WO2014170684A1

WO2014170684A1 - Nucleic acid analysis by sers and/or serrs

Info

Publication number: WO2014170684A1
Application number: PCT/GB2014/051207
Authority: WO
Inventors: Julie Green; William Ewen Smith
Original assignee: Renishaw Diagnostics Limited
Priority date: 2013-04-18
Filing date: 2014-04-17
Publication date: 2014-10-23
Also published as: GB201307013D0

Abstract

This invention concerns a method for detection of a targeted nucleic acid sequence in a sample. The method comprises, carrying out steps to: a) hybridise dye-labelled oligonucleotide probes to targeted nucleic acid sequences, if present in the sample, to form hybridised dye-labelled oligonucleotide probes; b) isolate hybridised dye-labelled oligonucleotide probes from unhybridised dye-labelled oligonucleotide probes; c) add additional oligonucleotides to dye-labelled oligonucleotide probes isolated in step (b); and d) detect dye-labelled oligonucleotide probes, to which the additional oligonucleotides have been added, with surface-enhanced resonance Raman spectroscopy(SERRS) or surface-enhanced Raman spectroscopy (SERS).

Description

NUCLEIC ACID ANALYSIS BY SERS AND/OR SERRS

This invention relates to a method for the detection of a targeted nucleic acid sequence such as may be present in a sample, for example from a patient. In particular, the invention disclosed herein relates to methods, kits, uses and reagents which may improve the performance of a SERRS or SERS assay for detecting dye labelled nucleic acid.

It is known to use Raman spectroscopy to identify a molecule in situ in a sample . However, Raman spectroscopy used in its basic form often lacks the sensitivity to identify molecules, particularly when attempting to detect multiple analytes simultaneously in solution in a single interrogation. To enhance the Raman signal, surface enhanced resonance Raman scattering (SERRS) may be used. SERRS uses the principal that the molecule to be identified is adsorbed on an active surface and contains a chromophore having an electronic transition with a frequency near to (preferably within 150nm) of the laser wavelength used to excite the plasmon on the enhancing substrate .

For a biological sample, to provide a sufficiently distinct chromophore for each type of molecule to be identified, the sample may be treated to attach different dyes to each type of molecule to be identified (e.g. different types of oligonucleotides). Examples of such techniques are described in WO09/022125 and US2006246460, which are incorporated herein by reference. An aim of the present invention is to provide improvements in the detection of specific nucleic acid sequences.

According to a first aspect of the invention, there is provided a method for detection of a targeted nucleic acid sequence in a sample comprising, carrying out steps to: a) hybridise dye-labelled oligonucleotide probes to targeted nucleic acid sequences, if present in the sample, to form hybridised dye-labelled oligonucleotide probes;

b) isolate hybridised dye-labelled oligonucleotide probes from unhybridised dye-labelled oligonucleotide probes; c) add additional oligonucleotides to dye-labelled oligonucleotide probes isolated in step (b); and

d) detect dye-labelled oligonucleotide probes, to which the additional oligonucleotides have been added, with surface-enhanced resonance Raman spectroscopy (SERRS) or surface-enhanced Raman spectroscopy (SERS).

Detection of a dye-labelled oligonucleotide probe may confirm the presence of the targeted nucleic acid sequence in the sample. The invention may improve the performance over the assay described in WO9705280.

Additional oligonucleotides

The additional oligonucleotides may be non-labelled; may not be dye-labelled; or may be dye-labelled with a different dye to the dye-labelled oligonucleotide probe. In an embodiment where multiple dye-labelled oligonucleotide probes are used, for example to target two or more different sequences, the additional oligonucleotide may be dye- labelled with a different dye relative to all the dye-labelled oligonucleotide probes. The additional oligonucleotides may be dye-labelled with a different dye relative to any dye-labelled oligonucleotide probe(s) targeting a nucleic acid sequence in the sample. The additional oligonucleotides may be arranged to not hybridise with the dye-labelled oligonucleotide probe under stringent conditions. In an embodiment where two or more, or a plurality of nucleic acid sequences are targeted for detection in a sample, the additional oligonucleotides may be arranged to not hybridise with any one of the dye-labelled oligonucleotide probes present, under stringent conditions. The additional oligonucleotides may be arranged to not hybridise with any target nucleic acid, and/or complementary sequences of the target nucleic acid, under stringent conditions. The additional oligonucleotides may be arranged to not hybridise with the target nucleic acid, and/or complementary sequences of the target nucleic acid, under stringent conditions. In an embodiment where two or more, or a plurality of nucleic acid sequences are targeted for detection in a sample, the additional oligonucleotides may be arranged to not hybridise with any one of the targeted nucleic acid sequences, or complementary sequences of the targeted nucleic acid, under stringent conditions. In an embodiment where two or more, or a plurality of nucleic acid sequences are targeted for detection in a sample, the additional oligonucleotides may be dye-labelled with a different dye to all dye-labelled oligonucleotide probes in the SE(R)RS reaction.

The additional oligonucleotides may comprise or consist of random nucleotide sequence. The additional oligonucleotides may comprise or consist of any nucleotide sequence. The additional oligonucleotides may comprise or consist of any nucleotide sequence that is not capable of hybridising with the target nucleic acid, or a complementary sequence of the target nucleic acid, under stringent conditions. The additional oligonucleotides may be non-labelled. The additional oligonucleotides may not be dye-labelled. The oligonucleotides may be dye-labelled with a different dye relative to the dye-labelled oligonucleotide probe. The additional oligonucleotides may comprise or consist of non-coding sequence . The additional oligonucleotides may not be complementary to the targeted nucleic acid sequence, or labelled oligonucleotide probe.

