CN1751129B - Identification, monitoring and treatment of infectious disease and characterization of inflammatory conditions related to infectious disease using gene expression profiles - Google Patents

Abstract

A method is provided in various embodiments for determining a profile data set for a subject with infectious disease or inflammatory conditions related to infectious disease based on a sample from the subject, wherein the sample provides a source of RNAs. The method includes using amplification for measuring the amount of RNA corresponding to at least 2 constituents from Table 1. The profile data set comprises the measure of each constituent, and amplification is performed under measurement conditions that are substantially repeatable.

Description

Utilize gene expression atlas to identify, monitor and treatment transmissible disease and sign transmissible disease related inflammation

Technical field and background

The present invention relates to the application of gene expression data, especially relate in transmissible disease identify, in monitoring and treatment and by transmissible disease, caused or the sign of relative experimenter's inflammation and assessment in the application of gene expression data.

Prior art has utilized gene expression data to determine existence or the disappearance of particular disorder when diagnosis special markers, has also described in some cases because specified disease marker is crossed and expressed and score accumulates so that the accuracy of diagnose or sensitivity raising.No matter from the efficiency aspect of hygiene department's medical practice, or with regard to improving diagnostic result, be conducive to regard to patient, about any symptom of particular patient and patient have become a major issue in clinical medicine today to the information of the reaction of therapeutical agent or nutrition agent type and dosage.

Summary of the invention

In first embodiment, the method of collection of illustrative plates (profile) data set of suffering from the experimenter of transmissible disease or transmissible disease related inflammation based on experimenter's sample determination is provided, described offering sample RNA source, described method comprises by augmentation detection corresponding to the amount of the RNA of at least two components in table 1 and obtains the observed value of each component, and the observed value that wherein spectrum data group comprises each component and wherein amplification are repeatably being carried out under measuring condition substantially.

In addition, experimenter may have the doubtful symptom (presumptive signs) of systemic infection, comprise with respect to medical science standard white blood counting rise, temperature raises, rhythm of the heart increase and elevation of blood pressure or be reduced at least one symptom, or the inflammation relevant to transmissible disease may be that at least one in blunt wound or penetrance wound, surgical operation, endocarditis, urinary tract infections, pneumonia or dentistry or Obstetric and Gynecologic Department inspection or treatment causes.

In other embodiments, substantially repeatably measuring condition can be repeatability higher than the degree of 5 percentage points in, or repeatable higher than 3 percentage points and all components amplification efficiency substantially similar, wherein said all components amplification efficiency is in two percentage points or be alternatively less than one percentage point.In this embodiment, sample can be selected from experimenter's blood, blood fraction, body fluid, cell mass and tissue.

In another embodiment, the method that provides sample based on experimenter to characterize the transmissible disease in experimenter or transmissible disease related inflammation, described offering sample the source of RNA, described method comprises the spectrum data group of assessing a plurality of members, each member is the quantified measures of the amount of certain distinct rna component in one group of selected component, thereby the observed value that makes described component can characterize the doubtful symptom of systemic infection, this observed value of wherein said each component is repeatably obtaining under measuring condition substantially.

In addition, experimenter may have the doubtful symptom of systemic infection, comprise occur with respect to medical science standard white blood counting increase, one of symptom such as temperature raises, rhythm of the heart quickening and elevation of blood pressure or reduction, or the doubtful sign of the relevant systemic infection of the inflammation that experimenter may have at least one reason causes in blunt wound or penetrance wound, surgical operation, endocarditis, urinary tract infections, pneumonia or dentistry or Obstetric and Gynecologic Department inspection or treatment.In this embodiment, assessment can further comprise by spectrum data group with the baseline collection of illustrative plates data set of this group setting is compared, baseline collection of illustrative plates data set wherein and transmissible disease to be characterized or the relevant inflammation-related with transmissible disease.

In other embodiments, the amplification efficiency of all components is substantially similar, and transmissible disease or the inflammation relevant to transmissible disease are from infected by microbes, more specifically that bacterium infects, or eucaryon parasitic infection or virus infection or fungi infestation, or relate to systemic inflammatory response syndrome (SIRS).More specifically, transmissible disease or the inflammation relevant with transmissible disease can be from microbemia, viremia or fungemias, or the septicemia being caused by the microorganism of any classification.In addition, transmissible disease or the inflammation relevant with transmissible disease can relate to experimenter's local organization, and sample can be from tissue or the fluid with the completely different type of this local organization.

Other embodiments comprise spectrum data group are stored in digital storage media, wherein store collection of illustrative plates data set and can comprise it is stored in database as record.

Also have another embodiment that the method for transmissible disease in first sample evaluating experimenter based on from experimenter or transmissible disease related inflammation is provided, described offering sample RNA source, described method comprises obtains first spectrum data group from first sample, spectrum data group comprises a plurality of members, each member (member) is the quantified measures of distinct rna group component in one group of selected component, thereby make can assess transmissible disease or transmissible disease related inflammation by the detection of these components, wherein the described observed value for each component is repeatably obtaining under measuring condition substantially.The method also comprises the calibration collection of illustrative plates data set producing for this group, wherein each member of calibration data set is each corresponding member of first spectrum data group and the function of the corresponding member of baseline collection of illustrative plates data set for this group, baseline collection of illustrative plates data set is wherein relevant with transmissible disease to be assessed or this transmissible disease related inflammation, using calibration spectrum data group as the comparison between first spectrum data group and baseline collection of illustrative plates data set, thereby experimenter's transmissible disease or transmissible disease related inflammation are assessed.

In relevant embodiment, experimenter has the doubtful symptom of systemic infection, comprise with respect to medical science standard white cell counting increase, temperature raise, at least one in the sign such as heart rate quickening and elevation of blood pressure or reduction, or transmissible disease or inflammation may relate to by inflammation that wherein at least one reason causes such as blunt wound or penetrance wound, surgical operation, endocarditis, urinary tract infections, pneumonia or dentistry or Obstetric and Gynecologic Department inspection or treatments.

In addition, baseline collection of illustrative plates data set can carry out comfortable one or more other samples that are different from the same experimenter who obtains under the condition of first sample, selectable condition is as follows: the time point that (i) first sample obtains, (ii) site that first sample obtains, experimenter's biology situation when (iii) first sample obtains.

In addition, described one or more other samples can be in for some time interval, between first sample and described one or more other samples, at least one month, obtain, or can be in for some time interval, between first sample and described one or more sample, at least ten two months, obtain, or can before Results or after Results, obtain them.In this embodiment, first sample can carry out autoblood and baseline collection of illustrative plates data set can be from tissue or the body fluid of the experimenter except blood.Or first sample is from experimenter's tissue or body fluid, and baseline collection of illustrative plates data set carrys out autoblood.

In other embodiments, baseline collection of illustrative plates data set can be from one or more other samples of same experimenter, experimenter, when from the sampling of first sample, under different biology situation, obtain, with regard in following factor with regard at least one: the exposure of reactivity, body weight and the environment of age, nutrition history, medical conditions, clinical indication, drug treating, health, and baseline collection of illustrative plates data set can be from one or more other samples of one or more not experimenters.

In addition, one or more different experimenters may have something in common at least one following factor: reactivity, body weight and the environmental exposure etc. of age, sex, race, geographical position, nutrition history, medical conditions, clinical indication, drug treating, health.In other embodiments, clinical indication can be used for assessing one or more different experimenters' transmissible disease or transmissible disease related inflammation, the calibration collection of illustrative plates data set that also can comprise at least one other clinical indication in explanation context, wherein said at least one other clinical indication is selected from hematochemistry, urinalysis, X ray or other radiation or metabolic imaging technology, other chemical assay and physical examination.

In described embodiment, transmissible disease or the inflammation relevant with transmissible disease can be from infected by microbes, bacterium infection, eucaryon parasitic infection, virus infection, fungi infestations, or transmissible disease or transmissible disease related inflammation can be from systemic inflammatory response syndrome (SIRS), from microbemia, viremia, fungemia or the septicemia that caused by the microorganism of any classification.

In other embodiments, funtcional relationship is mathematical function and is different from simple difference, comprises the quadratic function of the corresponding member of first spectrum data group and the corresponding member's of baseline collection of illustrative plates data set ratio, or logarithmic function.In relevant embodiment, if each member of spectrum data group after calibration has the numerical value differing higher than amount D, so just think that they have biology significance, D=F (1.1)-F (.9) wherein, and F is quadratic function.In this embodiment, the acquisition of first sample and first spectrum data group be quantitatively all positioned at first site, by network, enter the database that is stored in second position in digital storage media and produce the spectrum data group of calibration, wherein database is renewable to reflect quantitatively first spectrum data group from sample.In addition, use network to comprise and enter global computer internet.

In relevant embodiment, quantified measures is definite by increasing, and requiring measuring condition is that the amplification efficiency of all components differs and is less than about 2 percentage points, or is less than about 1 percentage point.

To also have another embodiment be first offering sample based on from experimenter, and certain refers to calibration method, this index is the indication that experimenter suffers from transmissible disease or transmissible disease related inflammation, first described offering sample RNA source, described method comprises from a spectrum data group of first sample acquisition, this spectrum data group comprises a plurality of members, each member is the measured value of certain distinct rna group component in one group of selected component, thereby the observed value that makes these components becomes the indication of the doubtful symptom of systemic infection, described group comprises at least two kinds of components in genetic expression group in table 1.In deriving the process of described spectrum data group, each described component detects and substantially repeatably under condition, completes, at least one observed value from spectrum data group is applied in a target function, this function provides the mapping (mapping) to the doubtful symptom of systemic infection observed value of at least one observed value from described spectrum data group, thereby has produced the index relevant with experimenter's transmissible disease or transmissible disease related inflammation.

In addition, experimenter can have the doubtful symptom of systemic infection, at least comprise one of following symptom: with respect to medical science standard, white count increase, temperature rising, heart rate quickening and elevation of blood pressure or reduction, or transmissible disease or inflammation may relate to by inflammation that wherein at least one reason causes such as blunt wound or penetrance wound, surgical operation, endocarditis, urinary tract infections, pneumonia or dentistry or Obstetric and Gynecologic Department inspection or treatments.

In relevant embodiment, target function is built into every linear summation, and form is:

wherein I is index, M _ithe value of spectrum data group membership i, C _ibe constant, P (i) is M _ithe power raising, all round valuess that summation is i are added and form, and i can reach the number of member in data set.In addition, use such as statistical methods such as latent class models and determine C _irelevant to given data with P (i) value, comprise clinical, test and originate and any other data relevant with the doubtful symptom of systemic infection.In alternate embodiment, the standardized value of target function is provided, it is measured with respect to relevant group of experimenter, thereby index can be described with respect to standardized value, wherein standardized value can comprise that thereby building target function makes standardized value be approximately 1, thereby or make standardized value be approximately 0, and deviation card since 0 target function is shown standard deviation units.In other embodiments, relevant group of experimenter has following at least one denominator: age, sex, race, geographical position, nutrition history, medical conditions, clinical indication, drug treating, physical activity, body weight and environmental exposure, or optionally there is following at least one denominator: age, sex, race, geographical position, nutrition history, medical conditions, clinical indication, drug treating, physical activity, body weight and environmental exposure.

In other embodiments, clinical indication can be used for assessing by the spectrum data group after explanation calibration in the background at least one other clinical indication transmissible disease or the transmissible disease related inflammation of relevant subject group, and wherein at least one other clinical indication is selected from hematochemistry, urinalysis, X ray or other radiation or metabolic imaging technology, other chemical assay and physical examination.In addition, quantitative measurment can be determined by amplification, measuring condition is the amplification efficiency of all components to be differed be less than about 2 percentage points, or they differ and are less than 1 percentage point, substantially repeatably measuring condition is within being better than the repeated scope of 5 percentage points, or is better than in the repeated scope of 3 percentage points.

In this embodiment, evaluated transmissible disease or transmissible disease related inflammation are with regard to experimenter's local organization, and first sample is from the tissue or the fluid that are different from described local organization type, transmissible disease wherein or transmissible disease related inflammation are from infected by microbes, being more specifically that bacterium infects, is more specifically also eucaryon parasitic infection, virus infection, fungi infestation or from systemic inflammatory response syndrome (SIRS).

A kind of finger calibration method that provides is provided another embodiment, further comprises:

From at least one other sample, derive at least one other spectrum data group, at least one described other spectrum data group comprises a plurality of members, each member is the quantitative assay value of distinct rna group component in one group of selected component, thereby makes the observed value of component become the indication of the doubtful symptom of systemic infection;

At least one other sample wherein, from same experimenter, extracts under the circumstances that is at least different from first sample extraction with regard to one of following factor: time, nutrition history, medical conditions, clinical indication, drug treating, physical activity, body weight and environmental exposure situation; With

At least one observed value from least one other spectrum data group is applied in target function, described target function provides under different situations the mapping of at least one observed value of described at least one other spectrum data group to an observed value of transmissible disease or transmissible disease related inflammation, thereby is created in the situation that is different from first sample at least one other index relevant with experimenter's transmissible disease or transmissible disease related inflammation.

Relevant embodiment comprises provides index, and target function wherein has 2,3,4 or 5 compositions, comprises the seriousness of disease condition, disease or the process of morbidity.In addition, target function is built into every linear summation, and form is:

wherein I is index, M _ithe value of spectrum data group membership i, C _ibe constant, P (i) is M _ithe power raising, all round valuess that summation is i are added and form, and i can reach the number for member in data set.In addition, use such as statistical methods such as latent class models and determine C _irelevant to given data with P (i) value, comprise clinical, test and originate and any other data relevant with the doubtful symptom of systemic infection.

Or, the standardized value of target function is provided, the relevant group of mensuration of carrying out with respect to experimenter, thereby at least one other index can be described with respect to standardized value, the standardized value wherein providing comprises that thereby building target function makes standardized value be approximately 1, thereby or make standardized value be approximately 0, and in target function, the deviation card since 0 is shown standard deviation units.Described embodiment also can comprise by clinical indication assesses transmissible disease or the transmissible disease related inflammation of relevant subject group by explaining the spectrum data group after calibration in the background at least one other clinical indication, and wherein at least one other clinical indication is selected from hematochemistry, urinalysis, X ray or other radiation or metabolic imaging technology, other chemical assay and physical examination.

