Patent Application
20160252459
This invention relates to spectroscopic apparatus and methods for determining components present in a sample. It is particularly useful in Raman spectroscopy, though it can also be used in other forms of spectroscopy, e.g. using narrow-line photoluminescence, fluorescence, cathode-luminescence, UV visible (UV Vis), nuclear magnetic resonance (NMR), mid infra-red (mid-IR) or near infra-red (NIR). The present has particular application to a multiplex assay and apparatus for carrying out the multiplex assay.
Multiplexing within a laboratory environment is defined as the detection of multiple analytes simultaneously in a single interrogation. In general, the advantages are that many more data points are generated and a higher degree of information can be gathered. This can lead to a large time reduction which can be attained by searching for multiple targets in a single test rather than carrying out multiple, serial assays. High throughput multiplex assays mean that large scale testing is easily facilitated. In addition, the physical volume of sample required for a full panel of tests is significantly reduced—this can be important in cases where the sample can be difficult to obtain and/or is only available in small volumes. A knock-on effect from this is that the amount of lab consumables and human resource is also significantly reduced, meaning a reduction in running costs and waste.
Many techniques can be used to detect multiple analytes. One of the most common techniques is fluorescence, where the analyte of interest is fluorescent or the analyte of interest is tagged with a fluorescent dye. There are several drawbacks to using fluorescence as a detection technique. The main problem is the broad nature of fluorescence emission spectra which lack uniquely identifiable features. This limits the number of analytes which can be simultaneously detected in a mixture due to the large spectral emission overlap that occurs between fluorophores.
Another molecular technique that gives molecularly characteristic spectra is Raman spectroscopy. Rather than the broad peaks observed with fluorescence, Raman produces narrow, well defined peaks which are molecularly specific and can be used to identify a molecule in situ in a mixture. These spectra are information rich and this specificity allows the opportunity for higher order multiplexing, however Raman scattering when used in its basic form lacks the sensitivity required for many real world multiplexed applications.
There are methods that can be used to enhance the signal. Surface enhanced Raman scattering (SERS), makes use of the plasmonic properties of metals to achieve enhancements in the Raman signal of up to 105-106. This allows multiplexing to occur at detection levels which may be of use in certain fields where direct identification of an analyte is required.
Surface enhanced resonance Raman scattering (SERRS) is another method of enhancing Raman signals, and uses the principles of SERS together with an analyte that contains a chromophore, where the chromophore has an electronic transition in the region of the laser wavelength being used to excite the sample. SERRS detection levels can surpass that seen for fluorescence (Faulds et al. Analyst, 129, 567-568 (2004)) by up to three orders of magnitude and combined with the selectivity of the technique make this an excellent technique for high order multiplexing. Each chromophore has a SERRS spectrum which is unique allowing it to be identified in situ.
Faulds et al. (Angew. Chem. Int. Ed., 46(11), 1829-1831 (2007)) have carried out a 5-plex of labelled oligonucleotide sequences where they identified 5 different dye labelled oligonucleotides in a mixture by careful choice of the dye and by using two excitation wavelengths. The sequences used corresponded to a range of different targets. FAM, Cy 5.5 and BODIPY TR-X were used to label a universal reverse primer, Rhodamine 6G (R6G) was used to label a probe for HPV, and ROX to label a probe to the VT2 gene of E. coli 157. The dyes were chosen since they generate distinctive, specific SERRS spectrum. However, since the dye labels have different absorbance maxima they will not all be in resonance with the same laser excitation frequency and this property can be exploited to produce a very sensitive and selective method for detecting each of these dyes within a mixture of the others using two different laser excitation frequencies.
Multiplexed assays with 6 dye labelled oligonucleotides using a single excitation source have also been carried out and show the difficulty of separating the spectra by eye. Faulds et al. (Analyst, 2008, 133, 1505-1512 (2008)) adopted a multivariate analysis approach where the whole of the SERRS spectrum is considered, rather than looking for specific discriminatory Raman bands. Using this approach the first multiplexed simultaneous detection of six different DNA sequences, corresponding to different strains of the E. coli bacterium, each labelled with a different commercially available dye label (ROX, HEX, FAM, TET, Cy3, or TAMRA) was reported. In this study, both exploratory discriminant analysis and supervised learning, by partial least squares regression, were used and the ability to discriminate whether a particular labelled oligonucleotide was present or absent in a mixture was achieved using partial least squares regression with very high sensitivity (0.98-1), specificity (0.98-1), accuracy (range 0.99-1), and precision (0.98-1).
In the above two multiplexed examples, the sample mixture was prepared using different types of labelled-oligonucleotides, all present at the same concentration. However, this will not be the case for many industrial and diagnostic applications, where some analytes may be present at relatively high concentrations and others at relatively low concentrations. In addition, many effective SERRS dyes share significant chemical similarity, leading to many shared features in their SERRS spectra and making them harder to resolve in mixtures. The difference in dye absorption cross-sections can also lead to a weak scattering dye being obscured by a strong scattering dye. Thus, for many practical applications, a method is required to provide both a high degree of multiplexing with each component identifiable over a range of concentrations and to enable the effective construction of a number of different multiplex combinations
A technique for analysing Raman spectra is the Direct Classical Least Squares method (DCLS). This standard technique analyses spectral data X of an unknown sample in terms of a set of, K, known component reference spectra Sk each having I data points (both may be subject to pre-processing). Component concentrations, Ck, for each component reference spectrum are determined by minimising the sum of the squared deviations of the spectral data from the reconstructed model,
where i represents the spectral frequency index. This results in a series of linear equations which are solved directly by matrix inversion for the component concentrations Ck.
DCLS will typically produce a solution in which the calculated concentrations of all components are non-zero, even those that are not present in the sample. In general, this is due to the noise present in the spectral data and differences between the reference spectra and data, which can arise due to sample environmental conditions or drift in the spectrometer performance for example. When all components in the sample are present at high concentration, it is a simple matter of comparing the calculated component concentrations to discriminate between components which are present and those that are not. However when some components are present in trace amounts it becomes more difficult to distinguish between components that are genuinely present, and those that have low Ck values due to noise etc.
It is desirable to determine which components are genuinely present in a sample, even when some of the components are present in trace amounts.
According to one aspect of the invention there is provided a method of determining components present in a sample from spectral data obtained from the sample. The method may comprise resolving a model of the spectral data separately for candidates from a set of predetermined component reference spectra. The method may comprise determining whether a component is present in the sample based upon a figure of merit quantifying an effect of including the candidate reference spectrum corresponding to that component in the model.