The additional oligonucleotides may comprise or consist of a series of a single type of nucleotide (e.g. A, T, G, C, or U; or variants or analogues thereof). The additional oligonucleotides may comprise or consist of a nucleic acid sequence selected from any of the group comprising poly guanine; poly cytosine; poly adenine; poly thymine; poly uracil; and analogues thereof; or combinations thereof. The additional oligonucleotides may comprise or consist of a series of alternating nucleotides. The additional oligonucleotides may comprise or consist of a l Omer, 20mer, 30mer, 40mer, 50mer, 80mer, or l OOmer of poly adenine. The additional oligonucleotides may comprise or consist of a l Omer, 20mer, 30mer, 40mer, 50mer, 80mer, or l OOmer of poly guanine.

The additional oligonucleotides may comprise or consist of a nucleic acid selected from any one of the group comprising DNA, RNA, and a nucleic acid analogue, such as PNA or LNA; or combinations thereof. The additional oligonucleotides may comprise or consist of DNA. The additional oligonucleotides may be at least about 8 nucleotides in length. The additional oligonucleotides may be at least about 10 nucleotides in length. The additional oligonucleotides may be at least about 12 nucleotides in length. The additional oligonucleotides may be at least about 15 nucleotides in length. The additional oligonucleotides may be at least about 20 nucleotides in length. The additional oligonucleotides may be at least about 30 nucleotides in length. The additional oligonucleotides may be about 20 nucleotides in length. The additional oligonucleotides may be no more than about 15 nucleotides in length. The additional oligonucleotides may be no more than about 20 nucleotides in length. The additional oligonucleotides may be no more than about 30 nucleotides in length. The additional oligonucleotides may be no more than about 50 nucleotides in length. The additional oligonucleotides may be no more than about 80 nucleotides in length. The additional oligonucleotides may be no more than about 100 nucleotides in length. The additional oligonucleotides may be no more than about 120 nucleotides in length. The additional oligonucleotides may be between about 8 and about 25 nucleotides in length. The additional oligonucleotides may be between about 8 and about 35 nucleotides in length. The additional oligonucleotides may be between about 8 and about 50 nucleotides in length. The additional oligonucleotides may be between about 8 and about 80 nucleotides in length. The additional oligonucleotides may be between about 8 and about 100 nucleotides in length. The additional oligonucleotides may be between about 8 and about 120 nucleotides in length. The additional oligonucleotides may be between about 15 and about 50 nucleotides in length. The additional oligonucleotides may be between about 15 and about 100 nucleotides in length. The additional oligonucleotides may be a mixture of different lengths, for example, the above mentioned lengths may refer to the average length in a population of additional oligonucleotides. The additional oligonucleotides may be substantially similar in length, or equal in length, to the dye-labelled oligonucleotide probe .

The additional oligonucleotides may be added to a concentration of up to 50ng/ml. The additional oligonucleotides may be added to a concentration of up to 30ng/ml. The additional oligonucleotides may be added to a concentration of up to 25ng/ml. The additional oligonucleotides may be added to a concentration of up to 15ng/ml. The additional oligonucleotides may be added to a concentration of up to 5ng/ml. The additional oligonucleotides may be added to a concentration of up to 2.5ng/ml. The additional oligonucleotides may be added to a concentration of between lng/ml and about 50ng/ml. The additional oligonucleotides may be added to a concentration of between about lng/ml and about 30ng/ml. The additional oligonucleotides may be added to a concentration of between about lng/ml and about 25ng/ml. In an embodiment where the additional oligonucleotides are between about 8 and about 100 nucleotides in length, the additional oligonucleotides may be added to a concentration of up to 5ng/ml. In an embodiment where the additional oligonucleotides are between about 8 and about 100 nucleotides in length, the additional oligonucleotides may be added to a concentration of up to 15ng/ml. In an embodiment where the additional oligonucleotides are between about 8 and about 100 nucleotides in length, the additional oligonucleotides may be added to a concentration of up to 25ng/ml. In an embodiment where the additional oligonucleotides are between about 8 and about 100 nucleotides in length, the additional oligonucleotides may be added to a concentration of up to 35ng/ml. In an embodiment where the additional oligonucleotides are no more than about 50 or 100 nucleotides in length, the additional oligonucleotides may be added to a concentration of up to 5ng/ml. In an embodiment where the additional oligonucleotides are no more than about 20 or 30 nucleotides in length, the additional oligonucleotides may be added to a concentration of up to 35ng/ml. In an embodiment where the additional oligonucleotides are no more than about 20 or 30 nucleotides in length, the additional oligonucleotides may be added to a concentration of up to 25ng/ml. In an embodiment where the additional oligonucleotides are no more than about 20 or 30 nucleotides in length, the additional oligonucleotides may be added to a concentration of up to 15ng/ml.

The additional oligonucleotides may be double and/or single stranded nucleic acid. The additional oligonucleotides may be triple stranded nucleic acid. In one embodiment the additional oligonucleotides may be single stranded nucleic acid.

The additional oligonucleotides may be unmodified but can be labelled with an additional label or be modified with a group such as biotin or another group to aid extraction or incorporation into the reaction mixture in a specific separation step.