As in other embodiments, quantitative measurment can be determined by amplification, measuring condition is the amplification efficiency of all components to be differed be less than about 2 percentage points, or they differ and are less than 1 percentage point, substantially repeatably measuring condition is within being better than the repeated scope of 5 percentage points, or is better than in the repeated scope of 3 percentage points.

In addition, transmissible disease or transmissible disease related inflammation are with regard to experimenter's local organization, and first sample is from the tissue or the fluid that are different from described local organization type.

Also have other embodiment to comprise for finger calibration method is provided, transmissible disease wherein or transmissible disease related inflammation are from infected by microbes, being more specifically that bacterium infects, is more specifically also to comprise at least two kinds of components in table 1 in eucaryon parasitic infection, virus infection, fungi infestation or the group from systemic inflammatory response syndrome (SIRS) and component.

Another embodiment provides the transmissible disease of first sample evaluating experimenter based on from experimenter or the method for transmissible disease related inflammation, first described offering sample RNA source, described method comprises from first sample and obtains first spectrum data group, first described spectrum data group comprises a plurality of members, each member is the quantitative assay value of the amount of certain distinct rna component in one group of selected component, thereby can assess transmissible disease or transmissible disease related inflammation by the observed value of these components, wherein each component detects and substantially repeatably under measuring condition, completes.The method also comprises that generation is for the spectrum data group after the calibration of this group, wherein calibrate the function that each member of collection of illustrative plates data set is the corresponding member of first spectrum data group and this corresponding member of group baseline collection of illustrative plates data set, wherein each member of baseline collection of illustrative plates data set is the stdn observed value of the amount of one of component in the group of mensuration with regard to relevant group experimenter, and described baseline collection of illustrative plates data set is corresponding with transmissible disease to be assessed or transmissible disease related inflammation, and the spectrum data group of calibrating is the comparison between first spectrum data group and baseline collection of illustrative plates data set, thereby can assess experimenter's transmissible disease or transmissible disease related inflammation.

In this embodiment, experimenter can have the doubtful symptom of systemic infection, at least comprise one of following symptom: with respect to medical science standard, white count increase, temperature rising, heart rate quickening and elevation of blood pressure or reduction, or transmissible disease or inflammation may relate to by inflammation that wherein at least one reason causes such as blunt wound or penetrance wound, surgical operation, endocarditis, urinary tract infections, pneumonia or dentistry or Obstetric and Gynecologic Department inspection or treatments.

In addition, relevant group of experimenter is health volunteer's group with one of following common trait: age, sex, race, geographical position, nutrition history, medical conditions, clinical indication, drug treating, physical activity, body weight and environmental exposure.As in other embodiments, quantitative measurment can be determined by amplification, measuring condition is the amplification efficiency of all components to be differed be less than about 2 percentage points, or they differ and are less than 1 percentage point, measuring condition is within being better than the repeated scope of 5 percentage points or to be better than in the repeated scope of 3 percentage points be substantially repeatably.

In this embodiment, evaluated transmissible disease or transmissible disease related inflammation are for experimenter's local organization, first sample comes from tissue or the fluid that is different from described local organization type, and spectrum data group can be stored in digital storage media, comprises it is stored in database as record.In addition, baseline collection of illustrative plates data set is being different from from same experimenter one or more other samples that obtain under first sample sampling condition, wherein one or more other samples obtain before Results or after Results, or one or more other samples obtain in certain time interval, this timed interval is at least one month of initial sample and this sample sampling interval, or interval at least 12 months.In addition, if first sample come autoblood baseline collection of illustrative plates data set come the experimenter outside autoblood to organize liquid or body fluid, or first sample is from experimenter's tissue juice or body fluid, baseline collection of illustrative plates data set carrys out autoblood.

Also have another embodiment to provide a kind of method in order to assess experimenter's transmissible disease or transmissible disease related inflammation, the method be take from first sample of experimenter and is basis from second sample of definite indicator cells group, described offering sample RNA source, the method comprises first sample or its part is added on definite indicator cells group.Described method also comprises by second sample derives first spectrum data group, first spectrum data group comprises a plurality of members, each member is the quantified measures of certain distinct rna or protein group component in one group of selected component, thereby by the detection of these components, can weigh the doubtful symptom of systemic infection, wherein the described observed value for each component is repeatably obtaining under measuring condition substantially, described method also comprises the spectrum data group producing for the calibration of this group, each member of the spectrum data group that its alignment is crossed is the corresponding member of first spectrum data group and function for the corresponding member of baseline collection of illustrative plates data set of this group, wherein each member of baseline collection of illustrative plates data set measures to the amount of one of component described in this group the stdn observed value obtaining, and wherein baseline collection of illustrative plates data set is relevant with transmissible disease or transmissible disease related inflammation to be assessed, the spectrum data group of calibrating is the comparison between first spectrum data group and baseline collection of illustrative plates data set, thereby can assess experimenter's transmissible disease or transmissible disease related inflammation.

In relevant embodiment, experimenter can have the doubtful symptom of the systemic infection that at least comprises one of following symptom,, with respect to medical science standard, white count increase, temperature rising, heart rate quickening, elevation of blood pressure or reduction, or, transmissible disease or inflammation may relate to by inflammation that wherein at least one reason causes such as blunt wound or penetrance wound, surgical operation, endocarditis, urinary tract infections, pneumonia or dentistry or Obstetric and Gynecologic Department inspection or treatments, and relevant group of experimenter is one group of health volunteer.

In addition, relative group of experimenter is health volunteer's group with one of following common trait: age, sex, race, geographical position, nutrition history, medical conditions, clinical indication, drug treating, physical activity, body weight and environmental exposure.In addition, clinical indication can be by explaining in the background at least another kind of clinical indication that the spectrum data group after calibration is used to assess transmissible disease or the transmissible disease related inflammation of relevant subject group, and wherein at least one other clinical indication is selected from hematochemistry, urinalysis, X ray or other radiation or metabolic imaging technology, other chemical assay and physical examination.

For other embodiments, quantitative measurment can be determined by amplification, measuring condition is the amplification efficiency of all components to be differed be less than about 2 percentage points, or they differ and are less than 1 percentage point, measuring condition is within being better than the repeated scope of 5 percentage points or to be better than in the repeated scope of 3 percentage points be substantially repeatably.In addition, evaluated transmissible disease is with regard to experimenter's local organization, and first sample is from the tissue or the fluid that are different from described local organization type, and transmissible disease or transmissible disease related inflammation are infected by microbes.

In relevant embodiment, baseline collection of illustrative plates data set is being different from from same experimenter one or more other samples that obtain under first sample sampling condition, wherein one or more other samples obtain before Results or after Results, or obtain in certain time interval, the described timed interval is at least one month of initial sample and this sample sample interval, or interval at least 12 months.In this embodiment, if first sample come autoblood baseline collection of illustrative plates data set come the experimenter outside autoblood to organize liquid or body fluid, or first sample is from experimenter's tissue juice or body fluid, baseline collection of illustrative plates data set carrys out autoblood.

In another embodiment of the present invention, provide take to be that from the sample of having used the target cell group of the first reagent basis assessment is subject to the target cell group's of described the first agents influence transmissible disease or transmissible disease related inflammation, described offering sample RNA source.Described method comprises derives first spectrum data group from sample, first spectrum data group comprises a plurality of members, each member is the quantified measures of certain distinct rna group component in one group of selected component, thereby can assess by the detection of these components transmissible disease or the transmissible disease related inflammation that is subject to described the first agents influence, wherein the described observed value for each component is repeatably obtaining under measuring condition substantially, described method has also produced the spectrum data group for the calibration of this group, each member of the spectrum data group that its alignment is crossed is the corresponding member of first spectrum data group and function for the corresponding member of baseline collection of illustrative plates data set of this group, wherein each member of baseline collection of illustrative plates data set measures to the amount of one of component described in this group the stdn observed value that obtains relevant group target cell group, and wherein baseline collection of illustrative plates data set is relevant with transmissible disease or transmissible disease related inflammation to be assessed, the spectrum data group of calibrating is the comparison between first spectrum data group and baseline collection of illustrative plates data set, thereby can assess the target cell group's who is subject to the first agents influence transmissible disease or transmissible disease related inflammation.Target cell group can have the doubtful symptom of the system infection that at least comprises one of following symptom, that is, with respect to medical science standard, white count increase, temperature rising, heart rate quickening, elevation of blood pressure or reduction.Transmissible disease or inflammation may relate at least one inflammation causing in the reasons such as blunt wound or penetrance wound, surgical operation, endocarditis, urinary tract infections, pneumonia or dentistry or Obstetric and Gynecologic Department inspection or treatment.Relevant group of target cell group can be the target cell group of one group of health.Or relevant group of target cell group can have one of following common trait: age, sex, race, geographical position, nutrition history, medical conditions, clinical indication, drug treating, physical activity, body weight and environmental exposure.In said case, clinical indication can be used for assessing relevant target cell group's transmissible disease or transmissible disease related inflammation, and the method is further included in the spectrum data group that at least one other clinical indication background, explanation was calibrated, the optional autoblood chemistry of at least one described other clinical indication, urinalysis, X ray or other radiation or metabolic imaging technology, other chemical assay and physical examination.Quantitative measurment can determine by amplification, and measuring condition is the amplification efficiency of all components to be differed be less than about 2 percentage points, or they differ and are less than 1 percentage point.Measuring condition is within being better than the repeated scope of 5 percentage points or to be better than in the repeated scope of 3 percentage points be substantially repeatably.In addition, evaluated transmissible disease or the inflammation relevant to transmissible disease are with regard to experimenter's local organization, and first sample is from the tissue or the fluid that are significantly different from described local organization type.Transmissible disease or transmissible disease related inflammation may be infected by microbes, bacterium infection, eucaryon parasitic infection, virus infection, fungi infestation, systemic inflammatory response syndrome (SIRS), microbemia, viremia, fungemia or the septicemia that caused by the microorganism of any classification.The related embodiment of described method also can comprise that storage collection of illustrative plates data set is in digital storage media.Storage collection of illustrative plates data set can comprise it is kept in database as record.This embodiment can comprise that restriction is that first sample comes autoblood and baseline collection of illustrative plates data set to come the experimenter outside autoblood to organize liquid or body fluid.Or first sample is from experimenter's tissue juice or body fluid, and baseline collection of illustrative plates data set carrys out autoblood.Equally, baseline collection of illustrative plates data set can be different from one or more other samples that obtain under first sample sampling condition from same experimenter.Such a or a plurality of other samples obtain before Results or after Results, or obtain after certain time interval, at least one moon of initial sample and this sample sampling interval.

Other embodiment of the present invention relates to respect to the transmissible disease or the transmissible disease related inflammation that are subject to the target cell group of the second agents influence for assessment, be subject to target cell group's transmissible disease or the method for transmissible disease related inflammation of the first agents influence, it take from used the first reagent target cell group first sample and from second sample having used the target cell group of the second reagent, be basis, described offering sample RNA source.The step that such method comprises has, from first sample, derive first spectrum data group, from second sample, derive second spectrum data group, first and second spectrum data groups comprise a plurality of members separately, each member is the quantified measures of a distinct rna group component in one group of selected component, thereby can assess with respect to the second reagent by the detection of these components, be subject to transmissible disease or the transmissible disease related inflammation of described the first agents influence, wherein the described observed value for each component is repeatably obtaining under measuring condition substantially, described method has also produced for this and has organized first spectrum data group of calibrating and second spectrum data group of calibrating, wherein each member of (i) first spectrum data group of calibrating is the corresponding member of first spectrum data group and function for the corresponding member of baseline collection of illustrative plates data set of this group, and (ii) each member of second spectrum data group of calibrating is corresponding member and the corresponding member's of baseline collection of illustrative plates data set of second spectrum data group function, wherein each member of baseline collection of illustrative plates data set measures with respect to relevant group experimenter, the stdn observed value of the amount of one of component described in this group, and wherein baseline collection of illustrative plates data set is relevant with transmissible disease or transmissible disease related inflammation to be assessed, first and second spectrum data group of calibrating are the comparisons between comparison between first spectrum data group and baseline collection of illustrative plates data set and second spectrum data group and baseline collection of illustrative plates data set, thereby can assess transmissible disease or transmissible disease related inflammation with respect to the target cell group of the second agents influence, be subject to the target cell group's of the first agents influence transmissible disease or transmissible disease related inflammation.Target cell group can have the doubtful symptom of the system infection that at least comprises one of following symptom, that is, with respect to medical science standard, white count increase, temperature rising, heart rate quickening, elevation of blood pressure or reduction.Equally, target cell group may have the doubtful symptom that relates at least one systemic infection causing inflammation in the reasons such as blunt wound or penetrance wound, surgical operation, endocarditis, urinary tract infections, pneumonia or dentistry or Obstetric and Gynecologic Department inspection or treatment.The first reagent can be the first medicine and the second reagent can be the second medicine.Or the first reagent is a kind of medicine and the second reagent is a kind of compounding mixture or nutrient drug.Quantitative measurment can determine by amplification, and measuring condition is the amplification efficiency of all components to be differed be less than about 2 percentage points, or they differ and are less than 1 percentage point.Measuring condition is within being better than the repeated scope of 5 percentage points or to be better than in the repeated scope of 3 percentage points be substantially repeatably.Evaluated transmissible disease or transmissible disease related inflammation are with regard to experimenter's local organization, and first sample is from the tissue or the fluid that are different from described local organization type.Transmissible disease or transmissible disease related inflammation may be infected by microbes, bacterium infection, eucaryon parasitic infection, virus infection, fungi infestation, systemic inflammatory response syndrome (SIRS), microbemia, viremia, fungemia or the septicemia that caused by the microorganism of any classification.Described method also can comprise stores first and the step of second spectrum data group in digital storage media.Storing first can comprise it is kept in database as record with second spectrum data group.Baseline collection of illustrative plates data set can be different from first sample sampling condition or be different from one or more other samples that obtain under second sample sampling condition from same experimenter.First sample carrys out autoblood and baseline collection of illustrative plates data set comes the experimenter outside autoblood to organize liquid or body fluid.Or first sample is from experimenter's tissue juice or body fluid, and baseline collection of illustrative plates data set carrys out autoblood.

