WO2009059447A1

WO2009059447A1 - A quantitative method for oligonucleotide microarray

Info

Publication number: WO2009059447A1
Application number: PCT/CN2007/003124
Authority: WO
Inventors: Zhen Hong Sun; Wendy Wang; Yuan Zheng; Hang Liao
Original assignee: Honeywell International Inc.
Priority date: 2007-11-05
Filing date: 2007-11-05
Publication date: 2009-05-14

Abstract

A system and method capable of real-time, simultaneous, quantitative measurement of a plurality of nucleic acids from one or more pathogens in a sample is provided. Fluorescently tagged amplicons of the target nucleic acids and the internal nucleic acid controls are localized on a substrate surface by hybridization to target nucleic acid probes and internal nucleic acid control probes that have been arrayed and tethered to the substrate surface in a pre-determined, two-dimensional pattern. The hybridized amplicons are detected by exciting their fluorescent tags using an evanescent wave of light of the appropriate wavelength. By measuring the fluorescence at various locations on the substrate surface, the abundance of hybridized amplicons of each of die target nucleic acids and the internal nucleic acid controls can be determined.

Description

A QUANTITATIVE METHOD FOR OLIGONUCLEOTIDE

MICROARRAY

FIELD OF INVENTIQN

The invention relates to systems and methods for the quantitative measurement of nucleic acids, and particularly to systems and methods for the real-time, simultaneous quantitative assay of a plurality of nucleic acids from one or more pathogens.

BACKGROUND OF THE INVENTION

The quantitative assay of nucleic acids is of considerable importance in basic biological research as well as in fields such as clinical microbiology. A quantitative assay is typically accomplished in two stages. A target nucleic acid in a sample is first amplified to produce a detectable amount of nucleic acid for use by quantifying tools. The detected amount of a target nucleic acid is used to calculate the amount of the nucleic acid that was initially present in the sample.

A polymerase chain reaction (PCR) is a powerful way of amplifying nucleic acids, particularly deoxyribonucleic acid (DNA). The key to practical PCR is the use of a thermostable DMA polymerase, that is, a protein capable of catalyzing I)NA replication that does not denature at the elevated temperatures used to separate a DNA helix into two single strands of nucleic acid.

PCR is initiated by placing a target double-stranded DNA in a buffer of nucleotides along with a supply of small sequences of single stranded DNA, known as primers, which are complementary to the target DNA, and a thermostable DNA polymerase. By cycling the temperature of the mixture through three stages, the target DNA can be exponentially amplified. The first stage is a high temperature (94 °C) denaturing stage, in which double-stranded DNA is separated into two single strands. The second stage is a low temperature (60 °C) annealing stage, in which the primers bind to the single stranded DNA. The final, extension stage occurs at an intermediate temperature (72-78 °C). In the extension stage, the DNA polymerase catalyzes the extension of primers that have annealed to single strands of target DNA, adding appropriate nucleotides until a complete, double-stranded DNA helix is formed. In each PCR cycle, the number of copies of the taigetDNA approximately doubles, allowing foτ rapid accumulation of the target DNA.

In principle, the quantity of target DNA produced at the end of a series of PCR cycles (also known as the "end product") is proportional to the number of copies of that target DNA in the initial sample. However, in practice, the exponential nature of the amplification, and subtleties of the primer annealing that initiates the replication, result in saturation and other effects that make the PCR end product a very unreliable estimate of the amount of a target DNA in the initial sample.

The real-lime polymerase chain reaction (real-time PCR) process was developed in the mid 1990's to improve the original PCR process in a way that avoids these difficulties and provides reliable, accurate quantitative measurements of the number of copies of any target DNA in the sample. In a real-time PCR, fluorogenic probes that are only active when bound to target DNA are added to the PCR buffer solution. These fluorogenic probes are single strands of DNA, with a middle portion having a sequence of nucleotides that is complementary to the target DNA. On cither side of this middle portion, are extension nucleotide sequences that are complementary to each other, so that an unattached probe will fold onto itself in a hairpin configuration. The fluorogenic probe has a fluorescent molecule at one end, and a fluorescence quenching molecule at the other end. An unattached, folded probe has a fluorescing and a quenching molecule adjacent to each other, and consequently no fluorescent light is emitted when the unattached probe is illuminated. When the fluorogenic probe is attached to its target DNA, however, it is unfolded, with the fluorescing and quenching molecules separated from each other. When the attached probe is illuminated with the appropriate wavelength of light, the fluorescent molecule emits fluorescent light.

By providing sufficient fluorogenic probes for a particular target DNA, and measuring the fluorescence from the bound probes at each stage of the PCR, the number of amplicons at each stage of the reaction can be measured. This measurement can be used to very accurately determine the number of copies of the DNA in the initial sample because of a straight line relationship between the fractional number of cycles for the number of amplicons to reach a pre-determined threshold and the logarithm of the number of copies in the initial sample. In this way, real-time PCR may be used to determine the amount of a target DNA in a sample with less than 2% error over a range of nine orders of magnitude, that is, it can count as few as five, and as many as five billion, strands of the target DNA copies in the initial sample.

Real-time PCR technology does have limitations, however, the most significant of which is that real-time PCR can only measure a small number of nucleic acids in one reaction tube due to a limited number of suitable fluorescent dyes with suitable corresponding, fluorescence exciting light sources.

For many applications, the simultaneous quantification of more than one kind of nucleic acid is highly desirable. What is needed is an apparatus and method that allows real-time PCR to be used to simultaneously quantify hundreds of different nucleic acids from a variety of pathogens using a small number of fluorescent dyes, and preferably only one fluorescent dye.

SUMMARY OF INVENTION

The present invention provides a method and apparatus for the simultaneous, quantitative measurement of a plurality of nucleic acids from one or more pathogen in a sample.

In an exemplary embodiment, the nucleic acids in the sample, along with one or more internal nucleic acid controls, are all amplified in a single reaction cell using a polymerase chain reaction (PCR), reverse transcription PCR, roll cycle replication, or T7 transcription linear amplification, in which the amplification buffer solution additionally contains fluoresceπtly tagged nucleotides or, preferably fluorescently tagged primers, so that the amplicons of the target nucleic acids and the internal nucleic acid controls are themselves fluorescently tagged. Typically, the one or more internal nucleic acid controls are present with a different copy number and at different concentrations, for example, internal nucleic acid control A is present at low concentration and internal nucleic acid control B is present at high concentration. The presence of an internal nucleic acid control at a low concentration and another internal nucleic acid control at a high concentration extends the detection range of the method. Further, the presence of more than two internal nucleic acid controls, for example, three, four, five, etc. at concentrations varying from low to intermediate to high, would allow for very accurate detection across a very wide range of concentrations.

During the annealing or the extension phases of the amplification process, the fluorescently tagged amplicons of the target nucleic acids and the internal nucleic acid controls are localized onto a substrate surface by hybridization with target nucleic acid probes and internal nucleic acid control probes that have been arrayed and tethered to the substrate surface in a pre-determined, two-dimensional pattern. The target nucleic acid probes and internal nucleic acid control probes have the same complementary, nucleotide sequences as the target nucleic acids and internal nucleic acid controls, respectively, and may be arrayed by robotic printing using commercially available microarraying technology.

The hybridized, fluorescently tagged target amplicons are detected by the fluorescence emitted when their fluorescent tags are exited by an evanescent wave of light of the appropriate wavelength. Because the evanescent wave decays exponentially as it enters the reaction cell, with an effective range of about 100-300 nm, it only penetrates far enough into the reaction cell to activate fluorescent tags very close to the substrate surface, that is, the fluorescently tagged target amplicons hybridized to the target nucleic acid probes and internal nucleic acid control probes tethered to the surface. The evanescent wave does not, therefore, activate the fluorescently tagged nucleotides in the remainder of the reaction cell.

By monitoring the strength of the fluorescence at the various locations on the substrate surface, the current abundance of hybridized amplicons of each of the target nucleic acids and the internal nucleic acid controls can be determined. This may be done in real-time as the PCR reaction progresses, and the analytic techniques of real-time PCR may be used to obtain accurate, quantitative measurements of the abundance of each of the target nucleic acids and the internal nucleic acid controls in the original sample.

