with magnetic particles. The aggregate is separated from the original solvent by applying a magnetic field and performing a washing step. After one washing step, the nucleic acid is dissolved in Tris buffer. However, this procedure has a disadvantage in that the precipitate is not selective for nucleic acids. On the contrary, various solid and dissolved substances are agglomerated as well. As a result, this procedure cannot be used to remove any significant amount of any inhibitor of specific enzymatic reactions that may be present. Magnetic porous glass containing magnetic particles in a specific porous glass matrix and covered with a layer containing streptavidin is also commercially available. This product can be used to isolate biological materials such as proteins or nucleic acids when they are modified in a complex preparation process such that they are covalently linked to biotin. Certain adsorbents that are magnetizable have proven very efficient and suitable for automated sample preparation. Ferrimagnetic and ferromagnetic as well as superparamagnetic pigments are used for this purpose. The most preferred method using MGP and magnetic glass particles is the method described in WO 01/37291. Particularly useful in the context of the present invention for isolation of nucleic acids is R. Boom et al. (J Clin Microbiol. 28 (1990),
495-503).

After the nucleic acid containing the target nucleic acid is purified and isolated from their natural environment, the target nucleic acid can be detected.
In one embodiment, the method of the present invention includes a step to avoid contamination. For example, enzymatic methods utilizing uracil-DNA glycosylase to reduce or eliminate contamination between one thermal cycler run and the next run are described in US Pat. Nos. 5,035,996, 5,683,896, and 5,945,313. Described in. In addition, standard laboratory containment practices and procedures are desirable when performing the methods of the present invention. Containment practices and procedures include, but are not limited to, separate work areas for different steps of the method, containment hoods, barrier filter pipette tips, and dedicated air displacement pipettes. Consistent containment practices and procedures by personnel are necessary for accuracy in diagnostic laboratories handling clinical samples.

The method described above is preferably based on fluorescence resonance energy transfer (FRET) between a donor fluorescent moiety and an acceptor fluorescent moiety. An exemplary donor fluorescent moiety is fluorescein, and exemplary corresponding acceptor fluorescent moieties include LC-Red 640, LC-Red 705, Cy5, and Cy5.5. Typically, the detection step involves exciting the sample at a wavelength that is absorbed by the donor fluorescent moiety and visualizing and / or measuring the wavelength emitted by the corresponding acceptor fluorescent moiety. According to the present invention, FRET is quantified after the detection. Preferably, the detection step is performed after each cycle step. Most preferably, the detection step is performed in real time. By using a commercially available real-time PCR instrument (eg, LightCycler ™ or TaqMan ™), PCR amplification and detection of the amplified product can be performed in a single sealed reaction compartment, eg, a cuvette. They can be combined and the cycle time is dramatically reduced. Since detection occurs simultaneously with amplification, real-time PCR eliminates the need for manipulation of amplification products and reduces the risk of cross-contamination between amplification products. Real-time PCR greatly reduces processing time and is an attractive alternative to conventional PCR techniques in clinical laboratories.

The following patent applications describe real-time PCR as used in the LightCycler ™ technology: WO 97/46707, WO 97/46714 and WO 97/46712. Li
The ghtCycler ™ instrument is a rapid thermal cycler combined with a microfluorometer that utilizes 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 alternately using heated air and ambient air. Due to the low mass of air and the high surface area to cuvette volume, very rapid temperature exchange rates can be achieved in the thermal chamber.

  TaqMan® technology utilizes a single stranded hybridization probe labeled with two fluorescent moieties. When the first fluorescent moiety is excited with light of the appropriate wavelength, the absorbed energy is transferred to the second fluorescent moiety according to the FRET principle. The second fluorescent moiety is generally a quencher 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 extension step, Taq or another suitable polymerase known to those skilled in the art, such as It is degraded by the 5 ′ to 3 ′ exonuclease activity of the preferred Z05 polymerase. As a result, the excited fluorescent and quencher moieties are spatially separated from each other. As a result, fluorescence emission from the first fluorescent moiety can be detected when the first fluorescent moiety is excited in the absence of the quencher.