In another embodiment of the present invention, first sample of usining from experimenter provides as basis as the index with experimenter's inflammation indication of the doubtful symptom of systemic infection, first described offering sample RNA source.Described method comprises from a spectrum data group of first sample acquisition, this spectrum data group comprises a plurality of members, each member is the measured value of certain distinct rna group component in one group of selected component, thereby the observed value that makes these components becomes the indication of inflammation, described group comprises at least two kinds of components in genetic expression group in table 1, in deriving the process of described spectrum data group, each described component observed value is repeatably obtaining under condition substantially, at least one observed value from spectrum data group is applied in a target function, this function provides the mapping to described inflammation observed value of at least one observed value from described spectrum data group, thereby produced the index with the inflammation-related of sample, wherein target function has been used the data from the baseline collection of illustrative plates data set for this group, each member of baseline collection of illustrative plates data set is the stdn observed value of the amount of one of component described in this group of measuring with respect to relevant group experimenter, baseline data group wherein relates to inflammation to be assessed.Experimenter can have the doubtful symptom of the systemic infection that at least comprises one of following symptom, that is, with respect to medical science standard, white count increase, temperature rising, heart rate quickening, elevation of blood pressure or reduction.Or the doubtful symptom of systemic infection relates at least one caused inflammation in the reasons such as blunt wound or penetrance wound, surgical operation, endocarditis, urinary tract infections, pneumonia or dentistry or Obstetric and Gynecologic Department inspection or treatment.The described observed value being applied at least one the spectrum data group in target function may be 2,3,4 or 5.

Also have other embodiment to provide by the index method that guiding treatment is intervened in transmissible disease or transmissible disease related inflammation patient, the index providing according to arbitrary in above-mentioned embodiment is provided the method, this index is compared to obtain difference value to this index of gained of standardized value measure to(for) relevant subject group, then by the difference between described index and this index standardized value, carry out guiding treatment intervention, Results is wherein microorganism specific treatment, or bacterium specific treatment, or fungi specific treatment, or virus-specific treatment, or eucaryon parasite specific treatment.

Another embodiment provides the method for pathogen type in one group of object pathogenic agent is distinguished transmissible disease or transmissible disease related inflammation patient, take at least one sample from experimenter as basis, this offering sample the source of RNA, described method comprises at least one spectrum data group of measuring for experimenter, by this spectrum data group at least one group of relevant group of sample in object kind pathogenic agent measured at least one obtained baseline collection of illustrative plates data set compare to obtain difference value, Pathogen category by this difference value at least one group of baseline collection of illustrative plates data set, distinguish for the pathogen type at least one spectrum data group of experimenter, wherein said Pathogen category is microorganism.Or the kind of pathogenic agent is bacterium, and this difference value is for making a distinction gram positive bacterium pathogenic agent and gram negative bacterium pathogenic agent.Or the kind of pathogenic agent is fungi, described difference value is used for distinguishing Candida (Candida) pathogenic agent and chronic Candida pathogenic agent.More particularly, the kind of pathogenic agent is virus, this difference value is used for distinguishing DNA virus pathogenic agent and RNA viruses pathogenic agent, or the kind of pathogenic agent is virus, and described difference is used for distinguishing Rhinovirus (rhinovirus) pathogenic agent and Influenza Virus (influenza) pathogenic agent.Also more particularly, this Pathogen category is eucaryon parasite, and described difference is used for distinguishing plasmodium (plasmodium) parasitosis substance and trypanosoma (trypanosomal) pathogenic agent.

Also having another embodiment to provide utilizes index from one group of object pathogenic agent, to distinguish the method for pathogen type in transmissible disease or transmissible disease related inflammation patient, take at least one sample from experimenter as basis, described method comprise provide according in above-mentioned embodiment any at least one index of experimenter, at least one index is compared to obtain at least one difference value to this index of gained of at least one standardized value measure to(for) at least one group of relevant subject group, then utilize described at least one index and at least one difference value between at least one standardized value of this index from a class pathogenic agent, to distinguish the type of pathogenic agent.

Accompanying drawing summary

Aforesaid feature of the present invention will be easier to understand with reference to following detailed description and accompanying drawing thereof, wherein:

Figure 1A has shown and during single male subject is suffered from optic neuritis process, has detected respectively at 8 days from the result of 24 genes of proinflammatory gene group (shown in table 1) originally.

1B has set forth the application of the inflammatory parameters that relates to Figure 1A data according to embodiment of the present invention.

Fig. 2 is the diagram of the identical inflammatory parameters that calculates in 9 different important clinical critical events.

Fig. 3 has shown the effect of the single dose 800mg Ibuprofen BP/EP processing characterizing by described index in single donor.

Fig. 4 has illustrated the calculating acute inflammation index of illustrated five different situations.

Fig. 5 has shown for monitoring the viral indicator reaction of upper respiratory tract infection (URI) process.

Fig. 6 and 7 use gene expression atlas have compared two kinds of different colonies (48 sites that relate to table 1 inflammation gene expression expression group).

Fig. 8 compares normal population and rheumatoid arthritis colony from longitudinal research direction.

Fig. 9 has compared two kinds of normal population, a kind of longitudinal, a kind of horizontal.

Figure 10 has illustrated the genetic expression value demonstrating for a plurality of individualities in normal population.

Figure 11 has shown the expression level of each gene in 4 genes (table 1 inflammation gene expression expression group), 8 every months in the middle of the month, single experimenter is detected.

The expression level of each in Figure 12 and 13 similar 48 genes that shown in each situation unique experimenter separately (take and feel good and do not take medicine as basis and select) in each situation (in table 1 inflammation gene expression expression group), in the situation of Figure 12, measure and continue 4 weeks weekly, be monthly to measure to continue 6 months in the situation of Figure 13.

Figure 14 has shown and has used within a certain period of time the impact that anti-inflammatory steroid is expressed inflammation gene expression in single people experimenter, by the inflammation gene expression expression group of table 1, detects.

Figure 15 is to have been shown and to have been used within a certain period of time the impact of single dose prednisone on 5 genes (in the inflammation gene expression expression group of table 1) expression by the whole blood sample available from people experimenter with the similar mode of Figure 14.

Figure 16 has also shown and has used within a certain period of time the impact that TNF inhibition compound is expressed inflammation gene expression in single patient with rheumatoid arthritis, but expression described herein is represented as the comparison of carrying out with the mean level (ML) of measuring in advance normal (that is, not yet diagnosed, healthy) the similar position of colony of (with Fig. 6 and 7 relevant).

Figure 17 A further describes the consistence that in Liao colony, inflammation gene expression is expressed.

Figure 17 B has shown available from the normal distribution of not making a definite diagnosis the desired value of colony.

Figure 17 C has illustrated the application of the index identical with Figure 17 B, and wherein the inflammation intermediate value of normal population is set as 0, and normal and ill experimenter is carried out to the standard deviation units mapping with respect to this intermediate value.

Figure 18 has made the population of subjects of trouble rheumatoid arthritis of two groups different (reactor is to nonresponders) gene expression atlas with identical in Figure 17 A 7 sites (every group of 6 people) to be similar to the mode of Figure 17 A.

Figure 19 illustrates the application of inflammation index in the single experimenter of assessment trouble rheumatoid arthritis thus, and described experimenter is to traditional methotrexate therapy poor response.

Figure 20 is similar has described the application of described inflammation index in 3 patient with rheumatoid arthritis of assessment, and described patient is to traditional methotrexate therapy poor response.

Figure 21-23 have shown respectively and have experienced three kinds of independently inflammation indexes of international group patient with rheumatoid arthritis after therapy.

Figure 24 has described inflammation index to assessing the application of single patients with inflammatory bowel.

Figure 25 shown with reference to other NSAID (non-steroidal anti-inflammatory drug) (NSAIDs), the gene expression atlas of 24 sites of the whole blood of processing with Ibuprofen BP/EP in vitro (in the inflammation gene expression expression group of table 1).

That Figure 26 has shown is how objective, quantitative, accurately and the repeatably relatively effect of two kinds of competitive anti-inflammatory compounds.

Figure 27 to 41 has illustrated the application of genetic expression group in the early stage evaluation of transmissible disease and monitoring.

Figure 27 utilizes the new bacteria genetic expression group of 24 genes of research and development to distinguish different bacterium situation in host living beings system.

Figure 28 has shown from three kinds of independent sources: the LTA of streptococcus pyogenes (S.pyogenes), subtilis (B.subtilis) and golden suis (S.aureus) is at the differential expression of Single locus IFNG.

Figure 29 and 30 shown respectively in whole blood inflammation 48A and 48B site (relating separately to above-mentioned Fig. 6 and 7) after two hours to applying the reaction of Gram-positive and gram-negative biological.

Figure 31 with 32 respectively corresponding to Figure 29 and 30 and similar to them, except monitoring is herein carried out after using 6 hours.

Figure 33 has compared the genetic expression of being brought out and being brought out by the intestinal bacteria filtrate of lifeless matter body by intestinal bacteria and has reacted.

Figure 34 and Figure 33 are similar, but the reaction of this place comparison is to the stimulation of the intestinal bacteria filtrate from independent with to carrying out oneself to add the reaction of stimulation of the intestinal bacteria filtrate of PXB.

Figure 35 has described the genetic expression reaction of bringing out after golden suis adds 2,6 and 24 hours.

Figure 36 to 41 compared different concns and the time intestinal bacteria and the genetic expression brought out of golden suis.

Figure 42 has described statistics T is detected and is applied to differentiate the potential member that can distinguish the signal gene expression group between normal subjects and unstable patient with rheumatoid arthritis.

Figure 43 has described the expression level of 8 bacteremic patients of doubtful trouble for the group of 17 genes.

Figure 44 has described statistics T is detected and is applied to differentiate effective member that can distinguish the signal gene expression group between normal subjects and microbemia patient.

Figure 45 has described the application of an algorithm (as shown in FIG.), has obtained the index of correlation of the rheumatoid arthritis (RA) when being applied to respectively normal subjects, patient with rheumatoid arthritis and microbemia patient.

Figure 46 has described the application of an algorithm (as shown in FIG.), has obtained the microbemia index of correlation when being applied to respectively normal subjects, patient with rheumatoid arthritis and microbemia patient.The detailed description of particular

Definition

Unless have other needs in context, otherwise following term should have below pointed implication:

" algorithm " is a rule for descriptive biology situation.Described rule can be special definition on algebraically, also can comprise and need alternative or a plurality of decision-points of specific domain knowledge, explanation or other clinical indication of specialty.

According to defined other term herein, " reagent " is " composition " or " stimulator ", or the combination of composition and stimulator.

" amplification " is the function of DNA replication dna number in quantitative RT-PCR test, can carry out quantitatively its concentration thus." amplification " is in sensitivity and the specificity of this specified amount detection technique.Therefore, amplification is that the observed value of concentration of component is provided under amplification efficiency and resultant sensitivity and repeated similar condition substantially for all components for detecting.

" baseline collection of illustrative plates data set " is, under expection biology situation for arithmetic stdn object, biological sample (or colony and a sample) is assessed to the numerical value that produced genetic expression group component is relevant.The biology condition of expection can be, for example, before being exposed to certain reagent or do not treating that disease exists or not ill condition under experimenter's (or colony or one group of experimenter) situation.Alternatively, or additional, the biology situation of expection can be experimenter or colony or one group of experimenter's healthy state.Alternatively, or additional, the biology situation of expection can be to based on the selected colony of one of following factor or the relevant situation of subject group: the reactivity of age, sex, race, geographical position, nutrition history, medical conditions, clinical indication, drug treating, health, body weight and environmental exposure situation.

One " group " or " group " sample or experimenter refer to limit or selected sample or subject group, wherein in this group or this group of sample or experimenter, between included member, have basic general character or association.

One " cell mass " refers to any groups of cells, wherein between the member of cell mass, exists basic general character with associated, for example, comprises that one group of cell is from an organism or from a collection of culturing cell or from Yi Ge tissue biopsy sample.

Experimenter's " biology situation " is the experimenter's situation under observing in relevant field, described field may comprise any aspect of the experimenter that can monitor its changed condition, such as healthy state, the disease that comprises cancer, wound, aging, infection, tissue deterioration, developmental process, healthy, obesity and mood.As observable, the situation within the scope of this may be chronic or acute or be only instantaneous.In addition, the biology situation of target can show maybe can be confined to (such as skin, heart, eyes or blood) in specific organ by organism or cell mass, but in either case, described situation all can be by the cell mass that is affected a sample and directly monitored, or carry out indirect monitoring by the sample from other position of experimenter.Term " biology situation " comprises " physiology situation ".

Experimenter's " body fluid " comprises blood, urine, spinal fluid, lymph liquid, mucous membrane juice, prostatic fluid, seminal fluid, hemolymph or other any body fluid known in the art of experimenter.

" the spectrum data group of calibration " is the function for first spectrum data group membership and the corresponding member of baseline collection of illustrative plates data set of given component in group.

" clinical indication " is separately or any physiological data of being combined with other data for assessment of cell mass or organism physiology situation.This term comprises clinical front indication.

" composition " comprises the complex mixture of combination, toxin, food, foodstuff additive, mineral and material in any physical condition or chemical compound, nutrient drug in the physical condition situation of combination, medicine, homeopathy preparation, allopathy preparation, naturopathy preparation, compound.

From sample, " derivation " spectrum data group comprises by following two kinds of modes and measures the one group numerical value relevant to the component of genetic expression group: (i) by the described component in direct-detection biological sample or (ii), by detecting, be exposed in primary sample or derived from the described component in the second biological sample in the material of primary sample.

" unique RNA or protein component " in one group of component is unique gene product of expressing, or RNA or protein." expression " product of gene comprises the gene product by the translation generation of messenger RNA(mRNA), or RNA or protein.

" genetic expression group (gene expression panel) " is one group of component examining through experiment, each component is the unique product of expressing of gene, or be RNA or for protein, wherein the selection of this group component is can detect target organism situation by their detection.