Dye-labelled oligonucleotide probe

The dye-labelled oligonucleotide probe may comprise a known/pre-determined sequence . The dye-labelled oligonucleotide probe may be complementary to the targeted nucleic acid sequence. The dye-labelled oligonucleotide probe may be 100% complementary to the targeted nucleic acid sequence. The dye-labelled oligonucleotide probe may be at least about 95%, or at least about 90% complementary to the targeted nucleic acid sequence. The dye-labelled oligonucleotide probe may be at least about 80% complementary to the targeted nucleic acid sequence . The dye-labelled oligonucleotide probe may be complementary to the targeted nucleic acid sequence along the whole length of the probe. The dye- labelled oligonucleotide probe may be complementary to the targeted nucleic acid sequence along a length of at least about 8 consecutive nucleotides of the probe . The dye-labelled oligonucleotide probe may be complementary to the targeted nucleic acid sequence along a length of at least about 10 consecutive nucleotides of the probe . The dye-labelled oligonucleotide probe may be complementary to the targeted nucleic acid sequence along a length of at least about 15 consecutive nucleotides of the probe . The dye-labelled oligonucleotide probe may be complementary to the targeted nucleic acid sequence along a length of at least about 18 consecutive nucleotides of the probe . The dye-labelled oligonucleotide probe may be sufficiently complementary to the targeted nucleic acid sequence to be able to hybridise under stringent conditions. The dye- labelled oligonucleotide probe may hybridise to target nucleic acid under stringent conditions. The dye-labelled oligonucleotide probe may comprise or consist of nucleic acid selected from any one of the group comprising DNA, RNA, and a nucleic acid analogue, such as PNA or LNA; or combinations thereof. The dye-labelled oligonucleotide probe may comprise or consist of DNA. The dye-labelled oligonucleotide probe may be at least about 8 nucleotides in length. The dye-labelled oligonucleotide probe may be at least about 10 nucleotides in length. The dye-labelled oligonucleotide probe may be at least about 12 nucleotides in length. The dye-labelled oligonucleotide probe may be at least about 15 nucleotides in length. The dye-labelled oligonucleotide probe may be about 20 nucleotides in length. The dye-labelled oligonucleotide probe may be no more than about 15 nucleotides in length. The dye- labelled oligonucleotide probe may be no more than about 20 nucleotides in length. The dye-labelled oligonucleotide probe may be no more than about 30 nucleotides in length. The dye-labelled oligonucleotide probe may be no more than about 40 nucleotides in length. The dye-labelled oligonucleotide probe may be between about 8 and about 35 nucleotides in length. The dye-labelled oligonucleotide probe may be between about 8 and about 30 nucleotides in length. The dye-labelled oligonucleotide probe may be between about 8 and about 25 nucleotides in length. The dye-labelled oligonucleotide probe may be substantially similar in length, or equal in length, to the additional oligonucleotides. The dye-labelled oligonucleotide probe may further comprise an affinity tag. Dye label

The dye label may comprise any molecule which has surface enhanced Raman scattering (SERS) activity, or surface enhanced resonance Raman scattering (SERRS) activity.

The dye label may comprise benzotriazole monoazo dyes. The dye label may comprise fluorophores and/or chromophores. In an embodiment where SERRS is used for detection, the dye label may comprise a chromophore.

Examples of suitable SE(R)RS-active dye labels include fluorescein dyes, such as 5- (and 6-)carboxy-4',5 '-dichloro-2',7'-dimethoxy fluorescein, 5 -carboxy-2',4',5 ',7'- tetrachlorofluorescein and 5 -carboxyfluorescein; rhodamine dyes such as 5- (and 6- )carboxy rhodamine, 6-carboxytetramethyl rhodamine and 6-carboxyrhodamine X; phthalocyanines such as methyl, nitrosyl, sulphonyl and amino phthalocyanines; azo dyes, such as those listed in C H Munro et al, Analyst ( 1995), 120, p993 ; azomethines; cyanines and xanthines such as the methyl, nitro, sulphano and amino derivatives; and succinylfluoresceins. The dye-label may be substituted or un-substituted as required. The dye may be a positively charged dye .

Suitable dye labels may be selected from any one of the molecules shown in figure 9.

Suitable dye labels may be selected from any one of the group comprising TAMRA, Cy3, Cy3.5, FAM, ATTO520, ATTO 448, BODIPY FL, BODIPY 530/550, BODIPY TMR-X, HEX, JOE, TET, Rhodamine Green, Oregon Green 5 14, and TYE (of Integrated DNA Technologies Inc. sold under licence from Thermo Fisher Scientific (Milwaukee) LLC); or combinations thereof. Suitable dye labels may be TAMRA and/or Cy3.5.

Two or more, or a plurality of different dye labels may be used to differentiate between different target nucleic acid sequences.

Amplification The targeted nucleic acid may be amplified prior to hybridising the dye-labelled oligonucleotide probe . The sample may be provided with pre-amplified targeted nucleic acid. The amplification of targeted nucleic acid may comprise or consist of polymerase amplification. The amplification of target nucleic acid may comprise or consist of PCR. The PCR may use a pair of primers. One or both primer of the primer pair may be affinity tagged, thereby providing affinity tagged PCR product.

The targeted nucleic acid sequence may be affinity tagged. The affinity tag may comprise biotin.

Washing and Disassociation

The method may further comprise disassociating the hybridised dye-labelled oligonucleotide probe from the target nucleic acid sequence. The disassociation may be after the removal of unhybridised dye-labelled oligonucleotide probe . Disassociation may be by heating to melt double stranded nucleic acid to form single stranded nucleic acid. Disassociation may be by degradation of the target nucleic acid.