In one embodiment, the quantitative method for analyzing target nucleic acids, includes annealing one or more fluorescently tagged target amplicons to two or more target nucleic acid probes; annealing one or more fluorescently tagged internal control amplicons to one or more internal nucleic acid control probes; activating a first fluorescence response from the one or more fluorescently tagged target aroplicons hybridized to the two or more target nucleic acid probes; activating a siicond fluorescence response from the one or more fluorescently tagged internal control amplicons hybridized to the one or more internal nucleic acid control probes; and detecting the first and second fluorescence responses for a quantitative analysis of one or more target nucleic acids and one or more internal nucleic acid controls, wherein the two or more target nucleic acid probes and the one or more internal nucleic acid control probes are in close proximity to an upper surface of a substrate, and wherein the activating of the first and second fluorescence responses is by using an evanescent wave of a predetermined wavelength.

In another embodiment, the annealing occurs during a polymerase chain reaction. In yet another embodiment, the detecting of the first and second fluorescence responses occurs during the annealing step or an extending step of the polymerase chain reaction.

In one embodiment, the two or more target nucleic acid probes and the one or more internal nucleic acid control probes are printed onto a substrate using a micro-avray printer and are immobilized on the surface of a substrate. In another embodiment, the substrate is chemically modified with a reagent selected from silane, avidin, poly-L- lysine, streptavidin, polysaccharide, mercaptan, or a combination thereof. In yet another embodiment, the polymerase chain reaction is a real-time polymerase chain reaction.

In one embodiment, the one or more internal nucleic acid controls are linear double-stranded deoxyribonucleic acids, ringed double-stranded deoxyribonucleic acids, or combinations thereof. In another embodiment, the one or more internal nucleic acid controls have the same primer binding regions as that of the two or more target nucleic acids.

In yet another embodiment, the sequence length of the fluorescently tagged amplicons annealed to the one or more internal nucleic acid control probes is less than about one-thousand percent, preferably less than about five-hundred percent, and more preferably less than about two-hundred percent of the sequence length of the fluorescently tagged amplicons annealed to the two or more target nucleic acids.

In one embodiment, the sequence of the one or more internal nucleic acid controls has less than about twenty percent, preferably less than about ten percent, and most preferably less than about five percent cross reaction. In another embodiment, if two or more internal nucleic acid controls are present, the two or more internal nucleic acid controls are present in different concentrations. In yet another embodiment, if two or more internal nucleic acid control probes are present, the two or more internal nucleic acid control probes are present in the same concentrations as the two ov more target nucleic acid probes.

In one embodiment, the one or more internal nucleic acid controls has less than about twenty percent, preferably less than about ten percent, and most preferably less than about five percent cross reaction with the two or more target nucleic acids. In another embodiment, the first and second fluorescence responses are detected before the hybridization plateaus are reached. In yet another embodiment, the two or more target nucleic acids are derived from one or more pathogens, wherein the one or more pathogens is a virus, a bacterium, an archaea, a fungus, a protozoan, a mycoplasma, a prion, a parasitic organism, or combinations thereof.

In one embodiment, the one or more pathogens is Rickettsia, Chlamydia, Mycoplasma, Spirochete, Streptococcus, Staphylococcus, L. monocytogenes \ N. meningitides, E, coli, H. influenzae, B, burgdorferi, Leptospira, Proteus, Anaerobacter, Salmonella, M. tuberculosis, Enter ococcus, Poliovirus, Enterovirus, Coxsackievirus, HSV-I, HSV-2, or combinations thereof. In another embodiment, the two oi more target nucleic acid probes are selected from the group consisting of SEQ ID NOS: 13-52. In yet another embodiment, the target nucleic acids are E. coli and Salmonella, and the two or more target nucleic acid probes are selected from the group consisting of SEQ ID NOS: 14-17 and 22-26.

In another embodiment, the one or more pathogens are contained in tissue, cells, blood, sevum, cerebrospinal fluid, urine, cell lysate, plasma, excrement, sputum, blood cells, fine needle biopsy samples, peritoneal fluid, pleura fluid, or combinations thereof.

In one embodiment, the method further includes establishing a series of standard amplification curves for each of the two or more target nucleic acids and for each of the one or more internal nucleic acid controls, wherein each standard amplification curve is a dose - effect curve, wherein the dose is a number of amplification cycles and the effect is a hybridization signal. In another embodiment, the method further includes adjusting a detected concentration tor each of the two or more target nucleic acids by subtracting a difference in a detected concentration for the one or more internal nucleic acid controls and a predetermined concentration for the one or more internal nucleic acid controls.

The present invention provides a quantitative method for analyzing target nucleic acids, including annealing one or more fluorescently tagged target amplicons to two or more target nucleic acid probes; annealing one or more fluorescently tagged internal control amplicons to one or more internal nucleic acid control probes; activating a first fluorescence response from the one or more fluorescently tagged target amplicons hybridized to the two or more target nucleic acid probes; activating a second fluorescence response from the one or more fluorescently tagged internal control amplicons hybridized to the one or more internal nucleic acid control probes; and detecting the first and second fluorescence responses for a quantitative analysis of one or more target nucleic acids and one or more internal nucleic acid controls, wherein the two or more target nucleic acid probes and the one or more internal nucleic acid control probes are in close proximity to an upper surface of a substrate, and wherein the activating of the first and second fluorescence responses is by using an evanescent wave of a predetermined wavelength; establishing a series of standard amplification curves for each of the two or more target nucleic acids and for each of the one or more internal nucleic acid controls, wherein each standard amplification curve is a dose - effect curve, wherein the dose is a number of amplification cycles and the effect is a hybridization signal; adjusting a detected concentration for each of the two or more target nucleic acids by subtracting a difference in a detected concentration for the one or more internal nucleic acid controls and a predetermined concentration for the one or more internal nucleic acid controls.

The present invention also provides an apparatus for quantitatively analyzing target nucleic acids, includes: a substrate having an upper and a lower surface and a refractive index greater than a refractive index of water; a buffer solution substantially in contact with the upper surface of the substrate, the buffer solution being capable of sustaining an amplification reaction and nucleotide hybridization reaction and containing one or more fluorescently tagged primers, one or more optionally fluorescently tagged dNTPs, two or more target nucleic acids, and one or more internal nucleic acid controls; two or more target nucleic acid probes in close proximity to the upper surface of the substrate and within the buffer solution, the two or more target nucleic acid probes having a nucleotide sequence corresponding to, or complementary to, a nucleotide sequence of the two or more target nucleic acids; one or more internal nucleic acid control probes in close proximity to the upper surface of the substrate and within the buffer solution, the one or more internal nucleic acid control probes having a nucleotide sequence corresponding to, or complementary to, a nucleotide sequence of the one or more internal nucleic acid; a ray of light, having a wavelength chosen to activate one or more fluorescently tagged target amplicons, and one or more fluorescently tagged internal control amplicons, incident on an interface between the substrate and the buffer solution at an angle chosen so that an evanescent wave propagates into the buffer solution; a detector capable of detecting fluorescent light emitted by the one or more fluorescently tagged taTget ampUcons, and one or more fluorescently tagged internal control amplicons.

In another embodiment, the apparatus further includes a heating element capable of cycling a temperature of the buffer solution, thereby enabling the amplification reaction. In yet another embodiment, the amplification reaction is a real-time polymerase chain reaction.

These and other features will be more fully understood by references to the following drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention may be best understood by referring to the following description and accompanying drawings, which illustrate such embodiments. ϊn the drawings:

FIG. 1 illuscrates a cross-sectional view of an exemplary cartridge capable of evanescent wave detection of fluorescently tagged amplicons in a microarrayed PCR reaction in an initial stage of the PCR process.

FIG. 2 illustrates a cross-sectional view of an exemplary cartridge capable of evanescent wave detection of fluorescently tagged amplicons in a microarrayed PCR reaction at the end of the annealing and extension stage of the PCR process.

FIG. 3 illustrates a cross-sectional view of an exemplary cartridge capable of evanescent wave detection of fluorescently tagged amplicons in a rnicroarrayed PCR reaction at the denaturation stage of the PCR process.

FIG. 4 illustrates a cross-sectional view of an exemplary cartridge showing evanescent wave detection of fluorescently tagged amplicons in a microarrayed PCR reaction at the detection stage of the PCR process.

FIG. 5 illustrates exemplary plots of fluorescent intensity versus PCR cycle.

FlG. 6 is an exemplary plot of the logarithm of the number of copies of target nucleic acid strands, log[N], in the original sample versus the threshold cycle, CT, for the target nucleic acid.

FIG. 7 is an exemplary plot of the number cycle (x-axis) versus the internal number of amplicons (y-axis).

DEFINITIONS

As used herein, certain terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as one of skill in the art would understand. Such ordinary meanings may be obtained by reference to technical dictionaries, such as tlawley 's Condensed Chemical Dictionary 11 * Edition, by Sax and Lewis, Van Nostrand Reinhold, New York, N.Y., 1987, and The Merck Index, 11^th Edition, Merck & Co., Rahway NJ. 1989.