" gene expression atlas " is the one group numerical value relevant to genetic expression group component being produced by assessment biological sample (sample group or sample sets).

The value of the situation that " gene expression atlas inflammatory parameters " has been to provide gene expression atlas to the target function of the mapping of monodrome inflammation observed value.

Experimenter's " healthy state " comprises experimenter's intelligence, mood, health, spirit, allopathy, naturopathy and hahnemannian situation.

" index " is in order to help to simplify or to show or inform more complicated quantity information analysis and the arithmetic that carries out or the numerical characteristic of mathematical derivation.The index of disease or colony can be determined for several experimenters or the sample with common biological condition by applying specific algorithm.

" inflammation " is the same with the general medical science meaning of this word for the meaning herein, and can be acute or chronic, simple or pyogenic, local or reaction that scatter, cell or tissue, by any amount of chemistry, physics or biological reagent or agent combination, cause or maintain.

" inflammatory states " is used in reference to the corresponding biology situation of the experimenter who is produced by inflammation, or by the degree of inflammation, it characterized.

The number that " the large quantity " of the data set based on the common group of gene refers to data set is even as big as obtaining with respect to take the same group of significance,statistical result for basic routine data set.

" standard " state of the experimenter of composition to be administered refers to use front experimenter's state, even if experimenter is ill by chance, also can.

Gene " group " is the one group of gene that at least comprises two components.

The aliquots containig that can comprise individual cells or a plurality of cell or cell fragment or the body fluid of taking from experimenter from experimenter's " sample ", mode used comprises venipuncture, excretion, ejaculation, massage, slicer, syringe needle absorption, lavation sampling, scraping, surgical operation incision or intervention or alternate manner known in the art.

" characteristic spectrum (signature profile) " is the subset confirming through experiment in the gene expression atlas of selecting in order to distinguish the physiological mechanisms of biological condition, reagent or effect.

" feature group " is the subset of genetic expression group, and the selection of its component is in order to distinguish the physiological mechanisms of biological condition, reagent or effect.

" experimenter " is cell, tissue or people or the non-human being's body under observing, and is no matter in body, in vitro or external.When we mention the biology situation of the sample evaluating experimenter based on from experimenter, not only comprised and utilized blood or other tissue sample from people experimenter to carry out evaluator experimenter's situation, also comprised and used blood sample itself to assess for example therapy or the effect of certain reagent to this sample as tested product.

" stimulation " comprise (i) monitored with Physical interaction experimenter, for example ultraviolet light,long wave or B, or for the actinotherapy of seasonal emotional maladjustment, or treat melanoma by psoralene treatment psoriasis or with the radioactive substance of implantation, other radiation exposure, and (ii) experimenter's any monitored health, intelligence, mood or spiritual activity or inactive property.

" treatment " comprises that intention maintains or change all interventions of the monitored biology situation of experimenter, no matter is biological, chemical, physics, preternatural method or the combination of these preceding methods.

Be hereby expressly incorporated by reference, the patent application that herein contriver proposes and announcing in April 12 calendar year 2001, the PCT patent application publication number WO 01/25473 that is entitled as " system and the method for learning state or reagent with the gene expression atlas characterising biological of calibration " has announced the application of genetic expression group in following assessment: (i) biological condition (comprising health and disease) and (ii) one or more reagent the impact of biological condition (is comprised to healthy state, toxicity, therapeutical agent is processed and drug effect).

Especially, genetic expression group can be used for detecting therapeutic efficiency natural or synthetic composition or stimulator, and they can learn state preparation or combination or mixing separately for the target organism of certain limit; Toxicological action and the dosage effect of the mixture of contemplated composition or composition to certain individuality or a group or one group of individuality or a group cell, thereby determining how two or more different reagent of using in single therapy may interact detects any synergy, increase, negative, neutral or poisonous activity, carry out clinical before or clinical trial so that the preselected new standard that provides of experimenter carrying out according to informationalized spectrum data group for disclosing disease condition is carrying out carrying out the preparation dosetest for these patients before 1 phase and the test of 2 phases.These genetic expression groups can be used for sample from experimenter to assess their biological condition.

The selection mode of genetic expression group is to make the detection by quantitative of in group RNA or protein component form the detection to experimenter's biological condition.In a kind of arrangement, applied the spectrum data group of calibrating.Each member of the spectrum data group of calibrating is the observed value of unique ingredient in (i) genetic expression group and (ii) function of baseline amount.

We have found under can repeat condition (multiplicity of detection is better than 20%, preferably high 5 percentage points or higher, more preferably high 3 percentage points or higher) detection by quantitative of carrying out component can obtain valuable and unexpected result.For this object and following claim, we are used as the multiplicity of detection as the measuring condition that " substantially can repeat " is provided higher than 20%.Particularly, wish to obtain each time the observed value corresponding to certain component expression level in concrete sample, for the expression of substantially the same level, should obtain substantially the same observed value.In this way, genetic expression group can significantly compare at the expression level of certain component between sample and sample.Even for the expression level observed value of certain concrete component be inaccurate (for example, for example, low by 30%), the standard of repeatability means for this component, if had, depart from, to remain systematic departing from, and therefore the expression level observed value of this component can carry out significant comparison.Can in the situation changing, obtain valuable information and the correlated expression that compares component in this way.

Except repeated standard, also wish that second standard also can meet, that is, at all components amplification efficiency, substantially carry out the detection by quantitative of component under the condition of similar (in 1 to 2 percentage point, being conventionally less than 1 percentage point).When these two standards are all satisfied, in given sample, the expression level observed value of component can significantly be compared or compare between sample with the expression level observed value of another component.

The present embodiment relates to certain index or algorithm application in the following areas, and described index and algorithm produce the detection by quantitative from component, and can derive from addition analysis expert or calculation biology: (a) in complicated data set is analyzed; (b) between control or normalized sample or experimenter, in genetic expression value, do not provide impact information or other side subtle change; (c) simplify the sign of complex data group to compare with other complex data group, database or from index or the algorithm of complex data group; (d) monitoring experimenter's biological condition; (e) detect for certain limit target organism and learn situation and can prepare individually or the natural or synthetic composition of co-formulated or the therapeutic efficiency of stimulator or mixture; (f) estimate toxicological action and the dosage effect for individuality or a group or one group of individuality or the composition of cell mass or the mixture of composition; (g) thus determining how two or more different reagent of using in single therapy may influence each other detects any synergy, increase, negative, neutral or poisonous activity; (h) carry out clinical before and clinical trial so that experimenter that disease condition carries out according to informationalized spectrum data group is preselected to be provided new standard and is carrying out carrying out the preparation dose study for these patients before 1 phase and the test of 2 phases in order to disclose.

Gene expression atlas and for the application that concrete situation or reagent or the index of the two characterize can be used for reducing by 3 clinical trial phases expense and can be clinical in 3 phases outside use, for the medicine of approved marks, in a class medicine, be that concrete patient selects the direct suitable medicine for its unique physiology situation, diagnosis or determine may be before paresthesia epilepsy medical conditions or the prognosis of infection, or diagnosis is used relevant unfavorable negative interaction to certain therapeutical agent, the health care of body of managing patient, the quality of control different batches reagent or reagent mixture.

Experimenter

The method of herein announcing can be applicable to the cell of people, Mammals or other organism and carries out excessive experiment without those of ordinary skills, because all cells transcribe rna and be known in the art how to extract RNA from all types cell all.

The component of Select gene expression group

The universal method of Select gene expression group component has been consulted PCT application publication number WO01/25473.We have designed and have tested and have confirmed far-ranging series of genes expression group, and each group provides the quantified measures of the biology situation of coming autoblood or other tissue sample.For each group, the gene expression atlas that experiment has confirmed to utilize the component of this group to obtain provides the information of biology situation (when we have also proved biology condition information is being provided in other article, genetic expression group also can be used for other side, as detected treatment effect and providing target for Results).The summary of the example of genetic expression group and respectively component is referring to following appended form:

Table 1. Infectious Diseases or transmissible disease related inflammation genetic expression group

Other group can be built and test and examine according to the application's relative theory by those of ordinary skills.

Test design

We are conventionally by a sample quadruplicate experiment in group,, sample are divided into sample aliquot and for the concentration of each component in every a sample aliquot gene expression detection group that is.Altogether surpassed the test of 900 components, each test is quadruplicate carrying out, and we find that average coefficient of variation (standard deviation/mean value) * 100 is less than 2 percentage points in the result of each test, are conventionally less than 1 percentage point.This figure is the observed value of we so-called " variability between mensuration ".We test for different situations with same sample material.By 72 tests, produce self-contained 24 members' the concentration of component observed value of group and described concentration measurement and be at three kinds and measure different in the situation that, we find that average coefficient of variation is less than 5 percentage points, is less than 2 percentage points conventionally.We are called so-called " variability between mensuration " by this.

We have found to utilize quadruplicate detected result to identify and the data point of eliminating statistics what is called " outlier " is significant, described data point be differ percentage be greater than all four values mean value for example 3% and be not to depart from produced by being greater than for example any system of 1%.In addition,, if having more than one data point to get rid of by this step in one group of four value, all data for related component are just all removed so.

In group, the genetic expression of component detects

In order to detect in sample the amount of concrete RNA, we used method known to persons of ordinary skill in the art extract and quantitatively in sample the transcribe rna of the component of related gene expression group (detailed flow process is as follows.Also can consult the PCT application publication number WO98/24935 that is combined as the reference of RNA analytical procedure at this).In brief, RNA extracts in the samples such as substratum that may grow such as tissue, body fluid or experimenter's cell mass.For example, lysing cell and in being suitable for carrying out the solution of DNAse reaction eluted rna.Article one, the synthetic of chain can utilize reversed transcriptive enzyme to complete.Then can carry out gene amplification, the test of clearer and more definite quantitative PCR and for the size (Hirayama etc., Blood92,1998:46-52) such as marker alignment purpose genes such as 18S rRNA.At multiple double sample, for example, in 4 parts of repeat samples, detect sample.By the difference in the limit cycle between internal control and goal gene, determine the relative quantity of mRNA.In embodiments of the invention, with amplification, report reagent with carry out quantitative PCR purchased from the equipment of Applied Biosystems (Foster City, CA).Obtained definite object transcript amplification efficiency, can be directly relevant to the amount of specific courier's transcript in detected sample from the detectable point of the signal that is amplified To Template (as, cycle number).Similarly, such as fluorescence, enzyme work, per minute degradation rate, absorbancy etc. other quantitatively signal with known target template concentrations (for example, reference standard curve) relevant or while being normalized for standard due to limited mutability, can be used for the number of To Template in quantitative unknown sample.

Although be not limited to amplification method, quantitate gene expression technology can be utilized the amplification of target transcript.Alternatively or with the amplification of target transcript combine, can also utilize the amplification of report signal.The amplification of To Template can or complete by thermal cycling gene amplifications such as PCR by isothermal gene amplification strategy.

Wish to obtain between the target that is amplified or reporter gene and starting template concentration definable and dependency repeatably.We find by careful attention such as the ratio of constant primer-template and by experiment amplification efficiency be strict controlled in narrow can permission level in (for example relative efficiency of 99.0-100%, the normally relative efficiency of 99.8-100%) can achieve the above object.For example, when measuring the gene expression dose of relevant term single gene expression map, must make group all components primer template ratio (for example, in the scope of 10 times) and amplification efficiency (for example, being less than 1%) remain in the limited scope of phase Sihe to can carry out accurately and the relative determination of each component accurately.For this, describe and the object of following claim, if amplification efficiency differs and is no more than approximately 10%, we just think amplification efficiency " substantially similar ".Preferably they differ should be less than about 2%, more preferably less than about 1%.Within the scope of the whole to be detected concentration level relevant with corresponding biology situation, all should observe these constraint conditions.Although various embodiments herein therefore must meet detection substantially can repeat and wherein the specificity of all components complete this standard with under the substantially similar measuring condition of amplification efficiency, but, the test-results that does not directly meet these criterions by calibration reaches described measuring condition also still in the desired scope of the invention herein, compensating error in this way, thus after suitable calibration detected result, met described standard.

In practice, we test to guarantee that these conditions meet.For example, the many primer-probes pair of our common Design and manufactures, which right effect of measuring is best.Although use computer technology known in the art and general convention can improve design and the preparation of primer-probe, we still find that the confirmation of experiment is very useful.In addition,, in the process of experimental verification, we have linked together selected primer-probe combinations and series of characteristics.

Reverse primer should be complementary with DNA encoding chain.In one embodiment, intron-exon juncture should be crossed in primer location, three of reverse primer causes end and is no more than 3 bases and near-end exon complementation (if the base more than surpassing three is complementary, can trend towards competitive amplifying genom DNA).

In embodiments of the invention, primer probe answers amplification length to be less than the cDNA of 110 bases, and Ying Bucong relevant but on biologically irrelevant locus amplifying genom DNA or transcript or cDNA.

The suitable target of selected primer probe is article one chain of cDNA, and in one embodiment, it should be prepared as follows:

(a) utilize the in vitro assessment of whole blood to be subject to the biological condition of agents influence.

Venipuncture obtains human blood and sample is divided into baseline, the non-stimulated thing of at least three time points and has q.s stimulator for experiment.Common stimulator comprises lipopolysaccharides (LPS), phytoh(a)emagglutinin (PHA) and hot deactivation staphylococcus (HKS) or carrageenin and can use individually (conventionally) or combine use.By the whole blood aliquots containig of heparinization the non-stimulated thing in the situation that mixed be incorporated in containing the air of 5%CO2 in 37 ℃ of insulations 30 minutes.The stimulator that adds different concns, mixed lower 37 ℃ of the situation that pipe cap is ventilative that is incorporated in is placed 30 minutes.Now can add other test compound and place the different time, this time is depended on the pharmacokinetics of surveyed compound expection.In the time of appointment, centrifugal collecting cell, removes blood plasma and extracts RNA with various standard methods.