The hybridised dye-labelled oligonucleotide probe may be isolated from unhybridised dye-labelled oligonucleotide probe by washing. The unhybridised dye-labelled oligonucleotide probe may be removed by washing. Washing may comprise binding the targeted nucleic acid, having dye-labelled oligonucleotide probe hybridised thereon, to a solid support prior to washing away any unhybridised dye-labelled oligonucleotide probe. Any non-bound nucleic acid and/or other reagents from previous steps may be washed away in the wash step. A wash buffer may be used to wash away any unhybridised dye-labelled oligonucleotide probe .

The solid support for the wash may be a bead, such as a magnetic bead. The solid support may be a streptavidin coated bead. The binding may be by affinity tag binding. For example, binding to the solid support may be by biotin-avidin association. The binding may be by binding of affinity tagged targeted nucleic acid sequence. The affinity tag may comprise biotin.

The hybridised dye-labelled oligonucleotide probe may be released from the solid support after washing by disassociation from the targeted nucleic acid. Disassociation may be by heating to melt double stranded nucleic acid to form single stranded nucleic acid. Disassociation may be by degradation of the target nucleic acid. The targeted nucleic acid may remain bound to the solid support, or may be degraded. The targeted nucleic acid may be removed prior to the SE(R)RS detection.

SE(R)RS reagents may be added to the washed dye-labelled nucleic acid. Sample The sample may comprise a bodily fluid sample. In another embodiment, the sample may comprise an environmental sample, such as a water, air, or soil sample. The sample may comprise a food or beverage sample. The sample may comprise a cell culture sample. The sample may comprise a sample of pre-extracted nucleic acid. The sample may consist of nucleic acid and a solute.

Where the sample is a bodily fluid sample, it may be from a mammal. The mammal may be human. The sample may comprise a blood or blood plasma sample. The sample may be selected from any of the group comprising blood; blood plasma; mucous; urine; faeces; cerebrospinal fluid; tissue, such as organ tissue; lung aspirate; or combinations thereof.

Nucleic acid (in the sample)

The nucleic acid may comprise or consist of DNA or RNA. The nucleic acid may comprise a mixture of DNA and RNA. The nucleic acid may comprise genomic nucleic acid. The nucleic acid may comprise viral RNA; mRNA; ncRNA; small RNA; and siRNA; or combinations thereof. The nucleic acid may comprise mitochondrial nucleic acid. The nucleic acid may comprise or consist of chromosomal and/or non- chromosomal DNA.

The nucleic acid in the sample may comprise a mixture of mammalian and non- mammalian nucleic acid. The nucleic acid in the sample may comprise a mixture of mammalian and microbial nucleic acid. The nucleic acid in the sample may comprise a mixture of mammalian and bacterial and/or viral nucleic acid. The nucleic acid in the sample may comprise a mixture of mammalian and fungal nucleic acid. The nucleic acid in the sample may comprise a mixture of mammalian and pathogen nucleic acid. The nucleic acid in the sample may comprise a mixture of species and/or strains.

The nucleic acid may be extracted from the sample. For example, the nucleic acid in the sample may be purified or partially purified prior to hybridisation and/or amplification. The extraction of nucleic acid may be carried out by the skilled person by standard laboratory techniques.

Target nucleic acid sequence

The targeted nucleic acid sequence may comprise a species and/or strain specific sequence. The targeted nucleic acid sequence may comprise a pathogen's nucleic acid sequence. The targeted nucleic acid sequence may comprise microbial nucleic acid sequence. The targeted nucleic acid sequence may comprise fungal nucleic acid sequence. The targeted nucleic acid sequence may comprise nucleic acid sequence selected from any of the group comprising bacterial nucleic acid sequence; viral nucleic acid sequence; parasitic nucleic acid sequence; protozoan nucleic acid sequence; and fungal nucleic acid sequence; or combinations thereof. The targeted nucleic acid sequence may comprise a cell type and/or cell state specific sequence.

SERS/SERRS

Detecting the dye-labelled target nucleic acid using SERRS or SERS may comprise adding SERRS or SERS reagents to the dye-labelled target nucleic acid. The SE(R)RS surface may be provided by a naked metal or may comprise a metal oxide layer on a metal surface. SERS or SERRS reagents may comprise metallic nanoparticles. SERS or SERRS reagents may comprise silver, such as silver colloid. SERS or SERRS reagents may comprise gold, such as gold colloid. SERS or SERRS reagents may comprise silver and gold, or colloids thereof.

Where the SE(R)RS reagent is colloidal, the colloid particles may be aggregated. Suitable aggregating agents may be provided, such as acids (e.g., HN03 or ascorbic acid), polyamines (e.g., polylysine, spermine, spermidine, 1 ,4-diaminopiperazine, diethylenetriamine, N-(2-aminoethyl)- l ,3-propanediamine, triethylenetetramine and tetraethylenepentamine) and inorganic activating ions such as C1-, I-, Na+ or Mg2+. The colloid particles may be of any size so long as they give rise to a SE(R)RS effect. The colloid particles may be about 4-80 nm in diameter. The colloid particles may be about 20-36 nm in diameter. The colloid particles may be about 50- 100 nm in diameter. The skilled person will understand that the choice of metal may influence the size requirements of the colloid.

The SE(R)RS reagents may comprise silver or gold colloid particles, which are substantially hexagonal in shape. The SE(R)RS reagents may comprise silver or gold colloid particles, which are substantially rod shaped or triangular shape, or they may comprise hollow nanospheres.

The SE(R)RS surface may comprise a surface modifier. The surface modifier may comprise an organic coating such as citrate or a suitable polymer, such as polylysine or polyphenol, to increase its sorptive capacity.