It is believed that the invention is more effective in identifying trace components present in the sample than the standard DCLS method as described above. In particular, for distinguishing between components that are present in the sample and those that are not present, using the figure of merit based upon values of the model when resolved for one candidate reference spectrum separate from other candidate reference spectra may be more effective than comparing concentrations after the model has been resolved for the whole set of reference spectra. Once a subset of components has been identified, resolving the model for the component reference spectra of the subset, rather than all spectra, may more accurately determine concentrations of components in the sample than the standard DCLS technique.
The figure of merit may be determined in accordance with a merit function, which numerically scores a comparison between the resolved model and the spectral data. Determining that a component is present in the sample may be based upon whether the score for the candidate reference spectrum corresponding to that component meets a preset criterion. The figure of merit may be a measure of goodness of fit. Determining that a component is present in the sample may be based upon whether the inclusion of the candidate reference spectrum corresponding to that component in the model improves the measure of goodness of fit of the model to the spectral data above a preset limit.
The use of such a measure may be more effective in differentiating between trace components that are present in the sample and spurious components that are not present in the sample than the standard DCLS technique. In particular, even when resolving the model for a spurious minor component results in a significant concentration for the spurious component (relative to the concentration for a trace component), the improvement in a measure, such as goodness of fit, for that spurious component tends to be much less significant. Therefore, identifying components that are present in the sample based upon a measure such as goodness of fit, rather than a comparison of calculated concentrations, of the resolved component reference spectra may result in a more accurate solution.
A component reference spectrum may be a typical spectrum for a single chemical component or may be a typical spectrum for a group of different chemical components, such as a group of chemical components that are often found together. An advantage in using a component reference spectrum for a group of different chemical components is that it may reduce the number of fitting steps that have to be carried out relative to having separate spectra for each chemical component of the group. A component reference spectrum may also be specific to other factors, such as temperature and crystal orientation. A predetermined component reference spectrum may have been determined by spectral analysis of a material of a known chemical composition.
The method may comprise determining the components present in the sample in order of decreasing significance as determined by the figure of merit. This may be achieved through successive iterations. In each iteration, the model may be resolved separately for each candidate reference spectrum together with component reference spectra of greater significance as determined in previous iterations. For example, during each iterative step, the model is resolved separately for each candidate together with component reference spectra determined as present in the sample in a previous iteration, and it is determined whether a component is present in the sample based upon whether inclusion of the candidate reference spectrum in the model results in an improvement in the figure of merit greater than other candidate reference spectra and whether the improvement is above a pre-set limit. This process is repeated whilst improvements to the figure of merit remain above the pre-set limit. It will be understood however that component reference spectra or other spectra may be resolved before carrying out the iterative process. For example, the model may be first resolved for a background spectrum that represents features such as the contribution of the substrate that supports the sample, fluorescence and a baseline of the spectrometer. Furthermore, a user may know that certain components are present and the user may have the component reference spectra for these known components resolved before carrying out the iterative step.
In one arrangement, an iteration comprises determining whether a difference between the figure of merit for a most significant candidate reference spectra and the other candidate reference spectra is within a predefined threshold and splitting the iterative process into parallel iterations for each candidate reference spectrum that falls within the threshold, wherein for each parallel iteration the other candidate reference spectrum, rather than the most significant candidate reference spectrum, is considered as a next most significant spectrum in the order. In this way, if at a point in the iterative process a difference in the figure of merit between two or more candidate reference spectra does not merit selecting one of the candidate reference spectra over the other, the search is branched to explore all reasonable alternatives. Setting a narrow threshold will reduce processing as fewer branches will be explored, whereas setting a broad threshold will avoid dropping branches that may provide useful results. A component could then be deemed as present in the sample only if the component is determined as being present in the sample by all parallel iterations.
In an alternative arrangement, the method comprises initially resolving the model for all of the reference spectra of the set of predetermined component reference spectra and removing candidate reference spectra from the model based upon the figure of merit, for example whose inclusion improves a measure of goodness of fit below the pre-set limit. The removal of component reference spectra could be carried out as an iterative method, for example, the iterative step continuing until an improvement to the measure of goodness of fit is above the pre-set limit.
The measure of goodness of fit is a measure of a discrepancy between the spectral data and the resolved model, e.g. Σk=1K CkSk. The measure of goodness of fit may be lack of fit, R-squared, likelihood ratio test or other suitable measure. In one embodiment, the measure of goodness of fit is a lack of fit sum of squares, LoF, such as that given by:
The pre-set limit may be a proportional improvement in goodness of fit. For example, the proportional improvement in goodness of fit may be an improvement in goodness of fit relative to a baseline, for example a minimum or maximum goodness of fit, achievable for the spectral data and the set of predetermined reference spectra. The baseline may be a value for goodness of fit achievable for the spectral data and set of predetermined component reference spectra that is closest to a value for a perfect fit. For example, the baseline may be determined by calculating a measure of goodness of fit for the model resolved for all of the predetermined component reference spectra (as is the case in standard DCLS).
A system for carrying out the method may be arranged such that the limit for the improvement in the figure of merit/measure of goodness of fit can be set based on spectroscopy performance and/or other requirements. Increasing the limit will tend to improve specificity (freedom from false positive identifications) at the expense of sensitivity (freedom from false negative identifications). The method may comprise establishing a pre-set limit for a spectroscopy apparatus by obtaining spectral data for samples, wherein the components making up the samples are known, determining components of the sample using the method described above for two or more limit values and selecting a suitable limit for use in the analyses of an unknown sample based on accuracy of the solution (such as the number of false negatives or false positives). Each spectroscopy apparatus may be calibrated to determine a suitable value or range of values for the pre-set limit and the method may comprise setting the limit to the suitable value or a suitable value identified by the range.
The inclusion of a component reference spectrum in the model may automatically trigger the inclusion of one or more transformations and/or distortions of that component reference spectrum and/or one or more corrective spectra associated with that component reference spectrum. The inclusion of such terms can be useful to correct for components that are not adequately described by a single component reference spectrum. For example, such terms may take account of environmental and/or instrumental differences between the sample and reference spectra. The inclusion of such terms may be particularly applicable to a process where candidates are evaluated together with reference spectrum of components that have already been identified as present in the sample in light of any required distortion to those reference spectra.
Resolving the model may comprise calculating a concentration for the component (corresponding to the candidate spectrum) in the sample.
Determining that a component is present in the sample may be further based upon whether a positive concentration is calculated for the component. A negative concentration is a non-physical solution to the model and therefore, is to be avoided.
Reporting that a component is present may be further based upon whether the concentration for the component is above a predetermined (positive) minimum limit. The minimum limit may be set at a level that is deemed significant to a particular application.
The model may be a Direct Classical Least Squares analysis and the model may be resolved by minimising equation (1) above for particular reference spectra (e.g. the candidate reference spectrum plus the reference spectra of components that have already been selected).
The spectral data may be a Raman spectrum.