From cell mass to be tested or as purification of nucleic acid cell, tissue or the fluid of the clone of indicator, RNA and/or DNA.Preferably with various standard methods from nucleic acid mixture, obtain RNA (or RNA separation method RNA Isolation Strategies, 55-104 page, rNA methodology, separation with the lab guide RNA Methodologies characterizing, A laboratory guide for isolation and characterization, second edition, 1998, Robert E.Farrell, Jr., Ed., AcademicPress), what utilize at present is from Ambion (RNAqueous ^tM, without the total RNA separating kit of phenol Phenol-free Total RNA Isolation Kit, Catalog#1912, version 9908; Austin, Texas) to be filtered into basic RNA separation system.

According to a kind of method, the whole blood test of measuring for gene expression atlas is carried out as follows: people's whole blood is extracted in the 10mL Vacutainer pipe containing Vitrum AB sodium.Pipe gentleness is put upside down to mixed blood sample 4-5 time.This blood is used in 10-15 minute after splashing into.In experiment, blood is diluted 2 times, that is, every duplicate samples at each time point is, 0.6mL whole blood+0.6mL stimulator.Prepare the solution detecting and add suitable stimulator.

Then the whole blood of a certain amount of (0.6mL) is added in the polypropylene test tube of each 12x75mm.The 2X LPS (from e. coli serotype 0127:B8, Sigma#L3880 or serotype 055, Sigma#L4005,10ng/ml, changes with different batches) that adds 0.6mL in LPS pipe.Subsequently, in " contrast " pipe, add 0.6mL to detect liquid, each situation is all prepared to two parts of samples of repetition.Cap test tube is covered tightly.Put upside down test tube 2-3 time with biased sample.By pipe cap unclamp to the first lattice and by test tube at 37 ℃, 5%CO2 insulation 6 hours.In 6 hours, sample is mixed to the hemocyte that suspends gently, from each pipe, take 1mL (with thering is the micropipette rifle with the rifle head of baffle plate) away, and be transferred in " dolphin " Eppendorf tube (Costar#3213) of 2mL.

Then by sample centrifugal 5 minutes of 500xg room temperature (IEC whizzer or similar machine have the rotor that is applicable to various Eppendorf tubes), from each pipe, remove serum as much as possible and abandon it.Cell precipitation is placed on ice, with the fast as far as possible extraction RNA of Ambion RNAqueous test kit.

(b) amplification strategy

With information special primer or random primer amplifying specific RNA.The synthetic special primer of data according to available from public database (as, Unigene, national biotechnology information center, National Library of Medicine, Bethesda, MD), comprises the information available from genome and the cDNA library of people or other animal.Select the preferential amplification of primer from the special RNA of test group or indication group sample, consult, for example, rNA methodology, about lab guide RNA Methodologies separated and that characterize, A laboratory guide for isolation and characterizationthe 15th chapter, RT-PCR, second edition, 1998, Robert E.Farrell, Jr., Ed., Academic Press, or rNA separation and evaluation side method RNA isolation and characterization protocolsthe 22nd chapter 143-151 page of one book, molecular biology method Methods in molecular biology, volume 86, 1998, R.Rapley and D.L.Manning edit, Human Press, or the statistics of design of primers parameter improves the 14th chapter in Statistical refinement of primer design parameters mono-book, PCR application: for the 5th chapter 55-72 page of the method PCR applications:protocols for functional genomics of functional genomics, M.A.Innis, D.H.Gelfand and J.J.Sninsky edit, 1999, Academic Press).Amplification under isothermal condition, carry out or with thermal cycler (for example, available from Applied Biosystems, Foster City, the ABI 9600 of CA or 9700 or 7700; Consult nucleic acid detection method Nucleic acid detection methods 1-24 page, virus detects molecular method Molecular methods for virus detectionin one book, D.L.Wiedbrauk and D.H., Farkas edits, 1995, Academic Press) carry out.As described in for amplimer, with identify and synthetic from given data storehouse openly and with the detection primer of fluorescence labels detect the nucleic acid that is amplified (consult, for example, Taqman ^tMpCR reaction kit, method, version A, numbering 402823,1996, Applied Biosystems, Foster City CA.).In this situation, use available from the ABI Prism 7700Sequence Detection System detection of Applied Biosystems (Foster City, CA) and the DNA of quantitative amplification.That with sample to be tested, obtain or available from the amount of the special RNA of indicating clone can to relevant (the consulting of viewed fluorescence relative quantity, for example, quantitative PCR technique progress: 5 ' nuclease check Advances in quantitative PCR technology:5 ' nuclease assays, Y.S.Lie and C.J.Petropolus, physiotechnology general survey Current Opinion in Biotechnology, 1998, 9:43-48, or rapid thermal cycles and PCR kinetics Rapid thermal cyclin g and PCR kinetics, 211-229, PCR application: for the 14th chapter of the method PCR applications:protocols for functional genomics of functional genomics, M.A.Innis, D.H.Gelfand and J.J.Sninsky edit, 1999, Academic Press).

As the concrete implementation to method described herein, we have described synthetic article one cDNA chain in detail for the method for PCR.For whole blood RNA, from the RNA of culturing cell (that is, THP-1 cell), the two all can use the method with extraction.

Material

1.Applied Biosystems TAQMAN reverse transcription test kit (P/N 808-0234).Test kit forms: 10X TaqMan reverse transcription damping fluid, 25mM magnesium chloride, deoxyNTP mixture, random sexamer, RNase inhibitor, MultiScribe reversed transcriptive enzyme (50U/mL) (2) is without RNase/Dnase water (DEPC from Ambion (P/N 9915G) processes water or coordinator).

Method

1. RNase inhibitor and MultiScribe reversed transcriptive enzyme are placed on ice immediately.All other reagent then ice that can room temperature thaws is put.

2. from-80 ℃ of refrigerators taking-up RNA room temperature, melt, be then placed on ice immediately.

3. the following reversed transcriptive enzyme reagent mixture of preparation (for a plurality of samples, because the error of drawing need be prepared the mixture of additional quantity) for every 100mL reverse transcription reaction:

4. by each RNA sample of cumulative volume 20mL (for example, for THP-1RNA, take out 10mLRNA and use without RNase/Dnase water and be diluted to 20mL, for whole blood RNA, use the total RNA of 20mL) put into 1.5mL Eppendorf tube and add from step 5,2,3 reverse transcriptase reaction mixture 80mL.Upper and lower pressure-vaccum mixes.

5. by sample room temperature insulation 10 minutes.

6. sample is incubated to 1 hour at 37 ℃.

7. sample is incubated to 10 minutes at 90 ℃.

8. sample is turned fast in Eppendorf centrifuge to several circles.

9. if carry out immediately PCR, just sample is placed on ice, otherwise sample is placed in-20 ℃ standby.

10. when all reverse transcription samples being carried out to electrophoresis evaluation, all need with 18S and beta-actin calibration (consulting SOP 200-020).

As follows by the situation of the primer probe in detecting genetic expression group component with above-mentioned article one chain cDNA:

For setting up of the human gene expression group of 24 genes of inflammation.

Material

1. for the 20X primer/probe mixture of each goal gene.

2. for the 20X primer/probe mixture of the inherent object of reference of 18S.

The senior mixture of 3.2X Taqman universal PC R.

4. the cDNA coming from the rna transcription of cell from extraction.

5.Applied Biosystems 96-hole light reaction is dull and stereotyped.

6.Applied Biosystems optics cap or optical transparency film.

7.Applied Biosystem Prism 7700 sequential detection instrument.

Method

1. be prepared as follows primer/probe of containing for goal gene, for the inherent primer/probe of reference of 18S and each primer of the senior mixed solution of 2x PCR/probe mixture storage liquid.Enough excessive errors in order to drawing that solution need be prepared, for example, about 10% excessive.Following example has been described for certain gene and has been detected with quarter sample the system that two states (two flat boards) is prepared conventionally.

2. 95 μ l cDNA are diluted in and in 2000 μ l water, prepare cDNA target storage liquid.The amount of cDNA is adjusted to the Ct value that provides between 10-18, conventionally between 12-13.

3. draw the suitable hole that 15 μ l primer/probe mixture are put into Applied Biosystems 96 hole light reaction flat boards.

4. draw 10 μ l cDNA storage liquid put into Applied Biosystems 96 hole light reaction flat boards each hole.

5. clearly film phonograph seal is dull and stereotyped to use Applied Biosystems optics cap or printing opacity.

6. on AB Prism7700 sequential detection instrument, analyze dull and stereotyped.

Method herein also can be applicable to protein, wherein sensitive quantitative techniques such as enzyme-linked immunosorbent assay (ELISA) or mass spectrum is that this area can accomplish also well-known, for detection of the amount (consulting the WO98/24935 that is combined as reference herein) of protein component.

Baseline collection of illustrative plates data set

From single individuality, provide the spectrum data group library relevant to particular group or a series of groups with the sample analysis from a large amount of group of individuals.These spectrum data groups can be used as record and are stored in library to be used as baseline collection of illustrative plates data set.As term " baseline ", imply, the baseline collection of illustrative plates data set of storing is used as comparer to provide biological condition or reagent calibration collection of illustrative plates data set for information about.Baseline collection of illustrative plates data set can be stored in library and in the mode of cross reference classifies.Certain classification form can be depending on the feature of the group of derived data group.Another kind of classification form may be to classify by concrete biological condition.The concept of biology situation comprises the cell that can a time point in office finds or any state of cell mass.This state may reflect the regional distribution of sample, experimenter's sex or other any identification symbol.Some identification symbol may be crossover.Also in described library access and single experimenter or concrete clinical trial relevant record.The classification of baseline collection of illustrative plates data set can further be annotated with relevant concrete experimenter, medical conditions, the concrete medical informations such as reagent.

For setting up the selection of the baseline collection of illustrative plates data set that the spectrum data group of calibration carries out, be for example, with the object application (, monitoring drug development, quality control or other application) of to be assessed, monitoring or the biology situation of estimating and calibration group relevant.The baseline collection of illustrative plates data set of access can be different time points, be exposed to stimulator, medicine or complex compound and from first spectrum data group from same experimenter or from different experimenters, or can be from similar or dissimilar population of subjects or tested group.

Described spectrum data group can produce from same experimenter with first data set, and sample is wherein to obtain in that separate or similar time, difference or similar site or under difference or similar biological condition.For example, Fig. 5 provides the flow process of extraction sample before stimulation or after stimulating.Available from the spectrum data group of stimulated samples not, can be used as for the baseline collection of illustrative plates data set available from sample after stimulating.Baseline collection of illustrative plates data set also can be from the library of the spectrum data group of the tested group of containing some special characteristic of tool or biological condition or tested group.Baseline collection of illustrative plates data set also can to cultivate relevant in vitro or external feature corresponding to some and cell in vitro.Spectrum data group after the calibration producing after this can be with baseline collection of illustrative plates data set together with optional first spectrum data group or separate and be stored in (Fig. 6) in database or library as record, although first spectrum data group is incorporated in baseline collection of illustrative plates data set conventionally under suitable criteria for classification.Between gene expression atlas and given biological condition, significant consistence makes it be worth being stored as spectrum data, and it especially can be used for reaching makes frame of reference aims of standardization.Standardized frame of reference can be used for indicating experimenter to meet the degree of certain given biological condition (healthy or ill), or for clinical intervention provides target, or play this two kinds of effects simultaneously.

Selected baseline collection of illustrative plates data set also can be used as the standard of the conditions such as the efficiency, toxicity of judgement preparation batch.Wherein, in the situation that detecting therapeutical agent effect, baseline data group can be corresponding with the gene expression atlas of using before this reagent.When newly developing the quality control of product, baseline data group may be corresponding with the gold standard of this product.But, any suitable standardisation technique is all applicable.For example, average baseline collection of illustrative plates data set is available from the credible raw material of the herbal medicine class nutrient drug of spontaneous growth and compared different time and different batches is used the consistence between prepared compound batch or is lack of consistency to turn out to be.

The data of calibration

Under condition repeatably, we have completed the detection of genetic expression, i.e. the above-mentioned detection relevant with " gene amplification " to " genetic expression group ", and we infer that the difference occurring under condition is like this that difference by biological condition causes.We find that the spectrum data group of calibrating takes from the repeatability in same experimenter's sample with height under the same conditions thus.We find that the spectrum data group of calibrating is repeatably in the sample of duplicate detection equally.We also find wherein when experimenter's sample is exposed to compound in vitro, to obtain the repetition situation of spectrum data group of calibration and the alignment features data that ex vivo has been exposed to the sample in compound are comparable.Importantly, we also find, with the indicating clone of agent treated can provide in many cases with available from body or the spectrum data group of isolated cells group's the comparable calibration of data set.In addition, we find the sample from experimenter to be applied on indicator cells and can to provide about the abundant calibration collection of illustrative plates data set of the quantity of information of experimenter's biological condition, comprise experimenter's healthy state, morbid state, Results, aging degree or are exposed to the degree of environmental stimulus thing or toxin.

Calculating and the area of computer aided of the spectrum data group of calibrating

Spectrum data group available electron data sheet after calibration represents or uses graphical presentations such as histogram or tabulated form, also can three-dimensional space representation represent.About the function of baseline and spectrum data can mean the ratio into logarithm.Related components can be listed on x axle one by one, and logarithm standard can be on y axle.After calibration the member of data set can be expressed as for baseline, represent that genetic expression strengthens relatively on the occasion of or represent the negative value that genetic expression reduces relatively.

After calibration, each member of spectrum data group should be repeatably within the specific limits for the similar sample of experimenter under taking from simulated condition.The spectrum data group of for example, calibrating can repeat for the similar sample of experimenter under taking from simulated condition in the scope of an order of magnitude.More specifically, data can repeat in 50%, more specifically in 20%, can repeat, and conventionally in 10%, can repeat.According to embodiment of the present invention, in a plurality of gene locuss of surveying in genetic expression group, relative genetic expression increase, minimizing and the unconverted pattern of each can be used for the preparation spectrum data group of calibrate, it provide the biological effectiveness that related biological state, certain reagent treats condition information or for experimenter or sample colony or batch between relatively or for the comparison between cell mass.The pattern of this kind can be used for differentiating the possible candidate of drug test, can be used from diagnosis or the prognosis of related biological situation separately or with other clinical indication one, or can be used for instructing by preparation, detect and medicine that marketing is carried out or the exploitation of nutrient drug.