In both detection formats described above, the intensity of the emitted signal can be correlated with the number of original target nucleic acid molecules.
As an alternative to FRET, amplification products can be linked to fluorescent DNA binding dyes (eg
It can be detected using double stranded DNA binding dyes such as I® or SYBRGOLD® (Molecular Probes). Upon interaction with double stranded nucleic acids, such fluorescent DNA binding dyes emit a fluorescent signal after being excited by light of the appropriate wavelength. Double-stranded DNA binding dyes such as dyes that intercalate with nucleic acids can also be used. When using a double-stranded DNA binding dye, a melting curve analysis is usually performed to confirm the presence of the amplification product.

  Molecular beacons associated with FRET can also be used for detection of the presence of amplification products using the real-time PCR method of the present invention. Molecular beacon technology uses a hybridization probe labeled with a first fluorescent moiety and a second fluorescent moiety. The second fluorescent moiety is generally a quencher and the fluorescent label is typically located at each end of the probe. Molecular beacon technology uses probe oligonucleotides with sequences that allow secondary structure formation (eg, hairpins). As a result of secondary structure formation within the probe, both fluorescent moieties are in spatial proximity when the probe is in solution. After hybridization to the amplification product, the secondary structure of the probe is broken and the fluorescent moieties are separated from each other, and thus the emission of the first fluorescent moiety after excitation with light of the appropriate wavelength. Can be detected.

  Accordingly, a preferred method according to the present invention is the method using FRET as described above, wherein the probe comprises a nucleic acid sequence that allows spatial proximity between the first and second fluorescent moieties. .

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

Thus, in a preferred embodiment of the invention, the donor and acceptor fluorescent moieties are within a range of no more than 5 nucleotides of each other on the probe.
In a further preferred embodiment, the acceptor fluorescent moiety is a quencher.

  As described above, in the TaqMan format, the labeled hybridization probe binds to its target nucleic acid (ie, amplification product) during the annealing step of the PCR reaction, and is known to Taq or the skilled person during the subsequent extension step. Is degraded by the 5 'to 3' exonuclease activity of other suitable polymerases, such as the preferred Z05 polymerase.

Thus, in a preferred embodiment, the method according to the invention uses a polymerase enzyme having a 5 ′ to 3 ′ exonuclease activity for amplification.
The method according to the invention can be advantageously applied to viral nucleic acids. Thus, in a preferred embodiment of the invention, the microbial nucleic acid is a viral nucleic acid.

  Among viral nucleic acids, the method according to the invention can be advantageously applied to HCV. Accordingly, in a preferred embodiment of the invention, the microbial nucleic acid is an HCV nucleic acid. However, it should be understood that the present invention is applicable to any other microbial nucleic acid.

An example of how to calculate quantitative results in TaqMan format based on internal standards is described below. The titer is calculated from instrument corrected fluorescence input data from all PCR runs. PCR is performed on a set of samples containing a target nucleic acid, eg, a microbial nucleic acid, and a first control nucleic acid that serves as an internal quantitative standard nucleic acid on a thermal cycler using a temperature profile as specified. At a selected temperature and time during the PCR profile, the sample is irradiated with filtered light and filtered fluorescence data for the target nucleic acid and the internal quantification standard nucleic acid is collected for each sample. After the PCR run is complete, the fluorescence readings are processed to obtain a set of dye concentration data for the internal quantitative standard nucleic acid and a set of dye concentration data for the target nucleic acid. Each set of dye density data is processed in the same manner. After several plausibility checks, an elbow value (CT) is calculated for the internal quantitative standard nucleic acid and its target nucleic acid. The elbow value is defined as the point where the fluorescence of the target nucleic acid or the internal quantitative standard nucleic acid crosses a predetermined threshold (fluorescence concentration). The titer determination is based on the assumption that the target nucleic acid and the internal quantification standard nucleic acid are amplified with the same efficiency, and an equal amount of target nucleic acid and quantification standard nucleic acid amplicon copies are amplified at the calculated elbow value, Based on the assumption that it will be detected. Thus, (CTQS-CT target) is linear with respect to log (target concentration / QS concentration), where “QS” represents its internal quantitative standard nucleic acid. Then, for example, the following equation:
T ′ = 10 (a (CTQS-CT target) 2 + b (CTQS-CT target) + c)
The titer T can be calculated by using a polynomial calibration equation such as