According to a further aspect of the invention there is provided apparatus for determining components present in a sample from spectral data obtained from the sample. The apparatus may comprise a processor arranged to: —
According to a yet further aspect of the invention, there is provided a data carrier having stored thereon instructions, which, when executed by a processor, cause the processor to: —
The data carrier may be a non-transient data carrier, such as volatile memory, eg RAM, non-volatile memory, eg ROM, flash memory and data storage devices, such as hard discs, optical discs, or a transient data carrier, such as an electronic or optical signal.
According to another aspect of the invention there is provided a method of constructing a model of spectral data obtained from a sample comprising selecting one or more component reference spectrum from a set of predetermined component reference spectra based upon a figure of merit for including that candidate reference spectrum in the model.
According to a further aspect of the invention there is provided a method of indicating a likelihood that a component is present in a sample comprising resolving a model of spectral data of the sample for a set of predetermined component reference spectra, determining a figure of merit for including each component reference spectrum in the model and providing an indication of the relative likelihoods that components corresponding to the component reference spectra are present in the sample based upon the figure of merit.
The indication may be a display of the figure of merit associated with the component or alternatively, the indication may be a colour, symbol (non-textual) or the like associated with the component, for example colours red, amber and green to indicate components that are, respectively, least likely, are neither the least or most likely, and most likely to be included in the sample.
The present invention is based in part on a method for constructing effective combinations of dyes which can be used to detect various analytes which may be present over a concentration range in a sample and which can be discerned in a single wavelength multiplex assay.
According to a further aspect of the invention, there is provided a kit for use in a multiplex assay, the kit comprising a dye set consisting essentially of a plurality of dyes and an association of each dye to a reference concentration, wherein, using surface enhanced Raman spectroscopy (SERRS), each dye of the set is identifiable at better than 90% sensitivity and 90% specificity in the presence of any other dye of the set throughout a range of concentrations of each of the two dyes from 0.6 to 1.5 of the respective dye's reference concentration.
A kit according to the invention may be used for detecting different analytes in a sample, for example by attaching each dye to a ligand to form a dye-ligand conjugate, each ligand capable of binding to a specific analyte. The dyes may be used to detect analytes even when in the presence of another dye. This may be useful when detecting more than one disease in a sample from a patient. Furthermore, a kit according to the invention may provide a user with a higher level of confidence that an analyte will be detected because the dyes are identifiable in the presence of any other dye of the set across a range of concentrations. This may be important as a user may want to be confident that an analyte is detected even when a concentration of the dye is not precisely that of the reference concentration.
Preferably, each dye is identifiable at better than 95% sensitivity and/or 95% specificity, and more preferably, at better than 98% sensitivity and/or 97% specificity, in the presence of any other dye of the set throughout a range of concentrations of each of the two dyes from 0.6 to 1.5, and preferably 0.45 to 1.5 of the respective dye's reference concentration. Such high levels of sensitivity and specificity are desirable in medical diagnostics, wherein failure to identify an analyte or incorrect identification of an analyte may have serious repercussions.
It may be desirable to have different reference concentrations for the dyes of the set. In this way, the range of concentrations over which a dye can be identified may be increased by selecting lower reference concentrations of the dyes having more intense SERRS spectra for a specified excitation wavelength. A difference in the reference concentrations for any pair of dyes of the set may be less than 2 orders of magnitude and may be between 1×10−11 Molar and 1×10−9 Molar and further optionally may be between 4×10−11 Molar and 3×10−10 Molar.
It will be understood that the term “the dye set consisting essentially of a plurality of dyes” means that the dye set does not comprise any other dyes that are essential for detecting analytes in the multiplex assay beyond the plurality of dyes. However, the dye set may comprise other substances such as water, spermine and colloid.
The dye set may comprise a mixture made of the plurality of dyes. Alternatively, each dye of the set may be housed separately.
The association of each dye with the reference concentration may be an association of each dye to a reference SERRS spectrum used in the multiplex assay for identifying the dye, the reference SERRS spectrum obtained with the dye present in a sample at the reference concentration. For example, the kit may comprise a library of such reference SERRS spectra or alternatively, the kit may comprise identification of a source of reference SERRS spectra that should be used to analyse the dyes. Alternatively, the kit may comprise a reference sample for each dye, each reference sample comprising a mixture including the dye at the reference concentration. In use, a user may obtain reference spectra using the reference samples, the reference spectra for use in identifying dyes in a sample, for example using a Direct Classical Least Squares (DCLS) analysis.
The dye set may be made of a plurality of dyes for example 5, 6, 7, 8, 9, 10 or more dyes. The dye set may comprise of at least six dyes from any one of the following lists:
i) JOE, Rhodamine Green, ATTO520, BODIPY FL, BODIPY TMR-X, FAM, HEX, Cy3, Cy3.5, TAMRA and TYE563;
ii) JOE, Rhodamine Green, FAM. HEX. DY549, Cy3, Cy3.5, ATTO488, MAX and TYE563;
iii) BODIPY530/550, BODIPY FL, BODIPY TMR-X, CY3.5, CY3, FAM, HEX, Rhodamine Green, TAMRA and TYE563.
The dye set may comprise CY3.5, CY3, FAM, HEX, Rhodamine Green and TYE. In one embodiment the dye set may consist of 10 of the following dyes: JOE, Rhodamine Green, ATTO520, BODIPY FL, BODIPY TMR-X, FAM, HEX, Cy3, Cy3.5, TAMRA and TYE563. In another embodiment, the dye set may consist of JOE, Rhodamine Green, FAM, HEX, DY549, Cy3, Cy3.5, ATTO488, MAX and TYE563. In a further embodiment, the dye set may consist of BODIPY530/550, BODIPY FL, BODIPY TMR-X, CY3.5, CY3, FAM, HEX, Rhodamine Green, TAMRA and TYE563.
However, in accordance with the teaching described herein, the skilful addressee is able to identify other sets of dyes which may be used.
According to yet another aspect of the invention there is provided a kit for use in a multiplex assay comprising a dye set, the dye set consisting essentially of a plurality of dyes, each dye for use in identifying a separate analyte in a sample using surface enhanced resonant Raman spectroscopy when a concentration of the dye is less than 1×10−9 Molar, optionally, less than 5×10−10 Molar, and a concentration difference in the sample between the dye and any one of the other dye spans is at least 2×10−11 Molar.
It will be understood that the use herein of the term “surface enhanced Raman spectroscopy (SERRS)” in connection with the invention it intended to include arrangements wherein there is an overlap in the absorption and plasmon resonance profiles although the centre of each profile occurs at a different wavelength. The centre of each profile may be offset by less than 100 nm.