The digital material of expressing available from quantitate gene and standardized with respect to baseline collection of illustrative plates data set, from the digital material of genetic expression, can be stored in database or digital storage medium, or be used further to comprise the healthy state of managing patient or carry out clinical trial or identify in the various objects of medicine.Thereby these data can be transmitted and between remote geographical position, collect and converge (Fig. 8) by for example World Wide Web, e-mail or the Internet address or by hard copy on wired or wireless network.

In embodiments of the invention, one descriptive file is stored in independent database or a plurality of database, the data wherein stored comprises with the original gene expression data (first spectrum data group) before the conversion of baseline collection of illustrative plates data set and for generation of the baseline collection of illustrative plates data set record of the spectrum data group of calibrating, and for example comprises about whether baseline collection of illustrative plates data set is from the annotation of specific set of landmarks and other any annotation of conveniently explaining and using these data.

Because data have general format, so available computers is carried out data processing easily.Thereby get up to provide optional corresponding to the illustrated output of calibration data set by these data organizations.

For example, being at least unique samples a kind of in RNA or protein and can using P from experimenter _irepresent.From sample P _ifirst spectrum data group be expressed as M _j, M wherein _jto P _idistinct rna or the quantified measures of protein component.Recording Ri is that the ratio of M and P and other data of available relevant experimenter's situation are annotated, for example, the activity of age, diet situation, race, sex, geographical position, SOM, psychataxia, drug treating, health, body weight and environmental exposure factor etc.In addition, data processing further comprises that access is from containing data in the second status data storehouse of other medical data not having in the spectrum data group of current calibration.At this therebetween, data access can be undertaken by computer network.

Above-mentioned data storage on computers can provide user can obtain the information of form.Therefore, user can store these information into second position, comprises the described information of downloading.But, thus access can be confined to know password or other user who arranges safely can protect wherein contained medical records.Thereby the feature of this embodiment of the present invention is user can add new or being recorded to of annotating and make to be recorded as the part into biological information in data set.

By the mode of calibration collection of illustrative plates, be more specifically that the stdn that the mode by sign collection of illustrative plates is product provides a chance to the diagram of spectrum data group such as the relevant calibration of the products such as medicine.Described collection of illustrative plates can be used as the feature of the relative potency when comparing because of other medicines similar or that different application is got permission, difference of mechanism of action etc.

Various embodiments of the present invention also can be used as computer program for the use of computer system.Described product can comprise for deriving first spectrum data group and for generation of the program code of collection of illustrative plates of calibration.Such operation may comprise the series of computation machine instruction of being fixed on practical medium, such as computer-readable medium (for example, disk, CD-ROM, ROM or hard disk), or by adjusting detuner or other interface device, can be sent to the series of computation machine instruction of computer system, such as communications adapter connected to the network.Described network connects can be by for example optical cable or wire communication cable or for example, by certain combination of wireless technology (, microwave, infrared rays or other tranmission techniques) or these modes.This series of computer instructions preferably comprises all or part function of aforesaid relevant this system herein.Computer instruction described in those skilled in the art will be appreciated that can be write down for many computer architectures or operating system and use with many programming languages.In addition, such instruction can be stored in any memory storage, such as semi-conductor, magnetic, optics or other memory storage in, and available any communication technology propagates, such as optics, infrared rays, microwave or other tranmission techniques.Estimate that such computer program can (for example distribute by subsidiary removable media printed or electronic document page, the pre-packing software shortening), preloaded computer system (for example, system ROM or shaft collar), or divide the electronic bulletin board (for example, internet or World Wide Web) from server or network.In addition, computer system can be and derives first data set and calibration collection of illustrative plates data set and further provide included derivative group etc.

Described in WO01/25473, the algorithm of figure or the calibration collection of illustrative plates data set of tabulated form, relevant database and the index calculating or derivation and the information from extracting group, database, data set or index or algorithm are can be for together with various objects or separate sale.

index Establishment

Generally speaking, (i) cross over tested group or tested group or sample or cross over cell mass related biological situation gene expression atlas remarkable consistence and (ii) the specificity of group all components and amplification efficiency substantially use under similar measuring condition the genetic expression group that produces gene expression atlas is provided in component can repeated measures method, make likely to use certain index identified gene expression map therefore detection of biological situation.

With the numerical value in gene expression atlas can be set up to index to the target function of the single numerical value mapping that biological condition is relevant at hand.Numerical value in gene expression atlas is the amount with each component of the corresponding genetic expression group of gene expression atlas.These group components have formed spectrum data group, and target function has produced single numerical value-certain index from spectrum data group membership.

Target function can be built into every linear summation easily, and every is our so-called spectrum data group membership " Contribution Function ".For example, described Contribution Function can be spectrum data group membership's constant power.Therefore target function has following form:

Wherein I is index, M _ithe numerical value of spectrum data group membership i, C _ibe constant, and P (i) is M _ithe power improving, summation is the round values formation for all i, i can reach the number of members in data set.We have obtained a linear polynomial expression thus.

Numerical value C _iwith P (i) can measure in many ways, thereby make index I provide associated biomolecule to learn the information of situation.A kind of mode is that applied statistics learns a skill, and such as latent class model, makes spectrum data group and clinical data or experiment come source data or other Data relationship relevant with biology situation.In this contact, for example, can apply the Innovations from Statistical, Belmont, Massachusetts is called as Latent software.Consult the webpage that is combined as reference at this www.statisticalinnovations.com/lg/.

Or, can apply in a manner known in the art other simpler modeling technique.For example, the mode that inflammation (measuring according to the spectrum data group for the inflammation gene expression expression map) value larger with target function that can heavier degree is associated builds the target function of inflammation.Therefore, in a simple embodiment, each P (i) may be+1 or-1, this depends on that the heavy constituent that adds along with inflammation is to increase or reduce.As what below further discuss, we have built a significant inflammation index matching with expression

1/4{IL1A}+1/4{IL1B}+1/4{TNF}+1/4{INFG}-1/{IL10}，

Wherein component braces around points out it is the observed value of these components, and these components are subsets of table 1 inflammation gene expression expression group.

Can be as mentioned above for suitable stdn reference being provided and even can be used for setting up the spectrum data group of calibrating as baseline collection of illustrative plates data set, as mentioned above, based on standardized reference, the standardized value that the index of sign gene expression atlas also can have this target function is for setting up this index.This standardized value can be measured with relevant tested group or tested group or sample or relevant cell mass, thereby can think that this indicators and standards value is relevant.Relevant experimenter group or subject group or sample or relevant cell group can have at least one denominator in following characteristics: the range of age, sex, race, geographical position, nutrition history, medical conditions, clinical indication, drug treating, physical activity, body weight and environmental exposure factor.

For example, described index can be configured to, and the standardized gene expression atlas about for the tested group of health or tested group, makes reading be approximately 1 sign health volunteer's stdn gene expression atlas.We can further suppose, described index for biological condition be inflammation, the reading 1 in this embodiment is therefore corresponding to the gene expression atlas that meets health volunteer's standard.Those obvious higher readings may be differentiated the experimenter who is in inflammatory conditions.But, use 1 as definite standardized value, to be a possible selection, another is reasonably selected is to use 0 as definite standardized value.While doing this selection, index can standard deviation units represent 0 depart from, thus make-1 and+numerical value between 1 comprise 90% normal distribution with reference to tested group or tested group.Therefore we have found that gene expression atlas numerical value (and the index building based on them) trends towards normal distribution, and the index centered by 0 building is in this way the information that is highly rich in.Therefore facilitated the use of index in medical diagnosis on disease and therapeutic goal are set up.Select 0 as standardized value and use standard deviation units for example can consult, following Figure 17 B.

embodiment

embodiment 1: the acute inflammation index that helps to analyze large amount of complex data set.in one embodiment of the invention, desired value or algorithm can be used for complex data group to be reduced into the single desired value that experimenter's inflammatory conditions relevant information is provided.Described in Figure 1A and 1B.

Figure 1A is entitled as in a huge complex data group certain experimenter accurate inflammation collection of illustrative plates tracking results of originating.8 different dates that the figure illustrates during single male subject is suffered from optic neuritis are detected the result from 24 genes (as shown in table 1) of inflammation gene expression expression group.

Figure 1B has shown the application of acute inflammation index.The data of showing in above Figure 1A are shown in this figure after the target function calculating of using corresponding to following mathematic(al) representation: (1/4{IL1A}+1/4{IL1B}+1/4{TNF}+1/4{INFG}-1/{IL10}).

embodiment 2: acute inflammation index or algorithm are in monitoring sample or the experimenter's biology situation application.experimenter's inflammatory conditions has disclosed past process, future development, the information such as reaction to treatment of related biological situation.Acute inflammation index can be used for disclosing these information of relevant experimenter's biology situation.As shown in Figure 2.

The result (showing 24 genes of every a line in Figure 1A) that detects inflammation gene expression expression every day is shown as the single histogram after calculating.Index disclosed may the inflammatory conditions relevant to Results in trend (Fig. 2) clearly.

Fig. 2 is the diagram in the acute inflammation index from calculating in by 9 of the single patient blood of therapeutic treatment different significant clinical critical events because of optic neuritis.The change of the desired value of acute inflammation index is strongly relevant to the desired result of Results.Four clinical critical events have been marked the top of acute inflammation index in this figure, comprise that (1) is before using steroid therapy, (2) with 1 gram of IV Solu-Medrol, process every day, after (3) every day, then oral 60mg prednisone tapered to the treatment of 10mg every day and after (4) treatment.Data set is identical with Fig. 1's.Index and 1/4{IL1A}+1/4{IL1B}+1/4{TNF}+1/4{INFG}-1/{IL10} are proportional.Just as expected, acute inflammation index is with reducing rapidly after IV steroid therapy, during oral prednisone carries out processing that curative effect is lower, raise to some extent, and steroid no longer continue to add and completely metabolism after falling, got back to the level before treating.

Embodiment 3: utilize acute inflammation target setting dosage, comprise concentration and time, for the compound in exploitation or for compound to be tested in people and inhuman experimenter as shown in Figure 3.Acute inflammation index can be used as the common reference point with compound or Intervention Therapy without the treatment of acting in conjunction mechanism.Brought out by described index and shown the Gene response of this compound but the compound that do not improve known biology situation can compare from the different compounds in this biology situation for the treatment of with different effect.

Fig. 3 has shown the effect of using single dose 800mg ibuprofen in the single donor of identifying according to acute inflammation index.Single experimenter is at the time point picked-up 800mg of 0 time point and 48 hours OTC (over-the-counter) Ibuprofen BP/EP.The genetic expression value of measuring 5 inflammation genes involved sites of appointment in 2,4,6,48,50,56 and 96 hours as described below.Just as expected, inflammation index declines immediately and got back to baseline after 48 hour after picked-up non-steroidal anti-inflammatory Ibuprofen BP/EP.At 48 hours, take second dose in accordance with first dose of same dynamic law and within 96 hours, locate to get back to baseline at experiment terminal.

embodiment 4: utilize acute inflammation index to identify the effect of certain reagent, security and physiology work by pattern,it may be in developing and/or may be complicated at occurring in nature.As shown in Figure 4.

Fig. 4 shows that the acute inflammation index of illustrated calculating is for five kinds of different situations, comprise (A) untreated whole blood, (B) whole blood of processing with a kind of non-active carrier Compound D MSO in vitro, (C) whole blood stimulating without other of using in vitro dexamethasone (0.08ug/ml) to process, (D) use in vitro a kind of known proinflammatory compound lipopolysaccharides (LPS, 1ng/ml) whole blood stimulating and the whole blood of (E) using in vitro LPS (1ng/ml) and dexamethasone (0.08ug/ml) to process.Dexamethasone is to be conventionally medically used as the prescription drugs of anti-inflammatory steroid compound.From the gene expression dose of the inflammation genes involved of expressing of measuring, calculate acute inflammation index the people's whole blood available from single patient.For gene IL1A, IL1B, TNF, IFNG and IL10, the result of mrna expression is expressed as Ct ' s in this embodiment, but also can be expressed as, for example, relative fluorescence unit, copy number or other any can be quantitative, accurately with the form of calibrating.According to algebraic expression 1/4{IL1A}+1/4{IL1B}+1/4{TNF}+1/4{INFG}-1/{IL10}, by genetic expression value, determine proportional acute inflammation value.

embodiment 5: for colony's standardized value of gene expression atlas, derive and application.fig. 6 and 7 has shown available from two unique patient groups (patient's group) whole blood and for the arithmetical av of gene expression atlas (having adopted 48 sites of the inflammation gene expression expression group of table 1).These patient's groups are all normal or not yet diagnosed.Be confirmed as first patient's group of Bonfils (drawing point is represented by rhombus) by 17 compositions of the experimenters as blood donors of Bonfils Blood Center (Denver, crolla is many).Second patient's group has 9 blood donors, and its gene expression atlas is available from the assay of having carried out 4 times in surrounding.Experimenter's (drawing point is represented by square) in this second patient group be by transferee herein from Source Precision Medicine, in the employee of Inc., recruit.For each in 48 gene locuss of genetic expression inflammation group, calculate the average expression of each colony.As shown in Figure 6, and the result of site 25-48 (being below sometimes referred to as inflammation 48B site) as shown in Figure 7 for the result of site 1-24 (being below sometimes referred to as inflammation 48A site).

Between two different patient's groups, the consistence of gene expression dose is significant.The gene expression dose that two patients group all demonstrates in 48 sites each between two groups without significant difference.This observations shows that people's inflammation gene expression has the expression pattern of " normally ", the gene expression atlas that utilizes the inflammation gene expression expression group (or its subset) of table 1 and do has characterized described expression pattern, and colony-normal expression pattern can be used for, for example, instruct the medical intervention that causes any biology situation of normal expression patterns of change to be carried out.

Fig. 8 has shown in similar blood vessel equally the arithmetical av for gene expression atlas (48 sites of inflammation gene expression expression group in reusing table 1) available from two different patient groups (patient's group) whole blood.Therefore patient's group that its expression values is represented by triangle number strong point is 24 patients diagnoseds' (do not suffer from known inflammatory diseases) not normally.Another patient's group that its expression values is represented by diamond data points is 4 and suffers from rheumatoid arthritis and treat failed patient (therefore suffering from unsettled rheumatoid arthritis).