As used herein, the term "and/or" means any one of the items, any combination of the items, or all of the items with which this term is associated,

As used herein, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Therefore, for example, a reference to "a formulation" includes a plurality of such formulations, so that a formulation of compound X includes formulations of compound X.

As used herein, the term "about" means a variation of 10 percent of the value specified, for example, about 50 percent carries a variation from 45 to 55 percent, For integer ranges, the term about can include one or two integers greater than and less than a recited integer.

As used herein, the term "ambient condition" refers to the conditions of the surrounding environment (e.g., the temperature of the room or outdoor environment in which an experiment occurs). As used herein, the term "amplicon" refers to the products of polymerase chain reactions(PCR). Amplicons are pieces of DNA that have been syndiesized using amplification techniques (e.g., a double-stranded. DNA with two primers). The amplicon may contain, for example, a primer tagged with a fluorescent molecule at the 5' end as shown in Scheme 1 below.

As used herein, the term "aqueous" refers to a liquid mixture containing water, among other components.

As used herein, the terms "array" and "patterned array" refer to an arrangement of elements (i.e., entities) into a material or device, m another sense, the term "array" refers to the orderly arrangement (e.g., rows and columns) of two or more assay regions on a substrate.

As used herein, the terms "bacteria" and "bacterium" refer to all prokaryotic organisms, including those within all of the phyla in the Kingdom Procaryotae. It is intended that the term encompass all microorganisms considered to be bacteria including Mycoplasma, Chlamydia^ Actinomyces, Streptomyces, and Rickettsia. All forms of bacteria are included within this definition including cocci, bacilli, spirochetes, spheroplasts, protoplasts, etc. Also included within this term are prokaryotic organisms which are gram negative or gram positive. "Gram negative" and "gram positive" refer to staining patterns with the Gram-stairang process which is well known in the art. "Gram positive bacteria" are bacteria which retain the primary dye used in the Gτain stain, causing the stained cells to appear dark blue to purple under the microscope. "Gram negative bacteria" do not retain the primary dye used in the Gram stain, but are stained by the counterstain. Therefore, gram negative bacteria appear red.

As used herein, the term "biologically inert" refers to a property of material whereby the material does not chemically react with biological material.

As used herein, the terms "complementary" or "complementarity" are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence "A-G-T," is complementary to the sequence "T-C-A." Complementarity may be "partial," in which only some of the nucleic acids¹ bases are matched according to the base pairing rules. Alternatively, there may be "complete" or "total" complementarity between the nucleic acids, The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.

As used herein, the term "critical angle" is the angle of incidence above which the total internal reflection occurs.

As used herein, the term "diagnosed" refers to the recognition of a disease by its signs and symptoms (e.g., resistance to conventional therapies), or genetic analysis, pathological analysis, histological analysis, and the like.

As used herein, the term "disease" refers to pathologies and deleterious conditions, such as infections, inflammatory responses, cancer, autoimmune, and genetic disorders.

As used herein, the term "evanescent" refers to a nearfield standing wave exhibiting exponential decay with distance. As used in optics, evanescent waves are formed when sinusoidal waves are internally reflected off an interface at an angle greater than the critical angle so that total internal reflection occurs. As used herein, the term "fungi" refers to eukaryolic organisms such as molds and yeasts.

As used herein, the term "homology" refers to a degree of complementarity. There may be partial homology or complete homology (i.e., identity). A partially complementary sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid is referred to using the functional term "substantially homologous." The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous sequence to a target under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target that lacks even a partial degree of complementarity (e.g., less than about 30% identity); in liie absence of non-specific binding the probe will not hybridize to the second non-complementary target. One of skill in the art knows well that numerous equivalent conditions may be employed to comprise low stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe, and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.), and the concentration of the salts and other components (e.g., the presence or absence of foπnamide, dextraπ sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions. In addition, one of skill in the art knows conditions that promote hybridization under conditions of high stringency (e.g., increasing the temperature of the hybridization and/or wash steps, the use of fomiamide in the hybridization solution, etc.) (See definition below for "stringency").

When used in reference to a double-stranded nucleic acid sequence such as a cDNA or genomic clone, the term "substantially homologous" refers to any probe that can hybridise to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described above.

When used in reference to a single-stranded nucleic acid sequence, the term "substantially homologous" refers to any probe that can hybridize (i.e., it is the complement of) the single-stranded nucleic acid sequence under conditions of low stringency as described above.

As used herein, the term "hybridization" refers to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, the melting temperature (T_m) of the formed hybrid, and the G:C ratio within the nucleic acids. A single molecule that contains pairing of complementary nucleic acids within its structure is said to be "self-hybridized."

As used herein, the term "immobilization" refers to die attachment or entrapment, either chemically or otherwise, of material to another entity (e.g., a solid support) that restricts the movement of the material.

As used herein, the term "inhibitor" refers to a material, sample, or substance that retards or stops a chemical reaction.

As used herein, the term "reaction means inhibitor" refers to inhibitors that are capable of retarding or stopping the action or activity of a given reaction means (e.g., an enzyme).

As used herein, the term "internal nucleic acid control" refers to a polynucleotide that is added to the sample at a known concentration.

As used herein, the term "internal nucleic acid control probe" refers to a polynucleotide that is complementary to the internal nucleic acid control.

As used herein, the term "in vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments include, but are not limited to, test tubes and cell cultures.

As used herein, the term "in vivo" refers to the natural environment (e.g., an animal or a cell) and to processes or reaction that occur within a natural environment.

As used herein, the term "isolates" refers to nucleic acids separated from at least one other component present with the nucleic acids in its natural source. In one embodiment, the nucleic acid is found in the presence of (if anything) only a solvent, buffer, ion, or other component normally present in a solution. The terms "isolated" and "purified" do not encompass nucleic acids present in their natural source.

As used herein, the terms "material" and "materials" refer to, in their broadest sense, any composition of matter.

As used herein, the term "mercaptan" refers to a thiol (i.e., RSH) compound.

As used herein, the term "microarray" is a linear or two-dimensional microarray of discrete regions, each having a defined area, formed on the surface of a solid support, An oligonucleotide probe microarray complementary to the target nucleic acid sequence or subsequence thereof is immobilized on a solid support using one of the display strategies described below. The methods described herein employ oligonucleotide microarrays which comprise target nucleic acid probes exhibiting complementarity to one or more target nucleic acid sequences. Typically, these target nucleic acid probes are DNA and are immobilized in a high-density microarray (i.e., a "DNA chip") on a solid surface.

As used herein, the term "microorganism" refers to any species or type of microorganism, including, but not limited to, bacteria, archaea, fungi, protozoans, mycoplasma, and parasitic organisms. The present invention contemplates that a number of microorganisms encompassed therein will also be pathogenic to a subject.

As used herein, the term "nucleic acid molecule" refers to any nucleic acid containing molecule including, but not limited to, DNA or RNA. The term encompasses sequences that include, for example, any of the known base analogs of DNA and RNA including, but not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, asdridinylcytosiπe, pseudoisocytosine, 5-(carboxyhydτoxylmethyl)uracil, 5-fluorouracil, 5-bromouracϋ, 5-carboxymethylaminomethyl-2-thiouracil, 5- carboxymethylarainomethyluvacil, dihydrouracil, inosine,N6-isopentenyladenine, 1- methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2- dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-methyladenine, 7-methylguanine, 5-methyla-ninomelhyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-manτiosylqueosine, 5'- roethoxycarbonylmethyluracil, 5"methoxyuracil, 2-methy]thio-N6-isopentenyladenme, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thio\Jiacil, 2-tbiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

As used herein, the term "oligonucleotide," refers to a short length of single- stranded polynucleotide chain. Oligonucleotides are typically less than 100 residues long (e.g., between 15 and 50), however, as used herein, the term encompasses longer polynucleotide chains. Oligonucleotides are often referred to by their length. For example a 24 residue oligonucleotide is referred to as a "24-mer." Oligonucleotides can form secondary and tertiary structures by self-hybridizing or by hybridizing to other polynucleotides. Such structures can include, but are not limited to, duplexes, hairpins, cruciforms, bends, and triplexes.

As used herein, the term "organic solvents" refers to any organic molecules capable of dissolving another substance. Examples include, but are not limited to, chloroform, alcohols, phenols, ethers, or combinations thereof.