The constant of this polynomial and the concentration of its internal quantitative standard nucleic acid are known, so the only variable in the equation is the difference (CTQS-CT target).
An example of how to calculate qualitative results in TaqMan is described below: Analyze instrument corrected fluorescence input data from all PCR runs. PCR is performed on a thermal cycler using a temperature profile as specified, possibly against a set of samples containing a target nucleic acid, eg, a microbial nucleic acid, and a control nucleic acid that serves as a qualitative internal control nucleic acid. At a selected temperature and time during the PCR profile, the sample is irradiated with filtered light and filtered fluorescence data for the target nucleic acid and the qualitative internal control nucleic acid is collected for each sample. After the PCR run is complete, the fluorescence readings are processed to obtain a set of dye concentration data for the qualitative internal control nucleic acid and a set of dye concentration data for the target nucleic acid. Each set of dye density data is processed in the same manner. An elbow value (CT) is calculated for the qualitative internal control nucleic acid and, if present, the target nucleic acid. The elbow value is defined as the point where the fluorescence of the target nucleic acid or the internal quantitative standard nucleic acid crosses a predetermined threshold (fluorescence concentration). The qualitative internal control is effective if a CT within the specified range is obtained and the fluorescence exceeds a predetermined minimum fluorescence intensity. If no target CT is obtained, the qualitative internal control must be valid. If a target CT is obtained, it indicates the presence of the target nucleic acid in the sample and the qualitative internal control may be either valid or ineffective.

Any of the above methods are preferred within the meaning of the present invention, wherein the first and second control nucleic acids are provided in one control reagent.
For reliable qualitative detection and at the same time reliable quantification, the concept of a control reagent comprising different concentrations of the first and second control nucleic acids is advantageously used in any respective assay targeting microbial nucleic acids. be able to.

  Accordingly, another aspect of the present invention is the use of different concentrations of first and second control nucleic acids to simultaneously detect and quantify microbial nucleic acids by real-time PCR. The use described above is further preferred, wherein the first and second control nucleic acids are amplified with the same or different primer pairs but hybridize to different probes.

  The use of this concept in the process according to the invention is particularly preferred. Accordingly, in another aspect, the present invention relates to the use of different concentrations of first and second control nucleic acids for the simultaneous detection and quantification of microbial nucleic acids according to the method (s) described above.

  The present invention also provides a kit for the simultaneous detection and quantification of microbial nucleic acids in a biological sample by real-time PCR, which kit comprises different concentrations of first and second control nucleic acids, separate microbial nucleic acids as described above. One or more primer pairs that specifically hybridize to the sequence portion and to separate sequence portions of the first and second control nucleic acids, respectively, and sequences amplified by these one or more primer pairs, respectively Wherein the first and second control nucleic acids are provided in one control reagent.

  In a preferred embodiment, the present invention provides a kit for simultaneously detecting and quantifying microbial nucleic acids in a biological sample according to any of the methods described above, wherein the kit comprises first and second concentrations at different concentrations. One or more primer pairs that specifically hybridize to a control nucleic acid, to separate sequence portions of said microbial nucleic acid, and to separate sequence portions of said first and second control nucleic acids, and one or more thereof Probes that specifically hybridize to each of the sequences amplified by the primer pairs.

  A further preferred aspect of the invention is a kit as described above, wherein the first and second control nucleic acids are amplified by the same primer pair but hybridize to different probes, thus providing a competitive configuration. To do. However, it should be understood that kits for performing non-competitive or partially competitive assays and their respective methods are also included in the present invention.

  Such kits may further include plastic products that can be used during the sample preparation procedure, such as microtiter plates in 96 or 384 well format or conventional reaction tubes (such as Eppendorf (Germany), as is known in the art. And all other reagents for carrying out the process according to the invention. Thus, the kit can additionally contain a material having an affinity for nucleic acids, preferably the material having an affinity for nucleic acids comprises a material having a silica surface. Preferably, the material having a silica surface is glass. Most preferably, the material having an affinity for nucleic acids is a composition comprising magnetic glass particles. The kit can additionally or additionally include a protease reagent and a lysis buffer that allows lysis of the cells, eg containing chaotropic agents, detergents or alcohols or mixtures thereof. These components of the kit according to the invention may be provided separately in tubes or storage containers. Depending on the nature of their components, they can even be provided in a single tube or storage container. The kit additionally or additionally includes a washing solution suitable for the washing step of the magnetic glass particles when the nucleic acid is bound to the magnetic glass particles. This washing solution may contain ethanol and / or chaotropic agent in the buffer, as described above, or it may be a solution having an acidic pH without ethanol and / or chaotropic agent. Often, the wash solution or other solution is provided as a stock solution that must be diluted before use. The kit additionally or additionally contains an eluent or elution buffer, ie a solution or buffer (e.g. 10 mM Tris, 1 mM EDTA, pH 8.0) or pure water for eluting nucleic acids bound to the magnetic glass particles. May contain. There may also be additional reagents or buffers that can be used for the nucleic acid purification process.

