WO2010060728A1 - Procédé de calibrage et de contrôle - Google Patents

Procédé de calibrage et de contrôle Download PDF

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Publication number
WO2010060728A1
WO2010060728A1 PCT/EP2009/064366 EP2009064366W WO2010060728A1 WO 2010060728 A1 WO2010060728 A1 WO 2010060728A1 EP 2009064366 W EP2009064366 W EP 2009064366W WO 2010060728 A1 WO2010060728 A1 WO 2010060728A1
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WO
WIPO (PCT)
Prior art keywords
analyte
sub
calibration
assay
control
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Application number
PCT/EP2009/064366
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English (en)
Inventor
David Pritchard
Denise Barrault
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Iti Scotland Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Iti Scotland Limited filed Critical Iti Scotland Limited
Priority to EP09749063A priority Critical patent/EP2358896A1/fr
Publication of WO2010060728A1 publication Critical patent/WO2010060728A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions

Definitions

  • This invention relates to a method for the calibration and/or control of an assay for an analyte, which reduces the number and/or complexity of the separate processes required to perform and control (and/or calibrate) the assay.
  • the invention is particularly advantageous, since it allows separation of specific analyte-dependent and analyte-independent control (and/or calibration) processes. In other words, the invention splits the control process to allow at least part of that process to be directly integrated into the assay.
  • Many assay processes comprise a multiplicity of steps, sub-processes or processes, that are may carried out in a linear fashion (a sequential set of steps in serial with one another) or in parallel (two or more sets of such steps).
  • a sample may initially undergo preparation steps using certain starting materials; e.g. the analyte, or a component representative of the analyte, may be purified or captured.
  • the analyte may be processed in some manner, for example to react it with a label.
  • a result is reported based upon measurement.
  • assays which are comprised of these linear processes are more easily calibrated and controlled, because a single model process can be developed to mimic all of the steps of the assay, and provide the correct control and calibration adjustments when reporting the result.
  • the control and calibration processes are run in parallel with the assay processes, so that the components of the control and calibration process do not interfere with the assay result.
  • This although providing satisfactory control and calibration, requires two processes to be run in parallel (the assay and the control), with two sets of apparatus, two sets of reagents and in some cases two sets of detection equipment. The attendant space and cost implications can be problematic, particularly in the microfluidic and nanofluidic fields.
  • not all assays are as simple as the ones highlighted above.
  • some assays involve not only the detection of an analyte, but also involve characterisation of its type, effectively providing two separate pieces of information in the result.
  • These assays may need several different processes to determine the relevant pieces of information, and these processes may not necessarily be controllable using the same reagents, organisms or conditions for each process and sub-process.
  • some assays although providing only a single piece of information, require several successive processes and sub-processes that are themselves quite different; these processes and sub-processes may also not be compatible from a control perspective.
  • the control and calibration of such assays is potentially even more problematic, requiring more than one parallel control or calibration process, putting further strain on resources.
  • Such problems are particularly acute when an analyte comprising a nucleic acid is involved.
  • Such methods include, inter alia the detection of pathogens, such as bacteria and viruses, where the pathogen may be detected by probing for nucleic acid present in the pathogen.
  • pathogens such as bacteria and viruses
  • Such methods involve the amplification of nucleic acid, a sub-process which is very different from the preparation of the pathogen.
  • the present invention aims to solve the problems set out above, and in particular aims to provide an assay method for a complex assay that incorporates full calibration and control of the assay, preferably in a microfluidic or nanofluidic environment. It is a particular aim to provide an assay that enables point-of-care analysis of nucleic acids, a complicated process with many steps including sample preparation, target amplification and signal transduction. It is highly desirable to provide integrated control and calibration to ensure that each assay is valid and that erroneous results are not reported.
  • the present invention provides a method for the calibration and/or control of an assay for an analyte, wherein the calibration and/or control method comprises at least:
  • a second calibration and/or control process for calibration and/or control of a second assay process or assay sub-process; wherein the analyte is employed in either the first or the second calibration and/or control process, but is not employed in the other calibration and/or control process, and wherein the calibration and/or control process not employing the analyte is integrated with the assay.
  • the analyte is not especially limited.
  • the analyte may be a complex system having many components (such as a bacterium, a virus or other pathogen) or a molecule of interest (such as a protein, nucleic acid or a small molecule) or an entity indicative of the presence of another species (e.g. antibodies indicative of the presence of an infective agent).
  • a molecule of interest such as a protein, nucleic acid or a small molecule
  • an entity indicative of the presence of another species e.g. antibodies indicative of the presence of an infective agent.
  • an assay is directed at determining the presence or absence (or quantity or type) of a pathogen
  • the term "analyte" encompasses anything that is indicative of that pathogen, including: the entire pathogen (e.g. the entire bacterium or virus); any component of the pathogen (e.g.
  • analyte encompasses the HCV virus particle, HCV surface proteins, HCV RNA, DNA formed from HCV RNA by reverse transcription and anti-HCV antibodies.