As used herein, 1he term "pathogen" refers a biological agent that causes a disease state (e.g., infection, cancer, etc.) in a host. Pathogens may include, for example, viruses, bacteria, archaea, fungi, protozoans, mycoplasma, prions, and parasitic organisms. Specific pathogens may include, for example, Rickettsia, Chlamydia, Mycoplasma, Spirochete, Streptococcus, Staphylococcus, L monocytogenes, N. meningitides, E. colt, H. influenzae, B. burgdorferi, Leptospira, Proteus, Anaerobacter, M. tuberculosis, Enterococcus, Poliovirus, Enterovirus, Coxsackievirus, HSV-I, and HSV-2.

As used herein, the term "polymerase chain reaction" (PCR) refers to the method of K. B. Mullis, U.S. Patent Nos. 4,683,195, 4,683,202, and 4,965,188, which describe a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. This process for amplifying the target sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double-stranded target sequence. To effect amplification, the mixture is denatured and the primers annealed to their complementary sequences within the target molecule. Following annealing, the primers aϊe extended with a polymerase so as to form a new pair of complementary strands. The steps of denaturation, primer annealing, and polymerase extension can be repeated many times (i.e., denaturation, annealing, and extension constitute one "cycle;" there can be numerous "cycles") to obtain a high concentration of an amplified segment of the desired target sequence. The length of the amplified segment of (he desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the "polymerase chain reaction" (hereinafter "PCR"). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be "PCR amphlϊed "

With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-cnzyme conjugate detection; incorporation of ³²P-labeled deoxynucleotide triphosphates, such as dCΪP or dATP, into the amplified segment). In addition to genomic DNA, any oligonucleotide or polynucleotide sequence can be amplified with the appropriate set of primer molecules. In particular, the amplified segments created by the PCR process are, themselves, efficient templates for subsequent PCR amplifications.

As used herein, the terms "PCR product," "PCR fragment," and "amplification product" refer to the resultant mixture of compounds after two or more cycles of the PCR steps of denaturation, annealing, and extension are complete. These terms encompass the case where there has been amplification of one or more segments of two or more target sequences.

As used herein, the term "polysaccharide" refers to a carbohydrate that can be decomposed by hydrolysis into two or more molecules of monosaccharides.

As used herein, the terms "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances, However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are nol useful, and is not intended to exclude other embodiments from the scope of the invention.

As used herein, the terms "purified" or "substantially purified" means that the indicated material is present in the substantial absence of other biological macromoleculcs (e.g., polynucleotides, proteins, carbohydrates, and the like).

As used herein, the term "reaction" refers to any change or transformation in which a substance (e.g., molecules, membranes, and molecular assemblies) combines with other substances, interchanges constituents with other substances, decomposes, rearranges, or is otherwise chemically altered.

As used herein, the term "room temperature" refers, technically, to temperatures between approximately 20 and 25 °C. However, as used generally, it refers to the ambient temperature within a general area in which an experiment is taking place.

As used herein, the term "sample" is used in its broadest sense. A sample suspected of indicating a condition characterized by the dysregulation ofapoptotic function may comprise a cell, tissue, or fluids, chromosomes isolated from a cell (e.g., a spread of melaphase chromosomes), genomic DNA (in solution or bound to a solid support such as for Southern blot analysis), RlSA (in solution or bound to a solid support such as for Northern blot analysis), cDNA (in solution or bound to a solid support) and the like. A sample suspected of containing a protein may comprise a cell, a portion of a tissue, an extract containing one or more proteins and the like.

As used herein, the term "streptavidin" refers to a 60,000 dalton tetrameric protein purified from the bacterium Streptomyces avidinii.

As used herein, the terms "solid phase supports" or "solid supports," are used in their broadest sense to refer to a number of supports that are available and known to those of ordinary skill in the art Solid phase supports include, but are not limited to, silica gels, resins, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels, and the like.

As used herein, (he term "spectrum" refers to the distribution of light energies arranged in order of wavelength.

As used herein, the term "stability" refers Io the ability of a material to withstand deterioration or displacement and to provide reliability and dependability. As used herein the teim "stringency" refers to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. With "high stringency" conditions, nucleic acid base pairing will occur only between nucleic acid fragments that have a high frequency of complementary base sequences. Therefore, conditions of "weak" or "low" stringency are often used with nucleic acids that are derived from organisms that are genetically diverse, as the frequency of complementary sequences is usually less.

As used herein, the term "subject" refers to organisms to be treated by the methods of the present invention. Such organisms preferably include, but arc not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and most preferably includes humans.

As used herein, the term "substrate" refers to material capable of supporting associated assay components (e.g., assay regions, cells, test compounds, etc.). For example, in some embodiments, the substrate includes a planar (i.e., 2 dimensional) glass, metal, composite, plastic, silica, or other biocompatible or biologically unreactive (or biologically reactive) composition. Many substrates may be employed. The substrate maybe biological, nonbiological, organic, inorganic, or a combination of any of these, existing as particles, strands, precipitates, gels, sheets, tubing, spheres, containers, capillaries, pads, slices, films, plates, slides, etc. The substrate may have any convenient shape, such as a disc, square, sphere, circle, etc. The substrate is generally flat but may lake on a variety of alternative surface configurations. For example, the substrate may contain raised or depressed regions on which the synthesis takes place. The substrate and its surface can form a rigid support on which to carry out the reactions described herein. The substrate and its surface are also chosen to provide appropriate light-absorbing characteristics. For instance, the substrate may be a polymerized Langmuir Blodgett film, functionalized glass, Si, Ge, GaAs, GaP, SiO₂, SiN₄, modified silicon, or any one of a wide variety of gels or polymers, for example, (poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene, polycarbonate, or combinations thereof. Other substrate materials will be readily apparent to those of skill in the art upon review of this disclosure. In one embodiment the substrate is flat glass. As used herein, the term "substantially equivalent" or "substantially similar" refers to nucleotide sequences that vary from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences. Typically, such a substantially equivalent sequence varies by no more than about 35% (i.e., the number of individual nucleotide substitutions, additions, and/or deletions in a substantially equivalent sequence, as compared to the corresponding reference sequence, divided by the total number of nucleotides in the substantially equivalent sequence is about 0.35 or less). Such a sequence is said to have 65% sequence identity to the listed sequence.

In one embodiment, a substantially equivalent sequence varies from a reference sequence by no more than 30% (70% sequence identity), in a variation of this embodiment, by no more than 25% (75% sequence identity), in a further variation of this embodiment, by no more than 20% (80% sequence identity), in a further variation of this embodiment, by no more than 10% (90% sequence identity), and in a further variation of this embodiment, by no more that 5% (95% sequence identity). In one embodiment, the nucleotide sequence has at least about 65% identity. In other embodiments, the nucleotide sequence has at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity.

As used herein, the term "target nucleic acid" refers to a polynucleotide inherent to a pathogen that is to be detected. The polynucleotide is genetic material including, for example, DNA/RNA, mitochondrial DNA, rRNA, tRNA, mRNA, viral RNA₁ and plasmid DNA. By detecting the presence of a target nucleic acid that is unique to a pathogen, the presence of the pathogen can be inferred. Similarly, the presence of a target nucleic acid that is specific to a genus of pathogens indicates the presence of a member of the genus. Target nucleic acids are isolated from biological sample and test sample using standard techniques. The technique will be determined by what type of polynucleotide is to be isolated, foi example, DNA or RNA. Isolation techniques can be modified depending on the type of pathogen being investigated and the quantity of biological or test sample.

As used herein, the term "target nucleic acid probe" refers to a polynucleotide that is complementary to the target nucleic acid. As used herein, the term "T_m," refers to the "melting temperature." The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. The equation for calculating the T_n, of nucleic acids is well known in the art. As indicated by standard references, a simple estimate of the T_1n value may be calculated by the equation: T_m - 81.5 +0.41(% Cr+C), when a nucleic acid is in aqueous solution at 1 M NaCl (see, e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985)). Other references include, for example, more sophisticated computations that take structural as well as sequence characteristics into account for the calculation of T_m,.

As used herein, the term "virus" refers to minute infectious agents, which with certain exceptions, are not observable by light microscopy, lack independent metabolism, and are able to replicate only within a living host cell. The individual particles (i.e., virions) typically consist of nucleic acid and a protein shell or coat; some virions also have a lipid-containing membrane. The term "virus" encompasses all types of viruses, including animal, plant, phage, and other viruses.

As used herein, the term "visible spectrum" refers to light radiation that contains wavelengths from approximately 360 nm to approximately 800 nm.

DETAtLED DESCRIPTION OF THE INVENTION

The present invention provides a system and method capable of real-time, simultaneous, quantitative measurement of a plurality of nucleic acids from one or more pathogens in a sample.