Significant as the consistence of data between the different normal patient group of two shown in Fig. 6 and 7 is the systemic difference of data between the normal and ill patient's group shown in Fig. 8.Shown in 45 of 48 inflammation gene expression sites, the inflammation gene expression of unstable patient with rheumatoid arthritis is expressed mean level (ML), and (lower cycle threshold, Ct) higher than not ill experimenter.The data that obtain therefrom have further confirmed likely to utilize genetic expression to confirm whether the group with particular biological situation has carefully designed and controlled precision and the scale that need detect according to requirement herein.

Fig. 9 is to have shown equally the gene expression atlas arithmetical av of organizing 24 sites in the table 1 inflammation gene expression expression group of whole blood available from two different patients from the similar form of Fig. 8.Its expression values with patient's group of diamond data points representative be 17 as blood donors normally, do not make a definite diagnosis experimenter's (therefore not suffering from known inflammatory diseases).Its expression values is same normal and not yet diagnosed 16 patients by another patient's group of square shape data point representative, they monitored 6 months, and box-shaped data point representative for the mean number of these expression values.Therefrom the genetic expression value mean number of first health population and second the health population genetic expression value mean number of the longitudinal axis of transverse axis conform to very much, the expression values that detects difference on the corresponding basis of gene-gene be approximately 7% or still less.

Figure 10 has shown the genetic expression numerical value (having utilized 14 sites of table 1 inflammation gene expression expression group) (data that shown 10 experimenters) of normally not making a definite diagnosis blood donor's whole blood available from 44.In addition, in colony's (group), each member's genetic expression value conforms to the value of whole group very much, and the peak height that shows as each gene locus on figure is consistent.The result that other experimenter of this group and other gene locus outside site shown here present is with shown here consistent.

The inference of carrying out according to these principles and in many embodiments of the present invention, can be used for the biology situation of comparative assessment individual subjects for colony's standardized value of gene expression atlas, comprise healthy and/or ill.In one embodiment, standardized value for gene expression atlas can be used as baseline calculating for experimenter " calibration collection of illustrative plates data set " (starting defined according to this part), to disclose the deviation between described experimenter's genetic expression and colony's standard value.Colony's standardized value for gene expression atlas also can be used as baseline value when structure meets the target function of embodiment of the present invention.Therefore, for example, the target function of structure conventionally can not only disclose individual inflammation and express degree, but also relevant with standardized value.

embodiment 6: the time-independent consistence of genetic expression group component expression values can be used as raw thing is learned the reliable indication of situation.figure 11 has shown single experimenter each expression level in 84 genes that monthly detect the middle of the month (in table 1 in inflammation gene expression expression group).Visible expression level is significantly consistent within whole period.

Figure 12 is similar with 13 has shown respectively in different single experimenters (selecting based on feeling good and not taking medicine in each case) each expression level of 48 genes (in table 1 inflammation gene expression expression group), in Figure 12, be to detect weekly in surrounding, be monthly to detect in 6 months in Figure 13.In each situation, expression level is all significantly consistent equally and is similar between individuality within whole period.

Figure 14 has also shown when the inflammation gene expression expression group with table 1 detects the impact that proinflammatory gene in single people experimenter is expressed of using of within for some time anti-inflammatory steroid.24 sites in 48 sites have been shown in this case.From experimenter, extract benchmark blood sample to PAX RNA separator tube, then experimenter is used to 60mg single dose prednisone, a kind of anti-inflammatory prescription steroid.Within 2 hours and 24 hours after this independent drug oral once, extract other blood sample.The gene expression results of showing all three time points, wherein the numeric representation of authentic specimen is the unit of x axle.As desired, shown according to the histogram of using latter 2 hours, oral prednisone has caused most of inflammation genes involved site to express decline.But, use the histogram demonstration of latter 24 hours, most gene site gene expression dose in the time of 24 hours that genetic expression reduces in the time of 2 hours increases.

Although the baseline in Figure 14 is that to take genetic expression value before the dispenser relevant to single tested individuality be basis, but we are known from previous embodiment, healthy individual trends towards utilizing the colony's standardized value in the gene expression atlas that table 1 inflammation gene expression expression group (or its subset) does.From Figure 14, we infer in the trial of shown level in proinflammatory gene expression level is returned to Fig. 6 and 7 (normal or set level); disturbing normal expression to cause the compensatory genetic expression reaction of over-compensation for drug-induced reaction, may be because prednisone has significantly been metabolized to the form of non-activity or has cleared out from experimenter.

Figure 15 with the similar mode of Figure 14, by the whole blood sample available from people experimenter, shown and within for some time, used the impact that single dose prednisone is expressed 5 kinds of genes (from the inflammation gene expression expression group of table 1).When using prednisone (t=0) and use after 2 hours and 24 hours sampling.Every part of whole blood sample is by adding 0.1ng/ml lipopolysaccharides (Gram-negative intracellular toxin) and stimulates and measure the gene expression atlas of this sample after stimulation.Visible when using for the gene expression dose of (t=0), the sample of 2 hours demonstrates in inflammation gene expression expression group 5 locus genes and expresses significantly and reduce.Use latter 24 hours, the restraining effect of prednisone is no longer obvious, level when in fact 3 gene expression doses in 5 sites are higher than t=0, and this has quantitatively illustrated well-known bounce-back effect on molecular level.

Figure 16 has also shown and has used within a certain period of time the impact that TNF--Inhibitor is expressed proinflammatory gene in single patient with rheumatoid arthritis, but expression described herein and previous mensuration (with Fig. 6 and 7 relevant) normal (that is, do not make a definite diagnosis, healthy) patient organizes, the mean value in similar site compares to illustrate.As a part that relates to the relatively large international research of rheumatoid arthritis, the tracked monitoring of experimenter 12 weeks.It is because Planning Change therapy is also treated with TNF--inhibition compound immediately to the conservative pharmacotherapy of rheumatoid arthritis is reactionless that experimenter enters this research.Before starting, gets new therapy blood (access 1).After beginning new therapy, after latter 4 weeks of therapy change (access 2), new therapy start, 8 weeks (accessing 3) and 12 weeks (accessing 4) gets blood.Collect blood in PAX RNA separator tube, room temperature is placed 2 hours and is frozen in-30 ℃.

Centralab by frozen sample shipping to Source Precision Medicine, supplies at Boulder, and this paper transferee of Colorado measures the expression level of 48 genes in table 1 inflammation gene expression expression group.The step that blood sample Bing An manufacturers recommends of thawing is extracted RNA.RNA is transformed into cDNA and measures the expression level of 48 proinflammatory genes.Figure 16 has shown in 48 genes the expression of results of 11.When the expression of results from access 11 sites of 1 is compared with colony's mean value of U.S. normal donors, experimenter demonstrates sizable difference.Same, by after in doctor's access several times each time each locus gene expression level of gained compare with same normal mean value.From access 2,3 and 4 digital proof change the effect of therapy.In access each time after therapy changes, normal (that is, do not make a definite diagnosis, the healthy) patient who has the proinflammatory gene expression level in 10 sites to approach in 11 sites previously to have measured organizes the mean value in similar site.

Figure 17 A further illustrated one group 44 normally, do not make a definite diagnosis blood donor in (the inflammation gene expression expression group of the table 1) proinflammatory gene described herein in relevant 7 sites express consistence.For each independent site, the numerical value of demonstration is within the scope of the standard deviation of average expression values ± 2, and this is equivalent to 95% normally distributed population.Although the amplitude of fiducial interval large (95%), attractive be detected genetic expression value (Δ CT) still drop on mean value 10% in, regardless of relevant expression level.As what be below described in further detail, for given biology situation, can set index to detect described symptom.This may be the result of two kinds of situations associating: (i) between biology situation and colony, having gene expression atlas consistence and (ii) having the method that produces the genetic expression group component observed value that substantially repeatably can derive gene expression atlas, described detection is that specificity and the amplification efficiency of group all components carries out and therefore obtain the observed value of biology situation substantially under similar measuring condition.Therefore, in Figure 17 A, the function of representative component site expression values is used herein to derives inflammation index value, and it is by stdn, thereby makes reading 1 corresponding to health volunteer's component expression values, as shown in the right hand portion in Figure 17 A.

In Figure 17 B, measured the inflammation index value of not making a definite diagnosis each member in blood donor for one group 42, and the desired value shown in figure distributes and to be approximately similar to as seen normal distribution, although the size of subject group is relatively little.Desired value shows as with respect to take 0 as basic intermediate value, the deviation with standard deviation units calibration from intermediate value.Therefore 90% subject group drops in 0 value+1 and-1 scope.We have also set up the various indexs of showing analogue.

The application of the index that Figure 17 C is identical with Figure 17 B, is wherein made as 0 by normal population experimenter's inflammation intermediate value, and normal subjects and ill experimenter are all with the standard deviation units mapping with respect to this intermediate value.Measure normally, do not make a definite diagnosis the inflammation index value (black post) of each member in 70 individualities of colony.The desired value of the generation shown in visible Figure 17 C distributes and roughly approaches normal distribution.Same, from two ill batch totals, calculate desired value, (1) patient with rheumatoid arthritis for the treatment of and will treat with more effective medicine (as tnf inhibitor) with methotrexate (MTX), (2) use the patient with rheumatoid arthritis of rheumatism disease drug (DMARDS) treatment that improves symptom except MTX, they also will change into more effective medicine treat (as, MTX).Two groups of patients all present and compare the desired value (showing that inflammation increases the weight of) being tilted to normal distribution.Therefore this Figure illustrates to utilize and derive from the index evaluation disease condition of gene expression atlas data and objective and a therapeutic goal that can be quantitative is provided.These two groups of experimenters are being carried out after appropriate treatment, and the desired value of two groups is all returned to distribute more normally (data are not shown in this).

Figure 18 has drawn two groups of 6 different experimenters' patient with rheumatoid arthritis for the gene expression atlas in 7 sites identical in Figure 17 A in the mode similar from Figure 17 A.Yi Ge colony (being marked by the drawings " stable ") is the patient good to therapeutic response, and another group's (being marked with in the drawings " unsettled ") is bad to therapeutic response and the patient of the positive Planning Change of its therapy.Visible stable patient group's expression values drops in the scope of 95% fiducial interval, and for unstable patient group, in 7 sites, have the expression values of 5 to drop on outside this scope and on.The right hand portion of this figure shown, compares with normal not patient diagnosed group's numerical value 1, and the average inflammation index of unstable group is 9.3, and the average inflammation index of stablizing colony is 1.8.This index provides the examination criteria of a potential inflammation severity thus, and described inflammation is rheumatoid arthritis in this case.Therefore this index is except the observed value as biology situation, also can be used for detecting the effect for the treatment of and provides target for Results.

Figure 19 so for example clear inflammation index are being assessed the application in the single patient with rheumatoid arthritis of traditional methotrexate therapy poor response.This experimenter's inflammation index appears at ultra-Right side when starting new therapy (tnf inhibitor), within 2 weeks afterwards, 6 weeks and 12 weeks, continues to move to left.Visible this index is tending towards normal value, and the patient who observes with doctor is consistent to the response situation of new therapy.

Figure 20 has described equally when new therapy (also using tnf inhibitor) starts and with inflammation index, has assessed 3 patient with rheumatoid arthritis to traditional methotrexate therapy poor response in 2 weeks afterwards and 6 weeks.Index in visible various situation is tending towards normally conventionally again, and this patient who observes with doctor is consistent to the response situation of new therapy.

Figure 21-23 show respectively the inflammation index of the international group of Liao Yi patient with rheumatoid arthritis, and wherein everyone is treated doctor and is accredited as stable (that is, inexpectancy need change therapy).Figure 21 has shown each the index with 10 patients in the group of methotrexate for treatment, and known methotrexate can mitigation symptoms but do not touch the root of disease.Figure 22 has shown the index separately with 10 patients in Enbrel (a kind of tnf inhibitor) treatment group, and Figure 23 has shown the index separately with 10 patients in Remicade (another kind of tnf inhibitor) treatment group.In visible Figure 21, each patient's inflammation index increases with normal phase ratio, and in Figure 22, with the patient of Enbrel treatment, as a class, has with normal value and connect much closer inflammation index (80% in normal range).In Figure 23, although the patient's of the visible useful Remicade treatment of institute except a patient inflammation index is all in normal or lower than normally, wherein two patients have low inflammation index extremely, shown the immunosuppression of this medicine is replied.(in fact, studies show that Remicade is relevant with serious infection in some experimenter, immunosuppressive action is herein by quantitative.) equally in Figure 23, an experimenter has the inflammation index in the normal range of being significantly higher than.In fact, the anti-inflammatory steroid (prednisone) that this experimenter takes reduces gradually, and after inflammation index is measured, in about one week, experimenter once clinical symptom significantly strengthens.

Strikingly, these embodiment have shown that the blood testing by chemically examining from experimenter obtains the observed value relevant to experimenter's rheumatism.If this detection is relevant to inflammation degree, can expect that other take inflammation as basic symptom, comprise, for example, cardiovascular disorder, can monitor in a similar manner.

Figure 24 has described and has utilized inflammation index assessment with the single patients with inflammatory bowel of three doses of Remicade initial treatments.The figure illustrates and face first dose of inflammation index before using, and first dose used latter 24 hours, this index is returned in normal range.This index just improved before using second dose, but before using the 3rd dose in normal range.In addition, except the measurement standard of biology situation is provided, this index is used herein to the effect that detects therapeutical agent (Remicade), and provides target for the Results of dosage and two kinds of forms of progress.

Figure 25 shown with other NSAID (non-steroidal anti-inflammatory drug) (NSAIDs) and compared, the gene expression atlas of 24 sites in the whole blood of processing with Ibuprofen BP/EP in vitro (inflammation gene expression expression group in table 1).For the collection of illustrative plates of Ibuprofen BP/EP above.The visible all NSAID that comprise Ibuprofen BP/EP have a similar collection of illustrative plates substantially, and wherein the gene expression pattern between site is similar.Although have these similarities, each medicine has the characteristic of self uniqueness.