  • the assay of the present invention will typically employ only one of the above-described components and entities as the 'analyte'.
  • any assay process (or sub-process) that does not employ the analyte is analyte-independent
  • any assay process (or sub-process) that does employ the analyte is analyte-dependent.
  • a process for controlling the sample preparation steps which uses virus pseudoparticles containing GFP RNA will be analyte-independent (since it does not employ HCV RNA).
  • a process for controlling RNA reverse transcription and amplification that uses pre-prepared HCV RNA will be analyte-dependent (because HCV RNA is present).
  • processes that are analyte-dependent are ones in which the analyte that is detected is present. These processes will affect the result of the assay because they contain the detected analyte. In contrast, processes that are analyte-independent do not contain the detected analyte, and will thus not affect the result. Because of this, any analyte-independent control processes may be integrated with the assay itself, whilst analyte dependent processes should remain separate.
  • the analyte that is involved in detection may be controlled in a second separate (usually smaller or less complex) calibration or control process that is separated either spatially (performed in parallel with the integrated process, in separate channels and/or reaction zones) or temporally (performed in series with the assay i.e. in the same channels and/or reaction zones, but at a different time from the assay).
  • the invention has particular benefit for microfluidic devices where space (e.g. real estate on a chip) is at a premium, because the number of parallel channels and reaction zones can be reduced.
  • the invention relates to a method for the calibration and/or control of an assay for an analyte, which is capable of reducing the number of separate processes required to perform and validate the assay.
  • This is achievable because the method allows the calibration and control process that does not use the detected analyte to be integrated with the assay process itself: by removing the analyte that is involved in detection from this calibration or control process, it cannot interfere with the assay result, even when combined or integrated with the assay.
  • the analyte that is involved in detection is modelled in a second separate (and usually smaller) calibration or control process, to ensure that all of the elements of the assay have been modelled.
  • the calibration and/or control processes of the method may each independently comprise control steps, or calibration steps, or both.
  • a control step typically confirms the validity of the result.
  • a calibration step typically aids in determination of quantities, by using the assay measurement method on a known quantity of analyte, and then relating the measurement in the sample to that of the calibrator in order to determine the quantity of analyte within the sample.
  • the first and second calibration and/or control processes may model single steps in the overall assay (assay steps, or assay sub-processes), or multiple steps in the overall assay (assay sub-processes) or may model the entire assay process from start to finish with the exception of a small step or sub-process in the middle for which the detection analyte is required (assay process).
  • Figure 1 depicts an exemplary protocol for the control/calibration processes used in the present invention in respect of an assay for HCV.
  • Pseudoparticles with HCV viral proteins (El & E2) on the surface are automatically introduced into the sample.
  • the mechanism for capturing the HCV also captures the pseudoparticles and these are subjected to all of the stages that the virus is subjected to (i.e. purification, disruption, nucleic acid purification, reverse transcription, amplification and transduction).
  • RNA for green fluorescent protein (GFP) allows control and/or calibration of all stages in the process without affecting the result obtained for the patient sample, and if multiplexed detection is employed, sample and pseudoparticles can be analysed within the same fluidic channel.
  • the only part of the HCV assay that is not controlled by the GFP containing pseudoparticles are those that are HCV RNA specific (e.g. primers, hybridisation etc.), and this can be controlled by having a limited parallel channel where HCV RNA is introduced as the sample, and undergoes reverse transcription, amplification and transduction thus allowing control/calibration of these aspects of the process.
  • the reagents used in the control/calibration are the same as those employed in the assay.
  • first and second calibration and/or control processes are not especially limited, provided that they are complementary, as described above. They may be earned out in any desired order and thus may be simultaneous, or successive.
  • first calibration and/or control process is for calibration and/or control of an assay process
  • the second calibration and/or control process is for calibration and/or control of a sub-process of the assay process.
  • first and second calibration and/or control processes are for calibration and/or control of two different assay processes or two different assay sub-processes.
  • the assay for the analyte and the calibration and/or control process that does not employ the detected analyte are integrated with each other.
  • integrated is meant that the assay itself, and the control and/or calibration process that does not employ the detected analyte, are carried out simultaneously in the same reaction zones, channels, wells and conduits of the assay apparatus.
  • all of the reagents and starting materials for the assay and the calibration and control process are mixed together and processed with each other.
  • the assay for the analyte and the calibration and/or control process that does employ the detected analyte are carried out separately from each other, to ensure that the analyte employed for calibration and control is not detected as part of the assay itself.
  • the assay steps, sub-processes and processes are not especially limited, and may be any steps, sub-processes and processes depending on the specific nature of the assay involved,
  • the assay comprises the following assay processes or sub-processes: a sample preparation sub-process for preparation of the analyte; a reaction sub-process for reaction of the analyte; and the detection sub-process for detection of the analyte.
  • the sample preparation sub-process comprises a sub-process selected from any one or more of labelling, sorting, capture, isolation, purification, or disruption, of the analyte.