Preferably, the kit comprises a polymerase enzyme having 5 ′ to 3 ′ exonuclease activity. It is also preferred that the kit contains an enzyme having reverse transcription activity.
In another preferred embodiment, the kit comprises a polymerase enzyme having 5 ′ to 3 ′ exonuclease activity and reverse transcription activity.

  In a preferred embodiment of the invention, the method according to the invention is incorporated into a series of methods carried out in the analytical system, thereby preferably forming an automatable process.

Accordingly, preferred aspects of the present invention are:
An analytical system for simultaneously detecting and quantifying microbial nucleic acids in a biological sample by real-time PCR, the system comprising:
A sample preparation module comprising a lysis buffer and a vessel for isolating and purifying said microbial nucleic acid, an amplification and detection module comprising a reaction vessel in which the method described above is carried out, as described above kit.

  The sample preparation module advantageously comprises components for the sample preparation procedures described above, i.e., for example, magnetic glass particles and magnets for separating them from solution, protease reagents, chaotropic salt solutions, and crude One or more containers containing the sample and reagents necessary for sample preparation can be included.

  The reaction vessel in the amplification and detection module can be, for example, a microtiter plate, a centrifuge vial, a lysis tube, or any other type of vessel suitable for containing a reaction mixture according to the present invention.

In a preferred embodiment of the present invention, the analysis system includes a storage module that contains reagents for performing the method of the present invention.
The storage module can further include other components useful in the methods of the present invention, such as disposable items such as pipette tips or even containers for use as reaction vessels in amplification and detection modules.

  In a preferred embodiment of the invention, the analysis system includes a preanalytical system module for transferring a biological sample from the initial tube to a container usable on the analysis system.

In yet another preferred embodiment of the invention, the analytical system includes a transfer module for transferring a biological sample from the sample preparation module to the amplification and detection module.
Even though it is possible to carry out the movement manually, it is preferable to use an automated system in which the movement is carried out, for example by means of a robotic device, for example an electric mobile rack or a robot pivot arm.

  An automatable process means that the process steps are suitable for implementation with an apparatus or machine that can operate with little or no external control or influence by a person. An automated method means that the steps of the automatable method are performed using a device or machine that can operate with little or no external control or influence by a person. Only the preparatory steps for the method may have to be performed manually, e.g. the storage container must be filled and installed, sample selection and further steps known to those skilled in the art, e.g. operation of the control computer The person has to do. The device or machine can, for example, automatically add liquid, mix the sample, or perform a warming process at a specific temperature. Typically, such a machine or device is a robot controlled by a computer that executes a program in which a single process and command are specified.

Thus, preferably, the analysis system according to the invention further comprises a control unit for controlling the components of the system.
Such a control unit allows the various components of the analytical system to work and interact accurately and with precise timing, for example moving components such as samples to the reaction module in a coordinated manner. Software to ensure that can be included. The control unit may also include a processing unit that runs a real-time operating system (RTOS), which is a multitasking operating system intended for real-time applications. In other words, the system processor can manage real-time constraints, i.e., operational deadlines from event to system response, unrelated to system load. It controls in real time that the various units in the system operate and respond accurately according to the given instructions.

  Specific descriptions of all other preferred embodiments and embodiments of the uses, kits and analysis systems according to the present invention are those with respect to the method according to the present invention.

Example 1: First Control Nucleic Acid is Evaluated According to Different Criteria to Obtain Qualitative and Quantitative Results Output COBAS AmpliPrep / COBAS TaqMan HBV, HCV and HIV-1 tests are commercially available real-time PCR tests And they have been optimized for accurate quantification of the viral targets HBV, HCV and HIV-1. These assays are fully automated COBAS Amplis that include a docking station for automated transfer of the PCR reaction mixture from the sample preparation unit to the amplification / detection unit.
Used either on a Prep / COBAS TaqMan system or on a COBAS AmpliPrep / COBAS TaqMan system with manual transfer or on a COBAS AmpliPrep / COBAS TaqMan48 system.