That Figure 26 has described is how objective, quantitative, accurately and the repeatably relatively effect of two kinds of competitive antiphlogistons.In this embodiment, for each medicine in vitro various dose (0.08-250 μ g/ml) in whole blood detected in one group of two gene (table 1 is in inflammation gene expression expression group) each expression.The leading medicine of this listing demonstrates between dosage and proinflammatory gene are replied has complicated relation.Self-contradictory, in the case of the leading medicine going on the market, when dosage increases, the genetic expression in two sites starts all to decline and then raises again.For other compound, reaction result is more consistent, thereby when dosage increases, all more consistent decline of the genetic expression in two sites.

Figure 27 to 41 has described the application of genetic expression group in the early stage evaluation of transmissible disease and monitoring.The expression product that these figure have drawn the gene that indicates in whole blood to using the reaction of different infectious agents or infectious agent related products.In each figure, according to term defined herein, compare " calibration " gene expression dose to using the baseline expression level that applies relevant infectious agent whole blood assay before.In this, described figure is for example, with a plurality of figure of our patent application WO01/25473 mentioned below (, Figure 15) wherein similar in itself.After adding infectious agent or other stimulator, the change in concentration of corresponding time point monitoring shows with the form of measuring in proportion, and the baseline values 1 for concrete gene locus is corresponding to the described site expression level identical with expression level before interpolation stimulator.Change in concentration scale is in logarithmic coordinates.Rod below unified line represents density loss, and the rod more than unified line represents that concentration increases, and each excellent order of magnitude represents to change the order of magnitude of ratio.We prove, under suitable condition, can represent the gene expression atlas being exposed in vivo in corresponding stimulator in vitro by whole blood being exposed to the gene expression atlas obtaining in stimulator in WO01/25473 and other experiment.

Figure 27 has utilized one for distinguishing the new bacteria gene expression group of 24 genes that in host living beings system, various bacterium situations are researched and developed.Two different stimulator have been applied: fat teichoic acid (LTA), a kind of gram-positive cell wall fraction; And lipopolysaccharides (LPS), a kind of gram-negative cells wall fraction.The final concentration that stimulator is surveyed after using is 100ng/mL, 2 hours and the expression variation ratio of monitoring in 6 hours for level before using after each stimulator is used.In early by after using 2 hours, just can observe different expression as seen, for example, in IFNA2 site and other can distinguish other other site of Low Response between Gram-positive and gram negative bacterium.

Figure 28 has shown that IFNG place, single site is for the difference expression of three kinds of different sourcess (streptococcus pyogenes, subtilis and golden suis) LTA.Each stimulator application concentration is 100ng/mL, after using, after 1,2,4,6 and 24 hour, monitors response situation.Result shows that gene expression atlas can be used for the infectious agent of distinguishing different, in this case different types of gram-positive microorganism.

Figure 29 and 30 has shown that respectively inflammation 48A and the 48B locus (relevant to above-mentioned Fig. 6 and 7 respectively) in whole blood (in prescribed concentration, is respectively 10 after just having used to using golden suis stimulator and intestinal bacteria stimulator ⁷with 10 ⁶cFU/mL) reaction, with use before the baseline monitoring of comparing after using 2 hours.These figure demonstrate in latter two hours of infection has many reactions that have to coli-infection in described site.

Figure 31 with 32 respectively corresponding to Figure 29 and 30 and similar to them, so the monitoring that difference is carrying out after using for 6 hours.More site is for the reaction that has of infecting.Such as a plurality of sites such as IL2, demonstrate diacritic expression level between two kinds of infectious agents.

Figure 33 has shown that (concentration after just having used is also 10 for applying intestinal bacteria stimulator in inflammation 48A site ⁶cFU/mL) and apply containing intestinal bacteria product but without the reaction of the intestinal bacteria filtered liquid of coli somatic.Reaction is monitoring in 2,6 and 24 hours after applying.Visible, for example, site IL1B, IL18 and CSF3T are different to the reaction of intestinal bacteria and ECF in time.

Figure 34 is similar to Figure 33, but is to the stimulator of the ECF from independent and carrys out oneself to add the stimulator reaction of ECF of the microbiotic-PXB of a kind of combination lipopolysaccharides (LPS) in the reaction that this compared.For example, the demonstration of the reaction detection of IL1B, the existence of PXB does not affect the reaction of this site to ECF, thereby shows that LPS causes the factor of IL1B to ECF reaction.

Figure 35 has described in whole blood inflammation 48A site temporal evolution, and to golden suis stimulator, (concentration after just having applied is 10 ⁷cFU/mL) reaction, monitoring in 2,6 and 24 hours after applying.Visible time dependent reaction can comprise the trend of expression and the order of magnitude wherein changing.(consult, for example, IL5 and IL18.)

Figure 36 and 37 has shown respectively that in 6 hours the inflammation 48A of monitoring and 48B site are to (concentration after just having applied is 10 from intestinal bacteria ⁶with 10 ²cFU/mL) stimulation and (concentration after just having applied is 10 from golden suis ⁷with 10 ²the reaction of stimulation CFU/mL).Visible, in other cases, in a plurality of sites, such as B7 (Figure 36), TACI, PLA2G7 and C1QA (Figure 37), it is much remarkable that the reaction that intestinal bacteria produce produces than golden suis.The proof that these data are strong gene expression atlas can be used for the existence of highly sensitive evaluation Gram-negative bacteria and come with gram-positive microorganism difference.

Figure 38 and 39 has shown that respectively (concentration separately after just having applied is 1O to the golden suis of high density and intestinal bacteria separately for the inflammation 48B that monitors for 2,6 and 24 hours after using and 48A site ⁷with 10 ⁶cFU/mL) reaction stimulating.The reaction of passing in time in many site is included in the variation in quantity and direction.Figure 40 is similar to Figure 39, but demonstration is the reaction in inflammation 48B site.

Figure 41 has shown that (concentration separately after just having applied is 10 to the golden suis of high density and intestinal bacteria respectively for 24 hours monitor after using inflammation 48A site equally ⁷with 10 ⁶cFU/mL) reaction stimulating.The same with the situation of Figure 20 and 21, the reaction in some sites such as GRO1 and GRO2 is had any different between two types of infection.

Figure 42 has described statistics T-check has been applied to differentiate effective member that can distinguish the characterizing gene expression group between normal subjects and unstable patient with rheumatoid arthritis.Turn grey box and be illustrated in the gene of distinguishing between two groups of experimenters individually very effectively (the P value of t check marks in the box on each figure right side), thereby pointed out the effective member for the characterizing gene expression group of rheumatoid arthritis.

Figure 43 has described the expression level of one group of 17 gene for 8 doubtful microbemia patients.These data have reference value for the microbemia patient who finds the genetic expression of tool feature mode.

Figure 44 has described statistics T-check has been applied to differentiate effective member that can distinguish the characterizing gene expression group between normal subjects and microbemia patient.Turn grey box and be illustrated in the gene of distinguishing between two groups of experimenters individually very effectively (the P value of t check marks in the box on each figure right side), thereby pointed out the effective member for the characterizing gene expression group of microbemia.

Figure 45 has described the application of an algorithm (as shown in FIG.), has obtained the index of correlation of the rheumatoid arthritis (RA) when being applied to respectively normal subjects, patient with rheumatoid arthritis and microbemia patient.This index can be distinguished out from normal subjects and microbemia patient by RA patient easily.

Figure 46 has described the application of an algorithm (as shown in FIG.), has obtained the microbemia index of correlation when being applied to respectively normal subjects, patient with rheumatoid arthritis and microbemia patient.This index can be distinguished out from normal subjects and patient with rheumatoid arthritis by microbemia patient easily.

These Data supports our conclusion, that is, precision as described herein and the enough gene expression atlas (1) of calibration can detect has the individual subset that known organism is learned situation; (2) can be used for monitoring the reaction of patient to treatment; (3) can be used for effect and the security of assessment treatment; And (4) can make one or more relevant gene expression atlas more approach one group of target value to instruct the treatment for patient by adjusting therapy, described target value may be standardized numerical value or other expection or accessible numerical value.Even if we have proved that gene expression atlas also can provide significant information from the blood of ex vivo treatment or other tissue.We have also proved that the gene expression atlas from periphery whole blood provides the symptom information of wide region, no matter are direct or conventionally relevant to blood.

In addition, in embodiments of the invention, gene expression atlas also can be used in the sign and early stage evaluation (comprising the front state of symptom) of the transmissible diseases such as septicemia.Described sign comprises individuality, bacterium and virus infection, the pathogenic agent of specific hypotype, the stage of the natural history of infection (as early stage or late period) and the prognosis of the infected and uninfection of difference.Utilize above-mentioned algorithm and statistical method to complete described evaluation and distinguished in this way also in the scope in this paper multiple embodiments.

Claims (15)

1. in one group of component of quantitative assay, the reagent of the amount of distinct rna component characterizes the application in bacteremic diagnostic reagent in preparation between normal subjects and microbemia patient, described sign is that take available from first sample of experimenter is basis, this offering sample the source of RNA, wherein several members' spectrum data group can be used for assessment, each member is the quantitative assay value of the amount of distinct rna component in described one group of selected component, by the measurement of described component, can characterize the doubtful symptom of systemic infection, described group comprises one or more following component: MMP9 that are selected from, HSPA1A, IFI16, IL1R1, SERPINA1, ICAM1, TNFSF13B, IFNA2 and their any combination, wherein said each component observed value is repeatably obtaining under measuring condition substantially.

2. according to the application of claim 1, wherein said assessment further comprises:

Comparative map data set and for the baseline collection of illustrative plates data set of this group, wherein said baseline collection of illustrative plates data set is relevant with transmissible disease to be characterized or transmissible disease related inflammation.

3. in one group of component of quantitative assay, the reagent of the amount of distinct rna component is assessed the application in bacteremic diagnostic reagent in preparation between normal subjects and microbemia patient, described assessment is first sample based on available from experimenter, described offering sample RNA source, wherein can from first sample, derive first spectrum data group, this spectrum data group comprises a plurality of members, each member is the quantitative assay value of the amount of distinct rna component in described one group of selected component, measurement by described component can be assessed microbemia, described group comprises one or more following component: MMP9 that are selected from, HSPA1A, IFI16, IL1R1, SERPINA1, ICAM1, TNFSF13B, IFNA2 and their any combination, wherein said each component observed value is repeatably obtaining under measuring condition substantially, and

Can produce the spectrum data group for the calibration of this group, wherein each member of the spectrum data group of this calibration is the corresponding member of first spectrum data group and the corresponding member's of baseline collection of illustrative plates data set of this group function, wherein said baseline collection of illustrative plates data set is relevant with microbemia

The spectrum data group of calibrating is used between first spectrum data group and baseline collection of illustrative plates data set and compares, thereby the bacteremic assessment to experimenter is provided.

4. in one group of component of quantitative measurment, the reagent of the amount of distinct rna component characterizes the application in bacteremic diagnostic reagent in preparation between normal subjects and microbemia patient, described sign is first sample based on available from experimenter, described the first offering sample RNA source, wherein

Can obtain first spectrum data group from first sample, this spectrum data group comprises a plurality of members, and each member is the quantified measures of the amount of distinct rna component in described one group of selected component, the observed value of this component can become bacteremic indication, and described group comprises one or more following component: MMP9, HSPA1A of being selected from, IFI16, IL1R1, SERPINA1, ICAM1, TNFSF13B, IFNA2 and their any combination, and when obtaining spectrum data group

Substantially repeatably under measuring condition, obtaining the described observed value for each component, and

Come at least one observed value of spectrum data group since then to can be used for target function, the mapping of at least one observed value that this target function provides spectrum data group to the doubtful symptom of systemic infection observed value, thus produce relevant experimenter's bacteremic index.

5. according to the application of claim 4, wherein said target function has 2 kinds of compositions that comprise morbid state, being in a bad way property or disease process.

6. according to the application of claim 4, wherein said target function has 3 kinds of compositions that comprise morbid state, being in a bad way property or disease process.

7. according to the application of claim 4, wherein said target function has 4 kinds of compositions that comprise morbid state, being in a bad way property or disease process.

8. according to the application of claim 4, wherein said target function has 5 kinds of compositions that comprise morbid state, being in a bad way property or disease process.

9. in one group of component of quantitative measurment, the reagent of the amount of distinct rna component characterizes the application in bacteremic diagnostic reagent in preparation between normal subjects and microbemia patient, described sign is first sample based on available from experimenter, first described offering sample RNA source, wherein

Can obtain first spectrum data group from first sample, this first spectrum data group comprises a plurality of members, each member is the quantified measures of the amount of distinct rna component in described one group of selected component, from the observed value of this component, can assess microbemia, described group comprises one or more following component: MMP9 that are selected from, HSPA1A, IFI16, IL1R1, SERPINA1, ICAM1, TNFSF13B, IFNA2 and their any combination, wherein the described observed value of each component is repeatably obtaining under measuring condition substantially, and

Can produce the spectrum data group for the calibration of this group, wherein each member of the spectrum data group of this calibration is the corresponding member of first spectrum data group and the corresponding member's of baseline collection of illustrative plates data set of this group function, wherein said each member of baseline collection of illustrative plates data set is the stdn observed value of the amount of one of component in the group of measuring for relevant subject group, and described baseline collection of illustrative plates data set is relevant with microbemia

The spectrum data group of calibrating is to compare between first spectrum data group and baseline collection of illustrative plates data set, thereby the bacteremic assessment to experimenter is provided.

10. according to any one application in claim 1,3 or 9, wherein said quantified measures determines by amplification, and measuring condition is the amplification efficiency of all components to be differed be less than about 2%.

11. according to any one application in claim 1,3 or 9, and wherein said quantified measures determines by amplification, and measuring condition is the amplification efficiency of all components to be differed be less than about 1%.

12. according to any one application in claim 1,3 or 9, and wherein repeatably measuring condition is to be better than the degree of 5 percentage points with interior in repeatability substantially.

13. according to any one application in claim 1,3 or 9, and wherein repeatably measuring condition is to be better than the degree of 3 percentage points with interior in repeatability substantially.

14. according to any one application in claim 1,3 or 9, wherein

Spectrum data group can be stored in digital storage media.

15. according to the application of claim 14, and wherein spectrum data group can be stored in database as record.

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