According to a further aspect of the invention there is provided a kit for use in a multiplex assay, the kit comprising a dye set consisting essentially of a plurality of dyes, wherein, using surface enhanced Raman spectroscopy (SERRS), each dye of the set is distinguishable from any other dye of the set at better than 99% sensitivity and 99% specificity.
According to another aspect of the invention, there is provided use of a kit according to the first or second aspects of the invention for detecting one or more analytes present in a sample, using a single excitation wavelength.
According to yet another aspect of the invention, there is provided a method for conducting a multiplex assay on a sample, the method involving:
Preferably, identifying which analyte(s) is/are present in the sample involves irradiating the dye-ligand conjugates with a single excitation wavelength.
A number of different types of analytes that can be identified may be greater than 5, for example 6, 7, 8, 9, 10 or more and the ligand may be a peptide, an oligonucleotide an antibody or a protein for example.
The transduction technique is preferably a Raman based spectroscopy technique such as surface enhanced resonant Raman spectroscopy. However the transduction technique may also be a fluorescence technique. The transduction technique may also involve a single excitation wavelength for example a Raman excitation wavelength at 532 nm.
The analyte concentration may be extracted over a dynamic range of at least 1 order of magnitude for example 2, 3 or 4 orders of magnitude.
According to another aspect of the invention there is provided a method of selecting X dyes among N dyes comprising generating SERRS Raman spectra for dye sets, each dye set comprising one or more dyes selected from the N dyes, and calculating a figure of merit indicative of a chance of identifying, from the SERRS Raman spectra, correctly as present the one or more dyes in each set and/or incorrectly as present the dyes absent from each set and selecting the X dyes based upon the calculated figure of merit.
X and N represent integers, wherein X is less than N.
In this way, a “best” set of dyes are selected based upon the figure of merit.
The dyes may include dye sets comprising two or more dyes. In this way, the dyes may be selected based upon a chance of correctly identifying a dye and/or incorrectly identifying an absent dye as present when each dye is in the presence of one or more other dyes. The selected dyes may therefore be particularly suitable for identifying analytes when more than one analyte is present in a sample.
The method may comprise establishing a reference concentration for each dye, wherein the SERRS spectra generated for each dye set are based upon the dye set comprising one or more major dyes and a minor dye, wherein a ratio of a concentration of the or each major dye relative to the major dye's reference concentration is greater than a ratio of a concentration of the minor dye relative to the minor dye's reference concentration. Establishing the reference concentration may be based upon a limit of concentration of the dye when present as a minor dye in a dye set at which a sensitivity of the minor dye is above a set performance criteria, for example, a reference concentration may be chosen for each dye such that no one dye has a limit of concentration above a defined level, such as above 0.6, and preferably, above 0.45, of the reference concentration. Any dye for which a reference concentration cannot be identified for which the dye meets this criteria may be removed as an unsuitable dye. A further selection of the dyes may be based upon a limit of concentration determined for the established reference concentration, although other performance criteria, such as specificity, for these dye sets may first be considered as selection criteria. Using different reference concentrations for different dyes may allow the use of a dye that generates a weaker SERRS signal to be used with a dye(s) that generates stronger SERRS signals through appropriate shifting of the reference concentrations.
The method may comprise, for each dye set, determining specificity and/or sensitivity at at least one concentration, and preferably, at a plurality of different concentrations, of the minor and/or major dye and selecting the X dyes comprises selecting the dyes based upon the determined specificity and/or sensitivity. It may not be sufficient to check the specificity and/or sensitivity just at the limits of concentration of the dyes of each set but it may be necessary to check each dye set across a range of concentrations.
The method may comprise selecting a subset of dyes based upon a figure of merit calculated for m-plex dye sets then selecting dyes from the subset based upon a figure of merit calculated for p-plex dye sets formed from combinations of the subset of dyes, wherein each m-plex dye set comprises fewer dyes than each p-plex dye set. For example, the m-plex dye sets may be simplex dye sets and the p-plex dye sets may be duplex dye sets. The processing required to screen the dyes may increase as higher order plex dye sets are analysed if the number of dyes from which the selection is to be made remains the same. Accordingly, reducing the number of dyes by screening the dyes first though analysis of lower plex dye sets may reduce the time it takes to select the X dyes from the N dyes.
A further method for reducing the processing time to select the X dyes is to use core mixtures (such as duplexes, triplexes and quadruplexes, etc) that are known to meet the necessary requirements as a starting point for selecting the dyes, the method comprising determining dyes to add to the established core mixture.
The SERRS spectra may be simulated from data indicative of the variability of SERRS spectra obtainable from each dye of the set. For example, the variability data may comprise SERRS spectra for each dye obtained experimentally under different conditions. These SERRS spectra are different to a reference SERRS spectrum that may be used for identifying the dye. The SERRS spectra for each dye set may be generated by randomly selecting SERRS spectra from the variability data and applying scaling of the intensity as appropriate for the concentrations. In the case of dye sets comprising more than one dye, it may be necessary to remove features of the spectra that are duplicated when two or more spectra are combined, such as background.
According to another aspect of the invention there is provided a data carrier having instructions stored thereon, which, when executed by a processor, cause the processor to carry out the method of selecting dyes in accordance with the aspects described above.
According to a further aspect of the invention there is provided a computer system programmed to carry out the method of selecting dyes in accordance with the aspects described above.
Preferably, identifying a dye in a dye set involves analysing the spectrum using a multivariate analysis technique, such as a method based on a Direct Classical Least Squares method.
According to yet another aspect of the invention, there is provided a dye set obtainable by the method for selecting X dyes among N dyes described above.
According to yet another aspect of the invention, there is provided a dye-ligand set wherein the plurality of dyes is obtainable by the method for selecting X dyes among N dyes described above.
According to yet another aspect of the invention, there is provided a method for detecting analytes in a sample and a method for conducting a multiplex assay on a sample, wherein the dye-ligand conjugates are made of dyes selected according to the method for selecting X dyes among N dyes described above.
Various aspects of the invention will now be described by way of example only and with reference to the accompanying drawings, of which:
Referring to
The Raman spectrometer comprises an input laser beam 10 reflected through 90 degrees by a dichroic filter 12, placed at 45 degrees to the optical path. Alternatively a holographic dichroic filter may be placed at a low angle of incidence such as 10 degrees. The laser beam then passes to an objective lens 16, which focuses it at its focal point 19 on a sample 18. Light is scattered by the sample, and is collected by the objective lens 16 and collimated into a parallel beam which passes back to the dichroic filter 12. The filter 12 rejects Rayleigh scattered light having the same frequency as the input laser beam 10, and transmits the Raman scattered light. The Raman scattered light then passes to a Raman analyser 20.