The use of a polyamine such as poly(L-lysine) may be provided for enhancing sensitivity of the detection. This can help to control aggregation of colloid if present, and to enhance the interaction between the dye-labelled nucleic acid and the SE(R)RS surface. The polyamine may be a short-chain aliphatic polyamine such as spermine, spermidine, 1 ,4-diaminopiperazine, diethylenetriamine, N-(2-aminoethyl)- l ,3- propanediamine, triethylenetetramine and tetraethylenepentamine. The polyamine may be introduced in the form of an acid salt such as its hydrochloride . The polyamine may be provided at a concentration of between about 0.001 mol and about 1 mol. The polyamine may be provided at a concentration of between about 0.005 mol and about 0. 1 mol. The polyamine may be provided at a concentration of about 0.01 mol. Advantageously, when spermine or a similar agent is added, this alters the properties of the dye-labelled target nucleic acid and additional oligonucleotide, altering both charge and dielectric constant. The action of agents such as spermine and free nucleic acids may have beneficial impact on the formation of aggregates. Interaction of the dye-labelled oligonucleotide with the polyamine, such as spermine, may be kinetically favoured in solution over interaction on crowded reactions on the surface of the nanoparticle.

The order of addition of SE(R)RS reagents may be the addition of the polyamine to the additional oligonucleotide followed by addition of suspended nanoparticles.

SERRS uses signal enhancement from the surface and from the dye and requires the use of a laser excitation which effectively activates the surface plasmon and is close in frequency to an electronic transition of the dye . If the dye frequency is not close to the laser frequency used to excite the plasmon efficiently, there may be some appreciable SERRS enhancement but the effect is mainly surface enhanced Raman scattering (SERS) without any additional enhancement from the dye. Although the enhancement is lower, the SERS enhancements of specific molecules are widely different so effective labelling can still be achieved and assays run using SERS active labels will also be effective but in general at lower sensitivity.

The signal from the suspended nanoparticles used in some assays can come from the labelled particles alone but in many practical assays it can be advantageous to allow some aggregation of the particles. This is known in the art to increase signals by modifying the plasmon frequency and creating interactions between particles which increase the SERRS signal. The most intense regions are often known as "hot spots". The formation of the labelled particles or the use of reagents such as spermine to create aggregation can alter the nanoparticle properties so that the stable colloidal suspension is broken and a dynamic process begins which leads to precipitation. Under these circumstances, the rate of aggregation and precipitation is controlled so that a stable signal is obtained over a longer period than the measurement period. This period depends on the assay. For example a single analysis may take 1 - 10 seconds but since there may be some manipulation time such as adding the reagents and adding the sample to the reader, stability of a few minutes is desirable . However, to read a 96 well plate may take 20-60 minutes so stability of more than one hour is desirable. Controlling the surface of the particles using additional oligonucleotides can reduce the rate at which the intensity of the SERRS or SERS signal reduces compared to a corresponding assay wherein the additional oligonucleotides have not been added. This property may be referred to as longevity. Further, the signal is usually taken from a volume of a nanoparticle suspension defined by the interrogation volume created by the excited laser and the collection optics. Since in any one aggregated suspension the size of the clusters formed and the intensity of signal obtained from each varies, in semi-quantitative or quantitative analysis it is usual to measure an average of a number of Raman events from each of a number of particle clusters. Controlled addition of DNA can aid control of aggregate size and reduce the noise created by particles of widely different activities. Thus as well as increasing signal intensity, the noise can be reduced producing lower detection limits.

The order of addition of the dye-labelled nucleic acid, polyamine and colloid may be significant since interaction of the dye-labelled nucleic acid with the polyamine may be kinetically favoured in solution over interaction on crowded reactions on the surface . The order of addition of reagents to give the lowest detection limits may be polyamine added to the dye-labelled oligonucleotide probe followed by addition of suspended nanoparticles, such as silver colloid.

Reverse transcription In an embodiment where the nucleic acid comprises RNA, the RNA may be transcribed to cDNA by reverse transcriptase . RNA may be transcribed to cDNA prior to amplification of the target nucleic acid.

Multiple targets

Additional target nucleic acid sequences in the sample may be targeted in the same SERRS or SERS detection. The method may comprise detecting, or attempting to detect, two or more different target nucleic acid sequences in the sample. The method may comprise detecting, or attempting to detect, a plurality of different target nucleic acid sequences in the sample. The method may comprise detecting, or attempting to detect, three or more different target nucleic acid sequences in the sample. The method may comprise detecting, or attempting to detect, four or more different target nucleic acid sequences in the sample. The method may comprise detecting, or attempting to detect, five or more, six or more, seven or more, or eight or more, different target nucleic acid sequences in the sample. The method may comprise detecting, or attempting to detect, up to 15, or up to 10 different target nucleic acid sequences in the sample. For example, a plurality of different species or strains may be targeted for detection in the sample, by targeting their respective identifying sequences or expression of nucleic acid. A different dye may be used for each of the different nucleic acid sequences targeted, thereby facilitating their differentiation.

Additional aspects

According to another aspect of the invention, there is provided a method of enhancing SERRS or SERS detection of dye-labelled oligonucleotide probe, comprising addition of oligonucleotide to the dye-labelled oligonucleotide probe prior to detection, wherein the oligonucleotide is:

non-labelled;

not dye-labelled; or

dye-labelled with a different dye to the dye-labelled oligonucleotide probe.