In an exemplary embodiment, the target nucleic acids and the internal nucleic acid controls in the sample are amplified using the polymerase chain reaction (PCR). The PCR is a well-known method of amplifying one or more stands of deoxyribonucleic acid (DNA). PCR is begun by placing the target nucleic acids and the internal nucleic acid controls in a buffer containing the nucleotides adenine (A), thymine (T), cytosiπe (C) and guanine (G) (collectively referred to as dNTPs), a DNA polymerase, and primers. The primers are short strands of DNA, with sequences that complement the target nucleic acids and the internal nucleic acid controls to be amplified. The primers initiate replication of the target nucleic acids and the internal nucleic acid controls. In one embodiment, both the target nucleic acid primer and internal nucleic acid control primer may be fluorescently tagged with fluorescent molecules at the 5' end. In another embodiment, one of the target nucleic acid primer or the internal nucleic acid control primer may be fluorescently tagged with fluorescent molecules at the 5' end, In yet another embodiment, the dNTPs are fluorescently tagged.

The PCR process has three main steps: denaturation, annealing and extension. In the denaturation step, the mixture is heated to about 94 °C (Centigrade), at which point the target and internal control DNA separates into single strands. The mixture is quickly cooled. As the temperature falls to about 60 °C, the annealing step occurs, in which the primers, which are preferably fluorescently tagged, hybridize or bind to their complementary sequences on the target nucleic acids and the internal nucleic acid controls. The extension step may be performed at about 60 °C or may be raised to the 72- 78 °C range. In this step, the DNA polymerase uses the dNTPs in solution to extend the annealed primers, which are preferably fluorescently tagged, and forms new strands of DNA lαiown as an ampHcons. The mixture is briefly reheated back to about 94 °C to separate the newly created double helix stands into single strands of nucleic acid, which begins another cycle of the PCR process. With each cycle of the PCR process, the number of copies of the original target nucleic acids and the original internal nucleic acid controls roughly doubles.

In one embodiment, the PCR buffer additionally contains fluorescently tagged primers, that is, primers having a fluorescent dye molecule attached to them, so that upon completion of each PCR cycle, the amplicons produced are fluorescently tagged. The amplicons of the target nucleic acids and the internal nucleic acid controls are localized, using probe strands of DNA known as target nucleic acid probes and internal nucleic acid control probes, respectively. The target nucleic acid probes and internal nucleic acid control probes have the same complementary, nucleotide sequence as the target nucleic acids and the internal nucleic acid controls. The target nucleic acid probes and internal nucleic acid control probes are tethered to a substrate surface in a known, two- dimensional pattern, with the substrate surface forming part of the reaction cell containing the PCR ingredients. During the annealing and extension phases of the PCR process, the target amplicons hybridize to their corresponding target nucleic acid probes and internal nucleic acid control probes. The hybridi2ed, fluorescently tagged amplicoπs are illuminated with an evanescent wave of light of the appropriate wavelength to activate the fluorescent dye molecules of the fluorescently tagged primers or the fluorescently lagged dNTPs. This evanescent wave decays exponentially in power after entering the reaction cell via the substrate surface to which die target nucleic acid probes and internal nucleic acid control probes are tethered, with an effective penetration range of about 300 ran. This means that the evanescent wave penetrates far enough into the reaction cell to activate the fluorescently tagged amplicons hybridized to those target nucleic acid probes and internal nucleic acid control probes, but that it does not activate the fluorescently tagged molecules (e.g., the fluorescently tagged primers or the fluorescently tagged dNTPs) in solution in the main body of the reaction cell. By monitoring the strength of the fluorescence at various locations on the substrate surface, the current abundance of amplicons of the corresponding target nucleic acids and internal nucleic acid controls can be determined. This may be done in real-rime as the PCR progresses. The results are used to obtain a quantitative measure of the abundance of a specific target in the original sample, in a manner analogous to the real-time PCR calculation.

In one embodiment, the PCR utilizes prirαers including a polynucleotide selected from the group consisting of SEQ IDNO: 1-12 (Table 1). Some combinations of primers include, for example, SEQ ID NOS: 1 and 2; SEQ ID NOS: 1 and 4; SEQ ID NOS: 1 and 6; SEQ ID NOS: 3 and 2; SEQ ID NOS: 3 and 4; SEQ ED NOS: 3 and 6; SEQ ID NOS: 5 and 2; SEQ ID NOS: 5 and 4; SEQ ID NOS: 5 and (S; SEQ ID NOS: 7 and 8; SEQ ID NOS: 9 and 10 and/or SEQ ID NOS: 11 and 12.

In another embodiment, the target nucleic acid probes are designed to be complementary to polynucleotides isolated from a pathogen selected from the group consisting of bacteria, viruses, fungi, and protozoa. In yet another embodiment, the target nucleic acid probes are designed to be complementary to polynucleotides isolated from Rickettsia, Chlamydia, Mycoplasma, Spirochete, Streptococcus, Salmonella, Staphylococcus, L monocytogenes, N. meningitides, B. coli, H. influenzae, B. burgdorferi, Leptospira, Proteus, Anaerobacter, M. tuberculosis, Enterococcus, H. pohovirus 1, //. enterovirus 71, H, enterovirus 70, H. echovirus 2, H. echovirus 4, H. echovirvs 6, H. echovirus 9, H. echovirus 11, H. echovirus 12, H. echovirus 26, H. coxsackievirus A13, H. coxsackievirus A15, H. coxsackievirus Al 8, H. coxsackievirus A20, H. coxsachevirus A21 , if. coxsackievirus B3-A, H. coxsacldevints B3-C, ΗSV-1, and HSV-2. In another embodiment, the target nucleic acid probes are selected from the group consisting of SEQ TD NO: 13-52 (Table 2).

FIG. 1 is a cross-sectional view of an exemplary reaction cartridge capable of evanescent wave detection of fluorescently tagged ainplicoπs in a microarrayed PCR reaction, including a reaction cartridge 10, a substrate 12 having a substrate surface 13, a first nucleic acid target probe 14, a second nucleic acid target probe 15, a first internal nucleic acid control probe 14A, a second internal nucleic acid control probe 15A, a buffer solution 16, a first target nucleic acid strand 18, a second target nucleic acid strand 20, a first internal nucleic acid control 18A, a second internal nucleic acid control 2OA, dNTPs 22, optional fluorescently tagged dNTPs 24, fluorescently tagged primers 26, a thermostable DNA polymerase 28, a heating element 30, and a cooling element 31.

In one embodiment, the substrate 12 is comprised of a material that is optically denser than the buffer solution 16, so that evanescent wave detection can be used as described in detail below. The substrate 12 may for instance be glass, or a suitably coated plastic or polymer.

The first and second target nucleic acid probes 14 and 15 and the first and second internal nucleic acid control probes 14A and 15A are strands of DNA, each having a specific nucleotide sequence of one of the target strands of DNA 18 and 20 and the internal nucleic acid controls 18A and 2OA that they are used to detect, respectively. In one embodiment, these target nucleic acid probes and internal nucleic acid control probes are non-extendable. In other words, the nucleotides cannot be added to either end of the target nucleic acid probes and the internal nucleic acid control probes. The target nucleic acid probes 14 and 15 and the internal nucleic acid control probes 14A and 15A may be natural or synthetically fabricated polynucleotides, polynucleotides with artificial bases and/or artificial carbohydrates, peptide nucleic acids (PNAs), bicyclic nucleic acids (BNAs), or other nucleotide analogs, constructed using a commercially available oligonucleotide synthesizer, for example, the POLYPLEX synthesizer available from Genomic Solutions, Inc. of Ann Arbor, Mich. Alternatively, the probes may be, but are not limited to, a sequence chosen from a library of DNA sequences, such as a library of expressed sequence tags (EST) known to have some biological significance.

The target nucleic acid probes 14 and 15 and the internal nucleic acid control probes 14A and 15A are arrayed on a substrate surface 13. In one embodiment, tatget nucleic acid probes 14 and 15 and the internal nucleic acid control probes 14A and 15A are arrayed on the substrate as small spots by robotic printing using commercially available rnicroarraying technology, for example, the OMNIGRID microarrayer available from Genomic Solutions, Inc. of Arm Arbor, Mich.

The first and second target nucleic acid probes 14 and 15 and first and second internal nucleic acid control probes 14A and 15A may be immobilized on the substrate surface 13 by well-known techniques such as, but not limited to, covalently conjugating an active silyl moiety onto the target nucleic acid probes and internal nucleic acid control probes. Such silanized molecules are immobilized instantly onto glass surfaces after manual or automated deposition. Alternatively, the first and second target nucleic acid probes 14 and 15 and first and second internal nucleic acid control probes 14A and 15A may be immobilized by suitably electrically charging the surface, preferably by using a suitable coating, for example, a silane, poly-L-lysine, streptavidin, a polysaccharide, mercaptan, or a combination thereof.