The Raman analyser 20 comprises a dispersive element such as a diffraction grating. The light from the analyser 20 is focused by a lens 22 onto a suitable photo-detector. A photo-detector array is preferred. In the present embodiment the detector 24 is a charge-coupled device (CCD), which consists of a two-dimensional array of pixels, and which is connected to a computer 25 which acquires data from each of the pixels and analyses it as required. The analyser 20 produces a spectrum having various bands as indicated by broken lines 28, spread out in a line along the CCD 24.
Samples 18 may be mounted on an XYZ stage so that spectral data may be collected from each sample under control of the computer.
The computer 25 is programmed with software code on a suitable medium, such as memory 29, comprising instructions, which when executed by a processor of computer 25 cause the computer 25 to perform the analysis routines described below. Alternatively, the data on the Raman spectrum/spectra obtained may be transferred to a separate computer having such software for this analysis. In either case, as the analysis proceeds, the values determined are stored in the computer concerned, and may be further processed and output or displayed to show results of the analysis in terms of the components in the sample/samples. In the case where the analysis is performed by computer 25, memory 29 has stored thereon a databank of component reference spectra to be used for the analysis. Each component reference spectrum is a typical Raman spectrum for a different chemical component or group of chemical components.
Referring to
In this embodiment a Direct Classical Least Squares analysis is carried out of the spectral data, wherein equation (1) is resolved for each candidate spectrum of the predetermined set of component reference spectra, steps 103 to 108. A component reference spectrum is selected for inclusion in a final form of the model based upon whether the inclusion of that component reference spectrum improves a measure of goodness of fit of the model to the data above a preset limit.
An iterative process is carried out comprising selecting a component reference spectrum for inclusion in the final form of the model in each iteration in order of decreasing significance as determined by an improvement to the measure of goodness of fit.
In step 103, for each candidate of the set of predetermined component reference spectra, equation (1) is minimised for the candidate reference spectrum together with any component reference spectra that have already been selected, such as in a previous iteration. A measure of goodness of fit is calculated for the resolved components relative to the spectral data of the sample.
In this embodiment, the measure of goodness of fit is a measure of lack of fit (LoF) given by: —
This measure of lack of fit is compared to a previous measure of LoF calculated for the selected component reference spectra before the addition of the candidate reference spectrum to determine an improvement to the measure of LoF resulting from the addition.
In one embodiment, the improvement in the LoF, Llpr, is calculated as a proportional improvement in the LoF relative to a baseline LoF, Lmin, as given by: —
where Lold is the LoF value calculated for the selected component reference spectra before the inclusion of the candidate reference spectrum and Lnew is the LoF value calculated for the selected component reference spectra including the candidate reference spectrum.
In one embodiment Lmin may be set to zero. In another embodiment, the baseline, Lmin, is a minimum obtainable LoF calculated from the model when resolved for all predetermined reference spectra, as in conventional DCLS. In this way, rather than calculating Llpr against an absolute value of zero, Lmin is automatically adjusted to take into account data quality. As a consequence, the preset limit can be set relatively independent of data quality or pre-processing options.
In step 104, the process checks the resolved concentrations for the candidate reference spectra and removes from further consideration in the iteration (but not subsequent iterations) candidate reference spectra resolved as having a negative concentration.
In step 105, the improvements in the LoF, Llpr, for remaining candidate reference spectra are compared and the candidate reference spectrum associated with the greatest improvement in the LoF becomes the leading candidate reference spectrum for inclusion in the final form of the model.
A check 106 is made to determine whether the improvement in the LoF resulting from addition of the leading candidate reference spectrum is above a preset limit. If the improvement to the LoF, Llpr, for the leading candidate reference spectrum is above the preset limit, it is selected 107 as a component reference spectrum that is present in the final form of the model. The process 103 to 107 is then repeated for the remaining unselected component reference spectra.
If the improvement to the LoF, Llpr, for the leading candidate reference spectrum is below the preset limit, then the method is terminated and the final form of the model, comprising the model resolved for the component reference spectra selected up to that point, is output as an electronic signal, for example to memory 29 or to a display (not shown). The final form of the model will typically comprise a subset of the set of predetermined component reference spectra, these spectra being those of most significance as measured by lack of fit.
A determination can be made of components present in the sample based upon whether the component reference spectrum corresponding to that component is included in the final form of the model. The concentrations can be determined from the resolved component Ck. As the component reference spectra are filtered as part of the iterative process, all reference spectra in the final form of the model may represent components present in the sample. Accordingly, further filtering to remove spurious components may not be necessary.
However, in an alternative embodiment, components having a concentration Ck below a minimum limit in the final form of the model are not reported as present in the sample. The minimum limit may be set based upon the noise in the spectral data or a minimum concentration at which a component is of interest to the user.
The limit to the improvement in the LoF at which a component reference spectrum is selected for the final form of the model controls specificity and sensitivity of the technique and is likely to depend on the requirements of the application and spectroscopy performance. Accordingly, the apparatus may comprise an input for setting the limit for improvement to the LoF, such as an appropriate interface on computer 25. The Raman spectrometer may be calibrated to determine specificity and sensitivity at different limits. Such a calibration may be carried out by obtaining spectral data from samples of known components using the spectrometer, determining components of the sample using the analysis method described above for a plurality of limits and determining the proportion of false negatives and false positives at each limit. Armed with this information, a user can preset the limit used when analysing an unknown sample with the spectrometer for the specificity and sensitivity desired.
In one embodiment, the method comprises an additional step whereby the inclusion of a component reference spectrum in the final form of the model automatically triggers the inclusion of one or more transformations and/or distortions of that component reference spectrum and/or one or more corrective spectra associated with that component reference spectrum. The inclusion of such terms can be useful to correct for components that are not adequately described by a single component reference spectrum. For example, such terms may take account of environmental and/or instrumental differences between the sample and reference spectra.
In a further embodiment, the iterative process is modified to split into parallel iterations if specified criteria are met. In this embodiment, in step 105 a determination is made of whether a difference between the improvement in lack of fit for the leading candidate reference spectrum and each of the other candidate reference spectra is within a predefined threshold.
If this value for one or more of the other candidate reference spectra is within the threshold, the iterative process is branched into parallel iterations for each candidate reference spectrum that falls within the threshold. In each parallel iteration, a candidate reference spectrum that fell within the threshold is selected for the final form of this branch of the model in place of the leading candidate reference spectrum. Each branch of the iterative process including the main iteration is then progressed independently of the other branches and split again, if appropriate. Each branch is terminated when condition 106 is met, the final forms for the model from each branch are compared and components common to all branches could then be reported as present in the sample.