The COBAS AmpliPrep / COBAS TaqMan HCV test was used as an example to study an approach to provide simultaneous quantification and qualitative test results using a set of identical reagents. The test has a detection limit of 15 IU / mL and exhibits the highest level of sensitivity comparable to or better than the quantitative HCV test on the market as well as the qualitative HCV test. Because of this high sensitivity, it has been attempted to use the test as a qualitative HCV test. The main focus of quantitative testing is to provide accurate HCV titers, while the main focus of qualitative testing is to ensure high sensitivity of the assay. Since the COBAS AmpliPrep / COBAS TaqMan HCV test was originally developed to quantitatively monitor the success of the treatment, it can be used to provide reliable qualitative results without any modification. Analysis was carried out. A review of the data revealed that in rare cases of less efficient PCR, a sensitivity of 15 IU / mL was not guaranteed by the quantitative test in its current data analysis settings. An example of a PCR reaction and a failed target (2183 IU / mL) detection showing inhibited but valid QS results is shown below:
The inhibited PCR reaction that resulted in effective results because it was an effective QS, and the “no target detected” results are shown in FIG. 4a. The QS was effective because it exceeded the fluorescence threshold setting corresponding to 0.8 relative fluorescence units.

Repeat testing of the same sample showed a standard PCR reaction and an HCV titer result of 2183 IU / mL (Figure 4b).
Therefore, the quantitative COBAS AmpliPrep / COBAS TaqMan HCV test without any changes cannot be used as a qualitative test.

  Using the tests referred to above, the IC / QS control nucleic acid data analysis parameter settings were optimized to achieve a reliable qualitative result output. In order to demonstrate general applicability, the optimization was additionally performed for all three types of commercial HBV, HCV and HIV-1 assays on the COBAS AmpliPrep / COBAS TaqMan system. By significantly increasing the minimum relative fluorescence intensity (RFImin) threshold at that parameter setting, the sensitivity of all three types of quantitative tests can be used to ensure that they yield reliable qualitative output. Become. The relative parameter settings are shown in Table 1 below:

.
A qualitative RFImin setting of 9.0 would negate the inhibited PCR reaction in the example shown above for HCV.

  The above setting of RFImin for the output of qualitative results, as shown in FIG. 1a, is that the presence of the target can affect the fluorescence intensity of its IC / QS growth curve, so Cannot be used simultaneously for output. As a result, the qualitative RFImin setting results in a high percentage of IC / QS ineffective responses in the presence of high target concentrations. In contrast, since the IC must not be effective in the presence of the target, this does not affect the effectiveness of its qualitative output. However, the effectiveness of the quantitative reaction is significantly affected.

  Thus, the same test reagent with one IC / QS provides reliable qualitative results output with guaranteed sensitivity of the test and reliable quantitative testing with acceptable low level QS invalid results. When used to provide both results, this can only be achieved by analyzing the raw data using two different sets of parameter settings as proposed herein.

Example 2: Different concentrations of first and second control nucleic acids are used to obtain quantitative and qualitative results output with the same test reagent. The goal is to provide reliable qualitative results with the same set of test reagents. Providing both reliable output and reliable quantitative test results. In this approach to address this goal, the reagent containing the internal control nucleic acid comprises a formulation with two different armored RNA particles. The following reagents can be used for this experiment:
a) COBAS AmpliPrep / COBAS TaqMan HCV test sample preparation reagents (magnetic particle suspension; protease solution, lysis buffer; elution buffer);
b) QS armored particles at a concentration level of about 1000 copies / reaction, and IC armored particles at a concentration level of about 100 copies / reaction. The generation of armored RNA particles is shown in Figure 2 below. The transcript encapsulated by the armored particle has different primer binding sites and different probe binding sites. These probes were combined with black hole quencher BHQ along with fluorescently labeled HEX and C
Labeled with Y5;
c) Magnesium reagent;
d) Master mix reagents including:
-Z05 DNA polymerase and UNG
-3 different primer pairs: for target HCV, for first control nucleic acid, and for second control nucleic acid -3 different probes: for targets labeled with FAM and BHQ, labeled with HEX and BHQ Against the first control nucleic acid, and against the second control nucleic acid labeled with CY5 and BHQ,
-Aptamer-dNTP (dUTP, dATP, dCTP, dGTP, dTTP)
-Master mix buffer component (potassium acetate, glycerol, tricine, DMSO, betaine, IGEPAL, water).