The fluorescently tagged primers 26 or the optionally fhioresceutly tagged dNTPs 24 are nucleotides that may be tagged with a fluorescent dye, for example, fluorescein or Rhodamine Green dyes, or similar, related compounds having similar fluorescing characteristics, such as functionalized or intercalating dyes and luminescent, functionalized nanoparticles (quantum dots). The optionally fluorescenϊly tagged dNTPs 24 may have one, two, three or four of the four base nucleotides dGTP, dCTP, dATP and dTTP that are fluorescently tagged.

Heating element 30 may be any suitable resistive material, for example, carbon, that provides heat when an electric current flows though it. Heating element 30 need to be capable of heating the reaction cartridge 10 to a temperature of 94 °C within minutes. Cooling element 31 may be any suitable solid state cooling element, for example, a well known Peltier solid-state device functioning as a heal pump. The heating element 30 and cooling element 31 can also be located outside the reaction cartridge 10.

FIG. 2 is a cross-sectional view of an exemplary reaction cartridge 10 capable of evanescent wave detection of fluorescently tagged amplicons in a microarrayed PCR reaction at the end of the annealing and/or extension stage of the PCR process, further including the first, second, third, and fourth fluorescently tagged amplicons 32, 34, 32A, and 34A, respectively. First fluorescently tagged ampHcon 32 is a DNA strand having a nucleotide sequence that is a complementary copy of the first target nucleic acid strand 18, that is, for every adenine (A) nucleotide in the first target nucleic acid strand 18, there is a thymine (T) nucleotide in the first fluorescently tagged amplicon 32, and vice versa. Similarly for every cytosine (C) nucleotide in the first target nucleic acid strand 18, there is a guanine (G) nucleotide in the first fluorescently tagged amplicon 32.

Second fluorescently tagged amplicon 34 is a DNA strand having a nucleotide sequence that is a complementary copy of the second target nucleic acid strand 20, that is, for every adenine (A) nucleotide in the second target nucleic acid strand 20, there is a thymine (T) nucleotide in the second fluorescently tagged amplicon 34, and vice versa. Similarly for every cytosine (C) nucleotide in the second target nucleic acid strand 20, there is a guanine (G) nucleotide in the second fluorescently tagged amplicon 34. Third fluorescently tagged amplicon 32 A is a DNA strand having a nucleotide sequence that is a complementary copy of the first internal nucleic acid control 18A, thai is, for every adenine (A) nucleotide in the first internal nucleic acid control 18A, there is a thymine (T) nucleotide in the third fluorescently tagged amplicon 32A, and vice versa. Similarly foi every cytosine (C) nucleotide in the first internal nucleic acid control 18A, there is a guanine (G) nucleotide in the third fluorescently tagged amplicon 32A.

Fourth fluorescently tagged amplicon 34A is a DNA strand having a nucleotide sequence that is a complementary copy of the second internal nucleic acid control 2OA, that is, for eveiy adenine (A) nucleotide in the second internal nucleic acid control 20A, there is a thymine (T) nucleotide in the fourth fluorescently tagged amplicon 34A, and vice versa. Similarly for every cytosine (C) nucleotide in the second internal nucleic acid control 20A, there is a guanine (G) nucleotide in the fourth fluorescently tagged amplicon 34A.

At the end of the annealing and/or extension stage of the PCR process, the first, second, third, and fourth fluorescently tagged amplicons 32, 34, 32A, and 34A produced by extension of annealed fluorescently tagged primers 26, remain hybridized to their corresponding first and second target nucleic acid strands 18 and 20 and the first and second internal nucleic acid controls ISA and 2OA. Additionally, amplicons produced in previous cycles of the PCR process are hybridized to the tethered first and second target nucleic acid probes 14 and 15 and the first and second internal nucleic acid control probes 14A and 15A. For instance, at surface site 36, a second fluorescently tagged amplicon 34 is hybridized to a second target nucleic acid probe 15. Similarly at surface site 38, a first fluorescently tagged amplicon 32 is hybridized to a first target nucleic acid probe 15. The first and second target nucleic acid probes 14 and 15 and the first and second internal nucleic acid control probes 14A and 15A are designed not to be amplified in the PCR process by, for instance, being tethered by their 3 ' end to the substrate, or by having the 3 ' end modified by dideoxidation or by having a stable group added to the 3' end or by any other well known methods of making the target nucleic acid probes and the internal nucleic acid control probes not participate in a PCR process in the presence of specific primers.

FTG. 3 is a cross-sectional view of an exemplary reaction cartridge 10 capable of evanescent wave detection of fluotescently tagged amplicons in a microarrayed PCR reaction at the denaturation stage of the PCR process. In this stage, the mixture within the reaction cartridge 10 has been heated to close to 100 °C, and optimally to about 94 °C. At this temperature, the DNA is denatured, that is, it separates into individual, single strands. When cooled in the next stage of the PCR process, each of the first and second target nucleic acid strands 18 and 20 and the first and second internal nucleic acid controls 18A and 20A, as well as each of the first, second, third, and fourth fluorescently tagged amplicons 32, 34, 32A, and 34A will anneal with fluorescently tagged primers 26. The annealed fluorescently tagged primers 26 will be extended as the thermostable DNA polymerase 28 adds appropriate nucleotides, until each of the first and second target nucleic acid strands 18 and 20, each of the first and second internal nucleic acid controls 18A and 20A, and each of the the first, second, third, and fourth fluorescently tagged amplicons 32, 34, 32A, and 34A, will be hybridized to a new amplicon, which is a copy or a complementary copy of the original first and second target nucleic acid strands 18 and 20 and first and second internal control strands 18 A and 20A.

FIG. 4 is cross-sectional view of an exemplary reaction cartridge 10 showing evanescent wave detection of fluorescently tagged amplicons in a microarrayed PCR reaction at the detection stage of the PCR process, further including an incident beam of light 40, an angle of incidence 42, a reflected beam of light 44, an evanescent wave of light 46, a fluorescent beam of light 48 and a fluorescent light detector 50. The detection stage can be coincident with the annealing and/or extension stage.

The incident beam of light 40 is chosen to be of a wavelength suitable for exciting the fluorophore used to label the fluorescently tagged primers 16 or the optionally fluorescently tagged dNTPs 24. In one embodiment, the incident beam of light 40 is the 488 nm spectral line of an argon-ion laser, which closely matches the excitation maximum (494 nm) of fluorescein dye that is used to tag or label the fluorescently tagged primers 16 or the optionally fluorescently tagged dNTPs 24.

The angle of incidence 42 of the incident beam of light 40 is chosen to be greater than the critical angle of the substrate to buffer interface. The critical angle of incidence is the angle at which total internal reflection occurs and is dependent on the refractive indices of the materials forming the interface. From Snell's laws of refraction, the critical angle of incidence = sin^{- 1}n₁/n₁) where n, and n₂ are the refractive indices of the materials on either side of the interface. In one embodiment of the present invention, the substrate 12 is comprised of glass and has a refractive index of about 1.5, while the buffer solution 16 is comprised mainly of water having a refractive index of about 1.3, so that the critical angle of incidence is about 61 degrees.

When light is reflected off the substrate surface 13 at an angle of incidence 42 degrees greater than the critical angle so that total internal reflection occurs, an evanescent wave 46 is formed and propagates through the substrate surface 13. The intensity of the evanescent wave 46 drops by a factor of V (i.e., the mathematical constant) for each 130 nm increase in distance from the substrate surface 13. Therefore, only objects very near substrate surface 13 are illuminated by the evanescent wave 46. This property is used in one embodiment of the present invention to illuminate the first, second, third, and fourth fluorescently tagged amplicons 32, 34, 32A, and 34A that are hybridized to the first and second target nucleic acid probes 14 and 15 and the first and second internal nucleic acid control probes 14A and 15 A, respectively. The fluorescent light 48 emitted by the fluorescently tagged amplicons 32, 32A, 34, and 34A may be detected and analyzed by the fluorescent light detector 50. The fluorescent light detector 50 typically includes collection optics such as, a microscope objective lens, which focuses the light on to a detection system such as, a photomuliplier tube or a charge coupled device (CCD) camera or photodiodes.