Furthermore, if, as in the example, after splitting, two branches later converge with all selected reference spectra being common to both branches, it is only necessary to continue with one of the branches. This is illustrated by the cross in
The First step relates to the pre-selection of N dyes in a dye pool, where dyes are assessed based on the following criteria:
The assessment of these criteria may be carried out by eye, for example by viewing SERRS spectra generated by the dyes, and any dye that clearly fails one or more of these criteria may not be included in the dye pool. This pre-selection of dyes removes dyes that are clearly inappropriate, reducing the processing required in subsequent steps.
The second step is to measure spectra encompassing signal variability for each of the N dyes in the dye pool at a reference concentration. In this embodiment, the reference concentration(s) of the dye in a reference sample is/are selected to be between 1×10−11 Molar and 1×10−9 Molar. Dye spectrum variability is acquired according to a fractional factorial experimental design, and covers a plurality of variability parameters which may include: the operator (the person preparing and measuring the spectra), the batch of dye, the batch of colloid, the batch of spermine, preferably present as hydrochloride, and the time that has elapsed between sample preparation and measurement. For each dye, a series of SERRS spectra are measured along with a number of “blank” reference spectra. There is no lower or upper limit on the number of spectra measured for each dye, but each set of variability spectra should encompass factors affecting variation in the dye signal.
In some cases, the data collected during this step may indicate that the dye should not be included in the dye pool, for example, because it fails to meet the criteria for pre-selection outlined above in the First Step. This can occur if pre-selection choices are based upon a smaller and less representative set of spectra than are collected for the dye variability step.
The SERRS spectra of the variability data are filtered to remove spectra where the dye signal is either significantly weaker or stronger than the average. This is intended to remove outlying spectra which should not be part of the set.
The third step referred to as Simplex Screening, estimates the risk of a false positive result when analysing a SERRS spectrum of a single dye. In other words, when analysing the SERRS spectrum of a single dye, the risk of identifying another (absent) dye.
The true positive and false positive rates are defined as follows. When considering the analysis of a sample in which dye A is present and dye B is absent, if for example the sample is analysed one thousand times, and if the analysis detects the presence of dye A in 995 cases, the estimated true positive rate (TPR) of dye A will be 99.5%. Similarly, if another dye, B, absent in the sample is incorrectly detected by the analysis in 10 cases, the estimated false positive rate (FPR) of dye B will be 1%. The TPR corresponds to sensitivity, and the FPR corresponds to selectivity.
The false positive rate is simulated for each individual dye by carrying out the following steps a number of times in order to obtain a statistically acceptable measure of a dye's false positive rate.
A reference spectrum is randomly selected for each dye from the variability data and a spectrum is simulated for the “present” dye by randomly selecting a further spectrum, different to the reference spectrum chosen for that dye, from the variability data.
The simulated spectrum is analysed using the algorithm described below with reference to
These steps are then repeated an appropriate number of times to obtain a statistically significant measure of the estimated false positive rates. Any false positive rates significantly above a threshold would indicate that the corresponding pair of dyes (one present, one absent but falsely detected) represent a false positive risk, and hence are not a good pairing to use in an assay.
Alternatively, an extended Simplex Screening simulation can be performed. This is essentially the same as the basic Simplex Screening simulation described above, but with one modification. In the basic form of the simulation, it is assumed that all of the dyes in the dye pool could be present in the sample, and the spectrum is analysed accordingly. However, this presents a small risk of “hiding” some possible false positive risks. For example in the instance where a dye A is present, and dyes B and C both have a risk of being incorrectly “detected” under these circumstances. If dye B has a slightly better fit than dye C, it will be chosen preferentially over dye C every time, and thus potentially masks the risk between dyes A and C. So in this variation of the simulation, the analysis is repeated several times, covering every possible pairing in a two dye system. For example, when dye A is present, the data is analysed assuming only that dyes A & B could be present, then the analysis is repeated assuming that only dyes A & C could be present, then only A & D . . . etc. This approach helps find additional False Positive risks that might otherwise have been missed.
Following simplex screening, dyes that are associated with poor specificity results may be considered for removal from the dye pool. However, removal of any one dye from the pool may eliminate the need to consider removal of other dyes from the pool. For example, if dye A shows poor specificity when analysed with dye B also potentially present in the sample, then removal of either one if these dyes may eliminate the need to also remove the other, provided the other dye does not have additional specificity issues with a third dye. Often, there are multiple viable options for removing a small number of dyes from the pool in order to leave a smaller sized dye pool with no significant specificity issues.
The fourth step, referred to as Multiplex Screening, estimates the risk of a false negative result when analysing a SERRS spectrum of a sample containing two or more dyes, and also checks that the estimated risk of a false positive result is not significantly higher than for separate simplex samples of the dyes present in the multiplex sample.
Multiplex Screening is illustrated by discussing the Duplex case. Considering a duplex having a “minor” dye at low concentration relative to its reference concentration in presence of a “major” dye a set concentration at or above its reference concentration, this step of the method calculates the minimum concentration of the minor dye that is sufficient to achieve a set performance criteria, for example TPR above a required limit, for example >99%. The minimum concentration of the minor dye that meets the performance criteria is defined as the lowest detectable concentration (LDC) of the minor dye.
To do so, the Raman spectrum of a mixture containing a (fixed) high concentration of dye A+a low concentration of dye B is simulated by combining spectra randomly selected from the variability data for dye A and B, the spectra scaled as appropriate for the concentrations. An appropriately-scaled blank spectrum (also chosen randomly from the variability data) is either added or subtracted as appropriate, to keep the overall “blank contribution” to the simulated spectrum appropriate. As with the simplex screening, a reference spectrum is selected for each candidate dye and the simulated spectrum analysed using the DCLS algorithm as described below with reference to
These steps are then repeated an appropriate number of times to obtain a statistically significant measure of the estimated true positive and false positive rates.
Assuming that the required performance criterion is not met, the concentration of dye B is increased by a small amount (for example 1%), but the concentration of dye A is kept the same. The method is repeated until the TPR meets its required limit. The corresponding concentration is the lowest concentration of dye B which can reliably be detected if dye A is present at the set concentration at or above the reference concentration.
The overall FPR is estimated (for all absent dyes) for each duplex at a range of concentrations of the minor dye, in order to confirm that the duplex combination does not have a significantly higher False Positive risk than simplex samples containing the dyes that make up the duplex.
This approach can be extended to higher-order multiplex levels, for example triplex or four-plex. In such cases, when sensitivity is the main property of interest, a suitable approach is to simulate samples with multiple major dyes and a single minor dye. For example, a triplex simulation may contain two major dyes with concentrations at or above their respective reference concentrations, and a single minor dye below its reference concentration.
Multiplex combinations associated with poor performance (a high LDC and/or or high FPR) are then identified. The individual dyes associated with the multiplex may be considered for removal from the dye pool. Alternatively, if the poor performance of the multiplex is due to False Positives, the dye or dyes which are incorrectly identified as present (the False Positive dye or dyes) may be considered for removal from the dye pool. As is the case with the Simplex Simulation, removing any one dye from the pool may eliminate the need to remove other dyes, and there may be more than one way to eliminate all poor-performing multiplex combinations.