  • pseudoparticles representative of the pathogen of interest are employed in the process for calibrating and/or controlling the sample preparation sub-process to model the sample preparation sub-process.
  • the sample preparation sub-process employs a surface protein characteristic of the pathogen to prepare and/or purify the analyte for reaction.
  • the reaction sub-process comprises a sub-process selected from any one or more of reaction with a recognition agent, amplification, chemical conversion, enzymatic conversion, oxidation, reduction, and hybridisation of the analyte.
  • the reaction sub-process comprises nucleic acid amplification.
  • the nucleic acid amplification comprises PCR of DNA, or RT PCR of RNA (or in both cases qPCR (quantitative PCR) where amplification and detection are in tandem).
  • the pre-prepared analyte nucleic acid is synthetic, or is isolated from a natural source.
  • the detection sub-process comprises a sub-process selected from any one or more of determination of the presence or absence of the analyte, characterisation of the analyte, and quantification of the analyte.
  • the detection comprises detecting a nucleic acid analyte. This may also be an aspect and/or modification of the nucleic acid analyte, such as methylated nucleic acid, or may involve epigenetic detection components. Alternatively, other analytes may be employed, such as proteins, carbohydrates or lipids.
  • the method comprises a sample preparation sub-process and a detection sub-process calibrated and/or controlled by a process employing pathogen pseudoparticles comprising non-analyte nucleic acid, and a reaction sub-process calibrated and/or controlled by a process employing pre-prepared analyte nucleic acid.
  • the calibration and/or control process for the sample preparation sub-process and the detection sub-process is integrated with the assay, and the calibration and/or control process for the reaction sub-process is separate from the assay.
  • parts of the control process are performed using material added to the sample (pseudoparticles), whilst other parts are performed independently (e.g.
  • sample preparation step comprises purification of the analyte
  • sample detection step comprises detection of the analyte employing analyte nucleic acid
  • reaction sub-process comprises nucleic acid amplification
  • the non-analyte nucleic acid is not especially limited, provided that it does not interfere with the assay itself.
  • the non-analyte nucleic acid is green fluorescent protein (GFP) nucleic acid.
  • any analyte may be the subject of the assay.
  • the analyte is a cell, a bacterium, a yeast, a fungus, a virus, a food sample, or an environmental sample, or any components of these, or any entity indicative of the presence of these.
  • the analyte is a virus, such as hepatitis C virus (HCV), and preferably the analyte is HCV RNA.
  • HCV hepatitis C virus
  • the assay comprises a sample preparation sub-process for preparing the virus (HCV) from a blood sample, a reaction sub-process for amplification of the HCV RNA, and a detection sub-process for transduction of HCV RNA to detect the HCV.
  • HCV virus
  • a typical method of this preferred embodiment is one in which:
  • the sample preparation sub-process for preparing HCV comprises purification of HCV from a blood sample, disruption of the HCV to obtain HCV RNA and purification of the HCV RNA;
  • the reaction sub-process for amplification of the HCV RNA comprises reverse transcription of the HCV RNA and PCR amplification of the resulting DNA;
  • the detection sub-process for transduction of HCV RNA comprises detection of DNA
  • HCV pseudoparticles comprising GFP RNA are employed in the sample preparation sub-processes to model HCV.
  • Pre-prepared HCV RNA is employed in the reaction sub-process to model HCV RNA.
  • the assay for the analyte is carried out on a microfluidic or nanofluidic device.
  • the left hand flow diagram depicts a control/calibration process for detection of the presence/absence of the virus itself.
  • the right-hand flow diagram depicts a control/calibration process for the characterisation of the viral RNA.
  • the assay steps themselves involve purification of the virus, disruption of the virus, purification of the RNA, reverse transcription, amplification and transduction.
  • the diagram is simplified and does not show separate transduction steps for the viral detection and the RNA characterisation.
  • the first flow process involves mixing of blood plasma with viral pseudoparticles comprising GFP RNA, to model the HCV virus. All of the assay steps are then carried out in the normal fashion, to ensure that there is no difference from the real assay. This gives a control for the target analyte detection.
  • the second flow process takes the target RNA and only performs the reverse transcription, amplification and transduction steps on it, in order to provide a control for the target nucleic acid. Both controls can then be fed back into the real assay for validation, and increased accuracy and precision.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de calibrage et/ou de contrôle de titrage d’un analyte, le procédé de calibrage et/ou de contrôle comprenant au moins : (a) un premier processus de calibrage et/ou de contrôle pour le calibrage et/ou le contrôle d'un premier processus de titrage ou d’un sous-processus de titrage; et (b) un second processus de calibrage et/ou de contrôle pour le calibrage et/ou le contrôle d'un second processus de titrage ou d’un sous-processus de titrage; l'analyte étant employé dans le premier ou le second processus de calibrage et/ou de contrôle, mais pas dans l’autre processus de calibrage et/ou de contrôle, et le processus de calibrage et/ou de contrôle n’utilisant pas l'analyte étant intégré au titrage, et caractérisé en ce que : le premier processus de calibrage et/ou de contrôle sert pour le calibrage et/ou le contrôle d'un processus de titrage, et le second processus de calibrage et/ou de contrôle sert pour le calibrage et/ou le contrôle d'un sous-processus du processus de titrage; ou le premier et le second processus de calibrage et/ou de contrôle servant au calibrage et/ou au contrôle de deux différents processus et/ou sous-processus de titrage à partir de différents processus de titrage.
PCT/EP2009/064366 2008-11-28 2009-10-30 Procédé de calibrage et de contrôle WO2010060728A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP09749063A EP2358896A1 (fr) 2008-11-28 2009-10-30 Procédé de calibrage et de contrôle