Enhancing SERRS or SERS detection comprises increasing intensity, signal to noise ratio and/or longevity of the SERRS or SERS signal. According to another aspect of the invention, there is provided the use of oligonucleotides in a solution or suspension comprising dye-labelled oligonucleotide probe to increase intensity, signal to noise ratio and/or longevity of the SERRS or SERS signal from the dye-labelled oligonucleotide probe,

wherein the oligonucleotides are:

non-labelled;

not dye-labelled; or

dye-labelled with a different dye to the dye-labelled oligonucleotide probe.

According to another aspect of the invention, there is provided a SERRS or SERS reagent composition for detection of dye-labelled oligonucleotide probe, the composition comprising:

a metal colloid; and

oligonucleotides, wherein the oligonucleotides are:

non-labelled;

not dye-labelled; or dye-labelled with a different dye to the dye-labelled oligonucleotide probe.

The SE(R)RS reagent composition may further comprise a polyamine, such as spermine.

According to another aspect of the invention, there is provided a kit for SERS or SERRS detection of a target nucleotide sequence in a sample comprising:

a dye-labelled oligonucleotide probe;

a metal colloid suspension; and

oligonucleotides, wherein the oligonucleotides are non-labelled; not dye- labelled; or dye labelled with a different dye to the dye-labelled oligonucleotide probe.

The dye-labelled oligonucleotide probe may be arranged to be substantially complementary to the target nucleotide sequence, or part thereof. The dye-labelled oligonucleotide probe may be arranged to hybridise to the target nucleotide sequence. The hybridisation may be under stringent conditions.

The oligonucleotide may not be a primer. The oligonucleotide may not be a primer arranged to be used for polymerase amplification of the target nucleotide sequence. In an embodiment where polymerase amplification of the target nucleotide sequence is required, the oligonucleotide may not be the same or substantially similar to a primer used in such polymerase amplification. In an embodiment where polymerase amplification of the target nucleotide sequence is required, the oligonucleotide may be arranged to be added at a different stage in the SERS or SERRS detection relative to the primer used in such polymerase amplification. The oligonucleotide may not be complementary to the target nucleotide sequence, or not complementary to a complementary sequence of the target nucleotide sequence. The oligonucleotide may not be arranged to hybridise with the target nucleotide sequence under stringent conditions, or not arranged to hybridise with a complementary sequence of the target nucleotide sequence under stringent conditions. The oligonucleotide may comprise the same sequence as the dye-labelled oligonucleotide probe, but does not comprise a dye- label, or comprises a dye-label which is different to the dye-label of the dye-labelled oligonucleotide probe. In an embodiment where multiple sequences of nucleic acid are targeted by different dye-labelled oligonucleotide probes, the oligonucleotide may comprise a dye-label which is different to the dye-label of all dye-labelled oligonucleotide probes.

The kit may comprise a plurality of dye-labelled oligonucleotide probes having different sequence and/or dye labels. The kit may further comprise polyamine, such as spermine. The kit may further comprise a primer pair for polymerase amplification of the target nucleic acid sequence. One or both of the primer pair may comprise an affinity tag, such as biotin. The kit may further comprise beads, such as magnetic streptavidin beads. It will be apparent to the skilled person, various other reagents and buffers may be provided in accordance with standard protocols for SE(R)RS, PCR amplification and/or nucleic acid extraction.

The kit may further comprise reverse transcriptase.

The kit may further comprise instructions. The instructions may comprise instructions to use the kit in accordance with the method of the invention herein. The instructions may provide instructions to add the oligonucleotides prior to the SE(R)S detection, and after any removal of unhybridised dye-labelled oligonucleotide probe, for example after a wash step.

According to another aspect of the invention, there is provided the use of additional oligonucleotides in a nanoparticle suspension containing dye-labelled oligonucleotide probe to alter an aggregate size distribution of clusters of the nanoparticles.

The use may be to reduce noise in a SERRS or SERS signal from the dye-labelled oligonucleotide probe.

The additional oligonucleotides may be non-labelled; may not be dye-labelled; or may be dye-labelled with a different dye to the dye-labelled oligonucleotide probe.

According to another aspect of the invention, there is provided a method of altering an aggregate size distribution of clusters of SERRS or SERS active nanoparticles in a suspension containing dye-labelled oligonucleotide probe, comprising adding oligonucleotides to the suspension. The amount of oligonucleotides added may be based upon an expected or measured cluster size in the suspension. According to another aspect of the invention, there is provided a method of diagnosis of a disease comprising the use of the method, the SERRS or SERS reagent composition, or the kit, according to the invention, to detect a diseased state .

According to another aspect of the invention, there is provided a method of analysing the status, progress or severity of a disease comprising the use of the method, the SERRS or SERS reagent composition, or the kit, according to the invention.

The disease state may be an infection or cancer. The infection may be a microbial infection, such as a bacterial infection or a viral infection. The infection may be a fungal infection. The disease state may be a genetic abnormality.

Where there is a method of analysing the status, progress or severity of a disease, the disease may be pre-diagnosed. The pre-diagnosis may be by the same methods, reagent compositions or kits according to the invention herein, or by alternative methods, reagent compositions or kits.

Additional or alternative embodiments

The following numbered paragraphs refer to additional or alternative aspects of the invention.

1. Use of unlabelled DNA and/or RNA in a solution or suspension containing dye labelled oligonucleotides to increase intensity, signal to noise ratio and/or longevity of the SERRS or SERS signal from the dye labelled oligonucleotides.

2. In a solution or suspension containing oligonucleotides labelled with a first dye, use of DNA and/or RNA labelled with at least one different label to the first dye to increase intensity, signal to noise ratio and/or longevity of the SERRS or SERS signal from the oligonucleotides labelled with the first dye. 3. A use according to paragraph 2, wherein the different label is one or more further dyes.