In general, duplex screening is performed before triplex screening, which in turn is conducted before higher-order multiplex screening. This allows the results from the lower-order multiplex simulations to reduce the number of dyes in the dye pool that is used in the higher-order multiplex simulations, reducing the simulation complexity.
During multiplex screening, if it is found that different dyes in the dye pool have very disparate lowest detectable concentration values, then the dye reference concentrations may be adjusted and the process re-started. For example, if dye A has a limit of concentration of 5% of the reference concentration whereas dye B has a limit of concentration of 30% of the reference concentration, the reference concentration for dye A may be increased and/or the reference concentration for dye B reduced. This may require the gathering of new variability data at these new reference concentrations.
The fifth step involves the selection of X dyes from the remaining N dyes in the dye pool based on data generated in the preceding steps If there are more dyes remaining in the dye pool than needed for a specific application (i.e. N is greater than X), further selection is required in order to identify which set of dyes would achieve the best result. Depending on the application it could be a choice between better sensitivity, specificity and some combination of the two.
Referring to
where i represents the spectral frequency index. This results in a series of linear equations which are solved directly by matrix inversion for the component concentrations Ck.
An iterative process is carried out in which Equation (1) is resolved for each candidate dye using the selected reference spectrum together, steps 103 to 108.
In step 103, for each candidate dye, equation (1) is minimised for the dye's reference spectrum together with any dye reference spectra that have already been selected in a previous iteration. A measure of goodness of fit is calculated for the resolved components relative to the simulated spectrum.
The measure of goodness of fit can be a measure of lack of fit (LoF) given by: —
This measure of lack of fit is compared to a previous measure of LoF calculated for the selected dye reference spectra before the addition of the candidate dye reference spectrum to determine an improvement to the measure of LoF resulting from the addition.
The improvement in the LoF, Llpr, is calculated as a proportional improvement in the LoF: —
where Lold is the LoF value calculated for the selected dye reference spectra before the inclusion of the candidate dye reference spectrum and Lnew is the LoF value calculated for the selected dye reference spectra including the candidate dye reference spectrum.
In step 104, the candidate dye reference spectra resolved as having a negative concentration are removed from further consideration in the iteration (but not subsequent iterations).
In step 105, the improvements in the LoF, Llpr, for the remaining candidate dye reference spectra are compared and the candidate dye reference spectrum associated with the greatest improvement in the LoF becomes the leading candidate dye reference spectrum for inclusion in the final form of the model.
A check 106 is made to determine whether the improvement in the LoF resulting from addition of the leading candidate dye reference spectrum is above a preset limit. If the improvement to the LoF, Llpr, for the leading candidate dye reference spectrum is above the preset limit, it is selected 107 as a dye reference spectrum that is present in the final form of the model. The process 103 to 107 is then repeated for the remaining unselected dye reference spectra.
If the improvement to the LoF, Llpr, for the leading dye reference spectrum is below the preset limit, then the method is terminated and the final form of the model, comprising the model resolved for the dye reference spectra selected up to that point, is output. The final form of the model will typically comprise a subset of the set of predetermined dye reference spectra, these spectra being those of most significance as measured by lack of fit.
A determination can be made of components present in the sample based upon whether the reference spectrum corresponding to a dye is included in the final form of the model.
Further details regarding the above method and of the preferred apparatus for conducting this method are described above with reference to
A dye set identified using the above method may be supplied for use in a multiplex assay. The dye set should be supplied in association with a reference concentration for each dye such that the sample comprising the dyes is analysed using reference spectra obtained at the reference concentration. Such an association may be supply of the reference spectra themselves, information on where such reference spectra may be obtained, such as a website, etc, supply of reference samples wherein the dyes are at the reference concentration, a list of reference concentrations at which reference spectra should be obtained or/and supply of the dye set for use with a particular system, wherein the system comprises a library of reference spectra obtained at the reference concentrations.
Now referring to
The dyes are attached to oligonucleotide sequences that are complimentary to DNA sequences found in the pathogens to be identified. These dye labelled oligonucleotides are added to the biotinylated PCR products such that the labelled oligonucleotides hybridise to any complimentary sequences that are present.
Streptavidin beads are then added such that the biotinylated products attach to the beads whist leaving the dye labelled oligonucleotides that have not hybridised to complimentary sequences unattached. These unattached dye labelled oligonucleotides can then be washed away
The remaining dye labelled oligonucleotides are released from the streptavidin beads into a solution using an elution process, the solution comprising SERRS reagents for use in analysis. A SERRS spectrum of the solution is obtained and the spectrum is analysed using the technique described with reference to
In order to carry out the above method, the dye kit may provided as part of a system, as shown in
The kit 200 is a consumable and, as such, can be supplied to the consumer, as and when required. Furthermore, different kits for identifying different targets can be used. A kit may be provided for identifying causative agents of gastroenteritis. For example, the kit may be used in an assay to detect two or more of the bacterial targets ETEC, EPEC, VTEC, Salmonella, S. enterica, Campylobacter, Shigella, C. difficile A, C. difficile B and Yersinia. A kit may be provided for identifying two or more viral targets, such as one or more of Norovirus G1, Norovirus G2, Adenovirus, Rotavirus, Sapovirus and Astrovirus. A kit may be provided for identifying causative agents of fungal infections. For example, such a fungal kit may be used in an assay to identify two or more of A. fumigates, A. glaucus, A. flavus, A. terreus, A. Niger, A. ustus, A. candidus, A. versicolor. A kit may be provided for identifying causative agents of cerebrospinal fluid (CSF) viral infections. For example, the kit may used to identify two or more of the targets Herpes simplex virus 1, Herpes simplex virus 2, Varicella-zoster virus, Epstein-Barr virus, Cytomegalovirus, Enterovirus and poliovirus, John-Cunningham virus, Parechovirus. An alternative kit may be provided to detect two or more of the Candida species, such as two or more of C albicans, C. parasilosis, C. tropicalis, C. viswanthii, C. guilliermondii, C inconspicua, C. lustaniae, C. dubliniensis, C. kefyr, C. famata, C. krusei and C. glabrata. In each example, the same dyes may be used, with each dye attached to an oligonucleotide sequence that hybridises to a corresponding sequence on the target in the amplified PCR product.