The origin and intensity of the collected fluorescent light can be used to estimate the number of fluorescently emitting molecules and therefore the number of fluorescently tagged amplicons currently hybridized to a particular type of oligoprobe by using, for example, the well known quantification techniques employed in Real-Time or Kinetic PCR analysis. In the Real-Time or Kinetic PCR analysis, the reactions are characterized by the point in time during cycling when amplification of a PCR product is first detected, rather that the amount of PCR product accumulated after a fixed number of cycles. The greater the number of copies of a target nucleic acid in the initfal sample, the sooner a significant increase in fluorescence is observed.

In a further embodiment of the invention, the fluorescent signal may be detected by monitoring the reflected light and determining the amount of light absorbed by the fluorescent lags.

FIG. 5 shows exemplary plots 52, 54, and 56 of fluorescent signal Versus the cycle number for three target PNA strands, each having a different number of copies in the initial sample. There is a starting or baseline background fluorescence signal, which is detectable even when no PCR cycle has taken place. Ih the initial cycles of the PCR, there is little change in this fluorescence signal. An increase in fluorescence above the baseline indicates the detection of accumulated PCR product. By setting a fixed fluorescence threshold 58 above the baseline, a threshold cycle (CT) parameter can be defined as the fractional cycle number at which the fluorescence for a particular oligoprobe passes this fixed threshold, as indicated by the three fractional values C_ri, Cr. and C₁₃.

FIG. 6 is an exemplary plot of the logarithm of the number of copies of target nucleic acid strands, log[N], in the original sample versus the threshold cycle, CT, for the target nucleic acid. Because of the exponential nature of the PCR, a plot of the log of the initial target copy number versus CT is a straight line 60. By introducing a number of internal nucleic acids controls, which have a known number of copies in the initial sample, the fluorescence associated with their corresponding internal nucleic acid control probes can be used to produce a straight line calibration line 60 of log of initial copy number versus CT. By measuring the CT of a location on the reaction cell known to have a particular target nucleic acid probe, the number of copies of the target nucleic acid corresponding to that target nucleic acid probe can be deduced from the calibration curve.

FIG.7 is an exemplary plot of the number cycle (x-axis) versus the internal number of amplicons (y-axis). The equation N = 1000 * (l-f-X)ⁿ represents the exponential phase function of amplification reaction, where N is the number of amplicons. If X = 80% under standard conditions, but if X = 75% under actual conditions, to calculate the standard cycle number (n) to reach 100,000, n = log(100)/log(1.8) = 7.83. However, if the amplification efficiency is reduced, the actual cycle number (n') may be n' = log( 100)/log(l .75) = 8.23. In ideal testing conditions (not considering different kinetics for different hybridization reactions, the amplicons number can be replaced by the hybridization signal in this equation. In this case, the calibration factor is f = (1+X_standard)/(1+X_standard)- This calibration factor is universal for each internal nucleic acid control and each target nucleic acid. Further, this calibration factor may be calculated with the actual and standard hybridisation curve. For example, from the curve in FIG. 7, f can be calculated as f- (1+0.75)7(1+0.8) = 0.972. In addition, the hybridization signal of target molecules can also be adjusted to its standard signal by this calibration factor. If the function of the standard curve for target nucleic acid (1) is expressed as N₁=N₁₀x(1+X_Standard)ⁿ and the actual hybridization signal is N₁', then, the standard hybridization signal is N₁=N₁'/f'' =N₁₀x(1+X_standard)". Therefore, N\o could be calculated from the equation N₁₀=N_{1 '}/[f''x(1+X_standard)'']. If the calculated f is out of a control range, for example, 0.9 to 1.1, the test maybe regarded as invalid.

The present invention also provides a kit for detecting two or more target nucleic acids from one or more pathogens. The kit includes at least one primer pair and an oligonucleotide microanay that includes two or more target nucleic acid probes and one or more internal nucleic acid control probes.

Although the foregoing discussion has used DNA as an exemplary nucleic acid, it would be obvious to a person of reasonable skill in the art to apply the methods disclosed herein to other nucleic acids, including RNA sequences or combinations of RNA and DNA sequences.

Although the foregoing discussion has used PCR as an exemplary reaction, it would be obvious to one of ordinary skill in the art to apply the methods disclosed herein using any suitable amplification reaction such as, but not limited to, reverse transcription PCR, random primer amplification, roll cycle amplification or linear amplification "T7."

Aldiough the foregoing discussion has used fluorescent tagged primers to label the target nucleic acids and the internal nucleic acid controls, it would be obvious to one of ordinary skill in the art to use related structures such as, but not limited to, functional ized nanoparticles, or intercalating dyes to label the target nucleic acids and the internal nucleic acid controls.

Although the foregoing discussion has, for simplicity, been discussed in terms of two target nucleic acids and two internal nucleic acid controls, it would be obvious to one of ordinary skill in the art to use the methods and apparatus for the quantitative evaluation of one target nucleic acid, or for a multiplicity of target nucleic acids and a multiplicity of internal nucleic acid controls. Although the invention has been described in language specific to structural features and/or methodological acts, it is to be understood that the invention defined in the appended claims is not necessarily limited to the specific features or acts described. Rather, the specific features and acts are disclosed as exemplary forms of implementing the claimed invention.

It is to be understood that certain descriptions of the present invention have been simplified to illustrate only those elements and limitations that are relevant to a clear understanding of the present invention, while eliminating, for purposes of clarity, other elements. Those of ordinary skill in the art, upon considering the present description of the invention, will recognize that other elements and/or limitations may be desirable in order to implement the present invention. However, because such other elements and/or limitations may be readily ascertained by one of ordinary skill upon considering the present description of the invention, and are not necessary for a complete understanding of the present invention, a discussion of such elements and limitations is not provided herein.

EXAMPLES

The following Example is illustrative of the above invention. One skilled in the art will readily recognize that the techniques and reagents described in the Example suggest many other ways in which the present invention could be practiced. It should be understood that many variations and modifications maybe made while remaining within the scope of the invention.

Other than in the operating examples, or unless otherwise expressly specified, all of the numerical ranges, amounts, values and percentages such as those for amounts of materials, times and temperatures of reaction, ratios of amounts, and others in the following portion of the specification maybe read as if prefaced by the word "about" even though the term "about" may not expressly appear with the value, amount, or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Furthermore, when numerical ranges of varying scope are set forth herein, it is contemplated that any combination of these values inclusive of the recited values may be used.

Example 1 Quantitative Microarray for Twelve Kinds of Bacteria

The target group will include twelve different kinds of bacteria along with two internal nucleic acid controls. Internal nucleic acid control A will be at a known low concentration and internal nucleic acid control B will be at a known high concentration. The set of primers will include a forward primer and a reverse primer for all twelve kinds of bacteria species, as well as for interna] nucleic acid controls A and B. The set of probes will include probes for all twelve kinds of bacteria species, as well as for internal nucleic acid controls A and B. For example, target probe A will be specific for target nucleic acid A and target probe B will be specific for target nucleic acid B.

Typically, there should be no cross-reaction between the probes for all twelve kinds of bacteria species and the probes for internal nucleic acid controls A and B. Also, typically there should be no cτoss-reactiort between internal nucleic acid control probes A and B and the twelve target nucleic acids. The internal nucleic acid controls A and B will be specially designed to meet the demands of equivalent amplification efficiency as compared with the target nucleic acids.

The internal nucleic acid controls may be designed to be linear or ringed doubled stranded DNA, for example, in the format of plasmid. The complementary sequence to the primer pairs may be designed in its molecules. The fragment between primer binding regions will be with proper sequence and length to ensure fit for the original amplification conditions with equivalent PCR efficiency and fit for probe design, without cross reaction with bacteria DNA targets and probes. The internal nucleic acid controls may be designed and cloned by molecular biological techniques, such as PCR, restriction enzyme digestions, DNA ligation, transformation, etc.

The internal nucleic acid controls A and B will be co-amplified with other 12 kinds of bacteria DNA in the sample. IfPCR inhibitors exist in the nucleic acid sample, the amplification reaction will be inhibited. As a result, the hybridization signal with probe A/B will fall out of the control range in an appointed detection time (e.g., after amplifying 20 cycles). Therefore, the detection result for this sample should be regarded as invalid.

When some factors exist that will affect the amplification/hybridization system, the internal nucleic acids controls may be used for the calibration of end results. For example, internal nucleic acid control A may be used for calibration when the suspected bacteria in the sample are at low concentration. On the other hand, internal nucleic acid control B may be used for calibration when the suspected bacteria are at high concentration.