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0821834.9 2008-11-28
GB0821834A GB0821834D0 (en) 2008-11-28 2008-11-28 Calibration and control method

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WO2010060728A1 true WO2010060728A1 (fr) 2010-06-03

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GB (1) GB0821834D0 (fr)
WO (1) WO2010060728A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2723897A4 (fr) * 2011-06-24 2015-03-18 Nanostring Technologies Inc Dosages diagnostiques multivariés et procédés d'utilisation de ceux-ci

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US20020111324A1 (en) * 2000-08-17 2002-08-15 Keiya Ozawa Adeno-associated virus-mediated delivery of angiogenic factors
WO2002033129A2 (fr) * 2000-10-17 2002-04-25 Impath, Inc. Materiel de controle d'amplification d'acide nucleique
US20070004635A1 (en) * 2005-06-02 2007-01-04 Schering Corporation Method of treating interferon non-responders using HCV protease inhibitor

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COSTAFREDA M ISABEL ET AL: "Development, evaluation, and standardization of a real-time TaqMan reverse transcription-PCR assay for quantification of hepatitis A virus in clinical and shellfish samples", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 72, no. 6, June 2006 (2006-06-01), pages 3846 - 3855, XP002563513, ISSN: 0099-2240 *
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2723897A4 (fr) * 2011-06-24 2015-03-18 Nanostring Technologies Inc Dosages diagnostiques multivariés et procédés d'utilisation de ceux-ci

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EP2358896A1 (fr) 2011-08-24
GB0821834D0 (en) 2009-01-07

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