The system further comprises a sample processor 204 for automatically carrying out steps of attaching the hybridised dye labelled oligonucleotides-PCR product complex to the magnetic beads, introducing a washing buffer to wash away the excess dye labelled oligonucleotides that are not attached to the target, introducing an elution buffer to detach the hybridized dye labelled oligonucleotides from the magnetic beads and combining with the SERRS reagents, This may be carried out by a robot arm 205 that controls a plurality of pipettes 210 to transfer a set volume of products contained in the wells of microplate 203, inserted into the sample processor 204 by the user to a further microplate 211 to which the magnetic beads, wash buffer, elution buffer and SERRS reagents can be added. The sample processor 204 comprises a number of reservoirs containing the magnetic beads, wash buffers, elution buffers and SERRS reagents. In the example, only four reservoirs are shown 206, 207, 208 and 209, but more than four reservoirs are preferably at least for the reason that there are a number of SERRS regents, each of which is kept in a separate reservoir. The robot arm can control the pipettes to take a set volume of these solutions when required.
The system further comprises a spectrometer 212 comprising a Raman spectrometer 213 for scanning a sample 215, the spectrometer connected to a computer 214. The computer comprises a processor 216, memory 217, a display 218 and an input device, such as a keyboard 219. Stored in memory is a set of Raman reference spectra 220 to be used in the analysis of the Raman spectrum obtained from the sample. The memory associates each Raman spectrum to a target for each different kit that may be used in the system. For example, the same dye may be associated with different targets for different kits.
In use, through appropriate inputs, a user identifies to the computer 214 the kit being used and the computer analyses the Raman spectrum of the sample using the reference spectra 220 for the dyes associated with this kit. The computer 214 can then output the targets that are deemed present in the sample based upon whether the dye associated with this target has been identified. Accordingly, in some sense, the Raman spectrum is sent to the computer but it may not be possible to decode the Raman spectrum into identified targets unless the correct reference spectra are used. Because the user has identified the kit used to generate the Raman spectrum and the memory has stored therein reference spectra obtained at the required concentrations of the dyes, the computer can decode the Raman spectrum and provide an interpretation of the results, i.e. a list of all targets with detected and undetected stated alongside. Because the dyes and reference spectra have been selected such that, when using those reference spectra, any one of the dyes can be detected in the presence of any other one of the dyes across a range of concentrations around the reference concentration, multiple targets can be detected by the system, even if the targets are not present at the reference concentration. Without knowledge of the keys, i.e. reference spectra, to use to decode the Raman spectrum, the system may not be able to provide the technical operation of identifying targets in the sample.
The reference spectra are obtained from calibration plates comprising the dyes at the reference concentration. The user or a service engineer uses the Raman spectrometer 212 to obtain a Raman spectrum from each plate and these spectra are stored as reference in memory 217. These reference spectra may be updated at regular intervals to take into the performance of the Raman spectrometer.
As an alternative to the above, a solid assay may be performed using an array of dye-ligand spots bound on a SERRS active substrate, such as Klarite®. In this case a suitable set of dyes may be selected in order to provide surface enhancement from Klarite.
Use of a set of dyes selected in the manner described above ensures that there is a high level of confidence that a pathogen will be correctly identified as present or absent given that the exact concentration of the amplified DNA of the pathogen may not be exactly that of the desired reference concentration and, in particular, that the presence of the dye will not be masked by one or more other dyes that are also present.
It will be understood that the above multiplex assay technique is not limited to the identification of pathogens but other could be used to identify other organic matter.
The next section presents as an example, the selection of 10 dyes for a SERRS diagnostic application from a pool initially containing 15 dyes. The 15 dyes in the pool provided for the experiment are: ATTO488, ATTO520, BODIPY 530/550, BODIPY FL, BODIPY TMR-X, CY3.5, CY3, FAM, HEX, JOE, MAX, Rhodamine Green, TAMRA, TET and TYE563. The chemical structures for these dyes are shown in
Review of the dye variability data collected in the Second Step of the process indicated that ATTO488 and BODIPY 530/550 were unsuitable for this application, due to a larger-than-desired level of variability in the dye spectrum signal. They were therefore removed from the dye pool, leaving 13 dyes in the pool.
An extended Simplex Screening for False Positive risks was performed as described above (the Third Step), producing the results shown in
Each value in the table corresponds to the estimated rate of incorrectly detecting a dye that is absent (in other words, obtaining a False Positive). For example, looking at
The most significant False Positive risks identified are between HEX and TET, and Rhodamine Green and TET. Other False Positive risks (for example, between MAX and TAMRA) are considered to be at or below an acceptable level for this application. The identified risks can be avoided by removing TET from the dye pool, or by removing both HEX and Rhodamine Green from the dye pool. In this case the decision was made to remove TET because this retains more dyes in the dye pool for use in subsequent steps of the process.
The results of a Duplex Screening (the Fourth Step) are shown in
The duplex simulation also estimates the overall FPR (for all absent dyes) for each duplex when the minor dye is present at the corresponding LDC; these results are shown in
Examining
As there are more dyes in the pool than required for the application, we are able to avoid this pairing by removing (at least) one of these dyes. Examining
This leaves 11 dyes in the pool (the original 15, minus ATTO488, BODIPY 530/550, TET and MAX), whereas only 10 dyes are required for the application. The values in
Below is an example of a dye set, including the dyes and the dye's reference concentration, according to one embodiment of the invention.
The dye sets presented above can be used to perform multiplex diagnostic assays as described above with reference to
A skilled person will appreciate that variations of the disclosed arrangements are possible without departing from the invention. For example, although the description of the method is based on the selection of 10 dye candidates among a dye pool containing 15 dye candidates, the nature of the dye candidates is not limited to these 15 dyes and could include other dyes such as for example ATTO550, DY549, TEX and Oregon Green. Accordingly the above description of the specific embodiment is made by way of example only and not for the purpose of limitation. It will be clear to the skilled person that minor modifications may be made without significant changes to the operation described.
| Number | Date | Country | Kind |
|---|---|---|---|
| 11250530.0 | May 2011 | EP | regional |
| 12163369.7 | Apr 2012 | EP | regional |
| 1207821.8 | May 2012 | GB | national |
This is a Continuation-in-Part of (i) application Ser. No. 14/115,229 filed Nov. 1, 2013, which is a U.S. National Stage application under 37 U.S.C. §371 of PCT/GB2012/000435 filed May 16, 2012, which claims the benefit and priority to GB 11250530.0 filed May 16, 2011 and (ii) application Ser. No. 14/389,915 filed Oct. 1, 2014, which is a U.S. National Stage application under 37 U.S.C. §371 of PCT/GB2013/050861 filed Apr. 2, 2013, which claims the benefit and priority to GB 1207821.8 filed May 4, 2012 and EP 12163369.7 filed Apr. 5, 2012. The disclosures of the prior applications are hereby incorporated by reference herein in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14115229 | Nov 2013 | US |
| Child | 15149959 | US | |
| Parent | 14389915 | Oct 2014 | US |
| Child | 14115229 | US |