4. A use according to paragraph 2, wherein the different label is a label, such as biotin, to aid the separation of the oligonucleotides, DNA and/or RNA from a mixture.

5. A use according to any one of the preceding paragraphs, wherein the unlabelled DNA and/or RNA or DNA and/or RNA labelled with at least one different label has a concentration in the solution or suspension of l-3ng/mL. 6. A use according to any one of the preceding paragraphs, wherein the solution or suspension comprises an aggregating agent, such as spermine.

7. A use according to paragraph 6, wherein the solution has been formed by adding nanoparticles to the solution or suspension after the aggregating agent.

8. A detection method using SERRS or SERS detection in which DNA or RNA is added to increase the intensity, signal to noise ratio and/or longevity of the SERRS or SERS signal. 9. A SERRS or SERS assay for detecting dye labelled oligonucleotides comprising the step of adding to a solution or suspension containing the dye labelled oligonucleotides, unlabelled DNA and/or RNA to increase the intensity, signal to noise ratio and/or longevity of the SERRS or SERS signal from the dye labelled oligonucleotides. 10. A SERRS or SERS assay for detecting dye labelled oligonucleotides comprising the step of adding to a solution or suspension containing oligonucleotides labelled with a first dye, DNA and/or RNA labelled with at least one different label to the first dye to increase an intensity, signal to noise ratio and/or longevity of the SERRS or SERS signal from the oligonucleotides labelled with the first dye.

11. A SERRS or SERS assay for detecting dye labelled oligonucleotides comprising the steps of processing a solution of oligonucleotides to attach a first dye to target oligonucleotides and adjusting a concentration in the solution of unlabelled oligonucleotides and/or oligonucleotides labelled with a different label to the first dye in order to increase an intensity, signal to noise ratio and/or longevity of the SERRS or

SERS signal from the dye labelled target oligonucleotides. A SERRS or SERS assay using metal nanoparticles, preferably of silver or gold which are SERRS or SERS active, in which DNA or RNA is added to increase the intensity, signal to noise ratio and/or longevity of the SERRS or SERS signal. A SERRS or SERS assay according to paragraph 12 wherein an aggregating agent, such as spermine, is added. A SERRS or SERS assay according to paragraph 12 or 13 in which the added DNA is in a concentration range of l-3ng/mL. A kit for an assay system using SERRS or SERS detection comprising a buffer or other reagent into which a DNA or RNA has been added to increase the intensity, signal to noise ratio or longevity of the SERRS or SERS signal. A kit for use in a SERRS or SERS assay for detecting dye labelled oligonucleotides comprising a buffer or other reagent for increasing an intensity, signal to noise ratio or longevity of a SERRS or SERS signal from the oligonucleotides labelled with the first dye, the buffer or other reagent comprising unlabelled DNA and/or RNA and/or DNA and/or RNA labelled with at least one different label to the dye labelled oligonucleotide. A procedure for increasing an intensity, signal to noise ratio and/or longevity of a SERS or SERS signal in an assay comprising adding reagents in the order of an aggregating agent, such as spermine, to DNA or RNA followed by suspended nanoparticles in water, buffer or other medium. A step in an assay system using SERRS or SERS detection in which a labelled oligonucleotide probe is separated from a mixture such as PCR product in the presence of excess DNA or RNA in a manner in which the excess is carried through to a SERRS or SERS analysis procedure. A step in an assay system using SERRS or SERS detection in which DNA or RNA other than primers or probes or other sequences required in an amplification procedure is added to the assay and carried through to the SERRS or SERS detection step to increase the intensity, signal to noise ratio or longevity of the SERRS or SERS signal. Use of DNA and/or RNA in a nanoparticle suspension containing dye labelled oligonucleotides to alter an aggregate size distribution of clusters of the nanoparticles. 21. A use according to paragraph 20, wherein the DNA and/or RNA are used to alter an aggregate size distribution of clusters of the nanoparticles to reduce noise in a SERRS or SERS signal from the dye labelled oligonucleotides.

22. A use according to paragraph 20 wherein the DNA and/or RNA is unlabelled DNA and/or RNA.

23. A use according to paragraph 20 wherein the DNA and/or RNA is DNA and/or RNA labelled with a different dye to the dye labelled oligonucleotides.

24. A method of altering an aggregate size distribution of clusters of SERRS or SERS active nanoparticles in a suspension containing oligonucleotides labelled with a first dye, comprising adding DNA and/or RNA to the suspension.

25. A method according to paragraph 24, wherein an amount of DNA and/or RNA that is added is based upon an expected or measured cluster size in the suspension.

The term "hybridise under stringent conditions" may be understood by the skilled person to mean that two nucleic acid fragments hybridise with one another under standardized hybridisation conditions as described for example in Sambrook et al., "Expression of cloned genes in E. coli" in Molecular Cloning: A laboratory manual ( 1989), Cold Spring Harbor Laboratory Press, New York, USA. Such conditions are for example hybridisation in 6.0 > SSC at about 45° C. followed by a washing step with 2 x SSC at 50° C. In order to select the stringency the salt concentration in the washing step can for example be chosen between 2.0 > SSC at 50° C. for low stringency and 0.2 x SSC at 50° C. for high stringency. In addition the temperature of the washing step can be varied between room temperature, ca. 22° C, for low stringency and 65° C. for high stringency.