Ideally, bacteria nucleic acid target X and internal nucleic acid controls A and B may be co-amplified with the same amplification and hybridization efficiency by proper design of the molecule sequence. Although they may have the same amplification efficiency, equivalent hybridization efficiency may not be reached in many cases. This may be because the hybridization kinetics of different target-probe pairs maybe different at a fixed temperature. Further, a seri.es of calibration curves for every kind of target molecule should be constructed. For example, if the original copy number of internal nucleic acid controls before hybridization is 10^3.25, duplicate tests in standard conditions should be done to obtain a dose (amplification cycles number)- effect (hybridization signal) curve based ou the average hybridization signal at eveiy amplification cycle. In a similar fashion, dose-effect curve at 10², 10^2.5, 10³, 10^3.5, 10⁴ copies may be obtained.

Typically, dose-effect curves of every bacteria target molecules at 10², 10^2.5, 10³, 10^{3 s}, 10*, 10^4.5, 10^5. 10^5.5, 10⁶, and 10^6.5 maybe obtained. If the hybridization signal of internal nucleic acid control at amplification cycle n, n + 1, n + 2 is between the curve of 10^2.5 and 10³, both internal nucleic acid control and the suspected bacteria DNA maybe calibrated upwards by 10^0.5 fold. Typically, if the hybridization signals of target nucleic acid X at amplification cycle n, n + 1 , and n + 2 is between the curve of 10³.⁵ and 10⁴, the concentration should be between 10⁴ and 10^4.5.

Typically, internal nucleic acid control B (with large copy numbers) will also be used because the amplification of target A will be inhibited when the bacteria target molecules are with high concentrations, e.g., 10⁸. That is, before internal nucleic acid control A will fall into the detection range, the PCR material will be largely consumed. In contrast, internal nucleic acid control B (with high concentration, for example, 10 ), will not be inhibited by such bacteria target molecules. For internal nucleic acid control B, a set of calibration curves at 10⁵, 10^5.5, 10⁶, 10^6.5, and 10⁷ copies should be developed. Dose-effect curves of every bacteria target molecule should be built at 10⁶'⁵, 10⁷, 10⁷.⁵, 10⁸, 10^8.5, 10⁹, and 10^10.5. Although the detection time may be altered to cycle m, m + 1, and m + 2 (m < n), the calibration should be done in similar way. By setting internal controls at different concentration, the detection range of the system will be extended.

All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

What is claimed is:

1. A quantitative method for analyzing target nucleic acids, comprising annealing one or more fluorescently tagged target amplicons to two or more target nucleic acid probes; annealing one or more fluorescently tagged internal control amplicons to one or more internal nucleic acid conno! probes; activating a first fluorescence response from the one or more fluorescently tagged target amplicons hybridized to the two or more target nucleic acid probes; activating a second fluorescence response from the one or more fluoresceotly tagged internal control amplicons hybridized to the one or more internal nucleic acid control probes; and detecting the first and second fluorescence responses for a quantitative analysis of one or more target nucleic acids and one or more internal nucleic acid controls, wherein the two or more target nucleic acid probes and the one or more internal nucleic acid control probes are in close proximity to an upper surface of a substrate, and wherein the activating of the first and second fluorescence responses is by using an evanescent wave of a predetermined wavelength.

2. The quantitative method of claim 1, wherein the annealing occurs during a polymerase chain reaction.

3. The quantitative method of claim 2, wherein the detecting of the first and second fluorescence responses occurs during the annealing step or an extending step of the polymerase chain reaction,

4. The quantitative method of claim 1, wherein the two or more target nucleic acid probes and the one or more internal nucleic acid control probes are printed and immobilized onto a substrate using a micro-array printer.

5. The quantitative method of claim 4, wherein the substrate is chemically modified with a reagent selected from a silane, avidin, poly-L-lysine, streptavidin, a polysaccharide, a mercaptan, or a combination thereof.

6. The quantitative method of claim 1 , wherein polymerase chain reaction is a realtime polymerase chain reaction.

7. The quantitative method of claim 1, wherein the one or more internal nucleic acid controls are linear double-stranded deoxyribonucleic adds, ringed double-stranded deoxyribonucleic acids, or combinations thereof.

8. The quantitative method of claim 1 , wherein the one or more internal nucleic acid controls have the same primer binding regions as that of the two or mote target nucleic acids.

9. The quantitative method of claim 1 , wherein sequence length of the fluorescently tagged amplicons annealed to the one or more internal nucleic acid control probes is Jess than about five-hundred percent of the sequence length of the fluorescently tagged amplicons annealed to the two or more target nucleic acids.

10. The quantitative method of claim 1 , wherein a sequence of the one or more internal nucleic acid controls has less than about five percent cross reaction.

13. The quantitative method of claim I, wherein if two or more internal nucleic acid controls are present, the two or more internal nucleic acid controls are present in different concentrations.

12, The quantitative method of claim 1, wherein if two or more internal nucleic acid control probes are present, the two or more internal nucleic acid control probes are present in the same concentrations as the two or more target nucleic acid probes.

13. The quantitative method of claim 1, wherein the one or more internal nucleic acid controls has less than about five percent cross reaction with the two or more target nucleic acids.

14. The quantitative method of claim 1 , wherein the first and second fluorescence responses are detected before the hybridization plateaus are reached.

15. The quantitative method of claim 1, wherein the two or more target nucleic acids are derived from one or more pathogens, wherein the one or more pathogens is a virus, a bacterium, an archaea, a fungus, a protozoan, a mycoplasma, a prion, a parasitic organism, or combinations thereof.

16. The quantitative method of claim 15, wherein the one or more pathogens is Rickettsia, Chlamydia, Mycoplasma, Spirochete, Streptococcus, Staphylococcus, L. monocytogenes, N. meningitides, E, colU H. influenzae, B. burgdorferi, Leptospira, Proteus, Anaerobacter, Salmonella, M. tuberculosis, Enteracoccus, PoHovirus, Enterovirus, Coxsackievirus, HSV-J, HSV-2, or combinations thereof.

17. A quantitative method for analyzing target nucleic acids, comprising annealing one or more fluorescently tagged target amplicons to two or more taTget nucleic acid probes; annealing one or more fluorescently tagged internal control amplicons to one or more internal nucleic acid, control probes; activating a first fluorescence response from the one or more fhiorescentJy tagged target amplicons hybridized to the two or more target nucleic acid probes; activating a second fluorescence response from the one or more fluorescently tagged internal control amplicons hybridized to the one or more internal nucleic acid control probes; and detecting the first and second fluorescence responses for a quantitative analysis of one or more target nucleic acids and one or more internal nucleic acid controls, wherein the two or more target nucleic acid probes and the one or more internal nucleic acid control probes are in close proximity to an upper surface of a substrate, and wherein the activating of the first and second fluorescence responses is by using an evanescent wave of a predetermined wavelength; establishing a series of standard amplification curves for each of the two or more target nucleic acids and for each of the one or more internal nucleic acid controls, wherein each standard amplification curve is a dose - effect curve, wherein the dose is a number of amplification cycles and the effect is a hybridization signal; adjusting a detected concentration for each of the two or more target nucleic acids by subtracting a difference in a detected concentration for the one or more internal nucleic acid controls and a predetermined concentration for the one or more internal nucleic acid controls.

18. An apparatus for quantitatively analyzing target nucleic acids, comprising: a substrate having an upper and a lower surface and a refractive index greater than a refractive index of water; a buffer solution substantially in contact with the upper surface of the substrate, the buffer solution being capable of sustaining an amplification reaction and nucleotide hybridization reaction and containing one or more fluorescently tagged primers, one or more optionally fluorescently tagged dNTPs, two or more target nucleic acids, and one or more internal nucleic acid controls; two or more target nucleic acid probes in close proximity to the upper surface of the substrate and within the buffer solution, the two or more target nucleic acid probes having a nucleotide sequence corresponding to, or complementary to, a nucleotide sequence of the two or more target nucleic acids; one or more internal nucleic acid control probes in close proximity to the upper surface of the substrate and within the buffer solution, the one or more internal nucleic acid control probes saving a nucleotide sequence corresponding to, oτ complementary to, a nucleotide sequence of the one or more internal nucleic acid; a ray of light, having a wavelength chosen to activate one or more fluorescently tagged target amplicons, and one or more fluorescently tagged internal control amplicons, incident on an interface between the substrate and the buffer solution at an angle chosen so that an evanescent wave propagates into the buffer solution; a detector capable of detecting fluorescent light emitted by the one or more fluorescently tagged target amplicons, and one or more fluorescently tagged internal control amplicons.

19. The apparatus of claim 18, further comprising a heating element capable of cycling a temperature of the buffer solution, thereby enabling the amplification reaction.

20. The apparatus of claim 18, wherein the amplification reaction is a real-time polymerase chain reaction.