The sample preparation and amplification / detection steps are as follows: COBAS AmpliPrep / COBAS
Performed using sampling parameters and PCR files established for TaqMan HCV testing. Growth curves for these three different fluorescent dyes were determined and are shown in FIG.

  For HCV samples, to obtain a quantitative result output, the titer is determined as follows: After the PCR run is complete, the fluorescence readings are processed to 1 for the QS nucleic acid (HEX fluorescence label). A set of dye concentration data, a set of dye concentration data for the qualitative standard nucleic acid (Cy5 fluorescent label), and a set of dye concentration data for the target nucleic acid (FAM fluorescent label) are obtained. All three sets of dye density data are processed in the same manner. Elbow values (CT) are calculated for the quantitative and qualitative standard nucleic acids and the target nucleic acid. The elbow value is defined as the point where the target nucleic acid or those two internal control nucleic acids cross a predetermined threshold (fluorescence concentration).

For output of quantitative results, titer determination is performed by analyzing only dye concentration data for HEX and FAM. The titer determination is based on the assumption that the target nucleic acid and the QS nucleic acid are amplified with the same efficiency, and equal amounts of target nucleic acid and QS nucleic acid amplicon copies are amplified and detected at the calculated elbow value. Based on the assumption. Therefore, (CTQS-CT target) is linear with respect to log (target concentration / QS concentration). Then, for example, the following equation:
T ′ = 10 (a (CTQS-CT target) 2 + b (CTQS-CT target) + c)
The titer T can be calculated by using a polynomial calibration formula such as in where the only variable in the formula is the difference (CTQS-CT target).

  For output of qualitative results, only dye concentration data for CY5 and FAM is analyzed. By comparing the dye concentration data for CY5 and FAM, the software determines whether the PCR reaction is target negative or target positive. If the PCR reaction is target positive, the IC result is ignored, so it may be valid or invalid. If the PCR reaction is target negative, the result is valid only if the IC shows a valid result. In the examples shown above, all reactions contain low levels of HCV target—although not related to the effectiveness of their respective test results—all reactions show effective IC.

Claims (4)

A method for the simultaneous detection and quantification of microbial nucleic acids in a biological sample, comprising:
a) isolating and purifying said microbial nucleic acid;
b) different concentrations of the first and second control nucleic acids, wherein the first control nucleic acid is a quantitative standard nucleic acid and the second control nucleic acid is a qualitative internal control nucleic acid, in separate sequence portions of the microbial nucleic acid, and A reaction mixture comprising one or more primer pairs that specifically hybridize to separate sequence portions of a control nucleic acid and a probe that specifically hybridizes to each of the sequences amplified by the one or more primer pairs Wherein the first control nucleic acid is present at a concentration of 20 to 5000 times the detection limit of the microbial nucleic acid, and the second control nucleic acid is 1 to 10 times the detection limit of the microbial nucleic acid. The microbial nucleic acid and the first control nucleic acid and the second control nucleic acid hybridize to different probes having different labels ;
c) adding the isolated and purified microbial nucleic acid to the reaction mixture;
d) performing one or more cycle steps, wherein the cycle step comprises an amplification step, the amplification step comprising one or more amplification products derived from said microbial nucleic acid if present in said sample; And generating an amplification product derived from the first control nucleic acid and the second control nucleic acid, wherein the cycling step comprises a hybridization step, wherein the hybridization step comprises said primer pair Hybridizing the sequences amplified by the above probes, wherein the probes are labeled with a donor fluorescent moiety and a corresponding acceptor fluorescent moiety, each of the probes having a different fluorescent dye;
e) Detect and measure the fluorescence signals proportional to their concentration produced by the amplification product of the first control nucleic acid and the microbial nucleic acid, and at the same time produced by the amplification product of the second control nucleic acid. detecting a fluorescence signal, wherein the presence of said second control nucleic acid amplification product indicates that amplification occurs in the reaction mixture even in the absence of an amplification product on microbial nucleic acids of the, and the The amount of the microbial nucleic acid in the biological sample is determined by comparing the signal produced by the microbial nucleic acid and the first control nucleic acid . The method of claim 1 , wherein the amplification uses a polymerase enzyme having 5 'to 3' exonuclease activity. 3. The method according to claim 1 or 2 , wherein the first and second control nucleic acids are provided in one control reagent. 4. The method of any one of claims 1-3, wherein the first control nucleic acid and the second control nucleic acid have the same primer binding site but different probe binding sites.

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