The skilled person will understand that optional features of one embodiment or aspect of the invention may be applicable, where appropriate, to other embodiments or aspects of the invention. Embodiments of the invention will now be described in more detail, by way of example only, with reference to the accompanying drawings. Figure 1 shows steps in a multiplex assay;

Figure 2 is a graph showing signal intensity of the main dye peak plotted over time for an oligonucleotide labelled with the dye TAMRA;

Figure 3 shows the effect of additional unlabelled aspergillus primer and Candida primer intended for PCR on the SERRS signal intensity of the corresponding dye-labelled probe.

Figure 4 shows enhancement of SERRS peak height after the addition of various concentrations of 20mer poly A (Figure 5A), T (Figure 5B), G (Figure 5C) or C (Figure 5D).

Figure 5 shows enhancement of SERRS peak height after the addition of various concentrations of poly adenine in the form of a l Omer (Figure 6A), 20mer (Figure 6B), and 30mer (Figure 6C).

Figure 6 shows enhancement of SERRS peak height after the addition of various concentrations of poly guanine in the form of a l Omer (Figure 7A), 20mer (Figure 7B), and 30mer (Figure 7C).

Figure 7 shows enhancement of SERRS peak height after the addition of various concentrations of poly adenine in the form of a 50mer (Figure 7A), and l OOmer (Figure 7B), using two different methods of reagent addition.

Figure 8 shows SERRS enhancement by adding a randomly chosen primer sequence. C. glabrata nucleic acid was targeted with ATTO520 dye-label probe using two different addition methods

Figure 9a-n shows structures of dyes that may be used to dye-label the nucleic acid.

Referring to Figure 1 , in one form, an assay is constructed to detect disease states by attaching the dye to an oligonucleotide built to complement a target nucleotide sequence (target sequence) known to be unique to the causative organism(s) . It is then introduced to a sample containing DNA fragments. If the target sequence(s) are present in the sample the dye labelled oligonucleotide hybridises to it. By adding an oligonucleotide with biotin which also recognises the target sequence it is possible to separate the DNA complex containing the target sequence and the dye using streptavidin coated magnetic beads which attach the complex via the biotin / streptavidin interaction. The dye sequence is then released and attached to silver nanoparticles which, preferably when aggregated, act as the SERRS substrate for the dye giving very strong signals from an aqueous environment. It is a characteristic of SERRS that the spectrum consists of a sharp set of lines almost always exclusively from the dye. This is because the Raman scattering surface enhancement factor for the dye is very high compared to the enhancement factor from the rest of the oligonucleotide so other signals are very weak in comparison. These sharp lines are characteristic of the dye giving in situ identification and the sharp nature of the lines mean that mixtures of dyes can be identified without separation. This enables the detection of multiple labels in one vessel.

There are various ways in which this assay can be configured. For example, a suspension of gold rather than silver nanoparticles can be used as can nanoparticles of other materials with suitable surface plasmons. In addition different shaped nanoparticles such as rods or triangles and hollow nanospheres designed to give good surface enhanced Raman scattering can be used as can solid state surfaces such as designed surfaces such as Klarite, immobilised nanoparticles or rough deposited metal layers. Silver nanoparticles have been chosen as the lead example because of the high sensitivity and reproducibility currently being achieved with them.

A feature of effective SERRS is that the label to be identified attaches strongly to the enhancing substrate. This is not only to achieve sensitivity, it is also to achieve stability and reproducibility which are lacking in some otherwise promising SERRS and SERS assays.

WO9705280 discloses a method whereby an assay can be achieved which is sufficiently sensitive and reproducible to enable practical development of a commercially viable product in this assay, amines such as spermine are used as aggregating agents to create the most effective substrate. Figure 2 illustrates how the additional DNA affects the intensity of a SERRS signal from TAMRA over time. The dotted line is for an assay wherein no additional DNA has been added, the solid line for an assay wherein 1.25ng/mL of additional DNA has been added and the dashed line for an assay wherein 2.5ng/mL of additional DNA has been added. This data involved C. albicans target sequence with TAMRA dye-labelled probe. The additional DNA is double stranded (herring sperm).

When ssDNA is added to dye labelled probe in the form of unlabelled primer for PCR and the matching TAMRA probe is added, the increased signal is again observed as shown in figure 3.

Example 1 - Determining the effect of adding oligonucleotides to the SERRS reaction

Materials and Method

Each oligonucleotide (poly A, T, G, C) was ordered as a string of 20 repetitive bases e.g. ccc ccc ccc ccc ccc ccc cc. The effect of adding extra oligonucleotides to the SERRS reaction was tested by adding oligonucleotides after spermine addition. The following concentrations were tested: 50ng/ml, 25ng/ml, 12.5ng/ml, 5ng/ml and 1.25ng/ml.

C. albicans targeted primer and a TAMRA dye-label was used to test the effect. Note, 2.5 μ1 of TAMRA labelled C. albicans primer at 1.7xl 0^"3 M was spotted into wells before the addition of water, spermine, extra DNA and finally 120μ1 colloid.

Plates were left at room temperature for 5 minutes before being read (instrument was SA 1000, Renishaw).

The results are shown in figure 4. Discussion

This experiment shows enhancement of SERRS signal for all 20mer oligonucleotides of bases A, T, G and C. The greatest argumentation of SERRS signal was seen by addition of 'G' s (20 in a string) at 25ng/ml by about 9000 counts compared to normal SERRS reaction without additional DNA.

Example 2 - Determining the effect of adding different lengths of strings of adenines to the SERRS reaction.

Materials and Methods

Oligonucleotides with different lengths of adenines were provided. Specifically a string of 10, 20 and 30 adenines were provided.

All sequences were reconstituted in DEFC H₂0. Once reconstituted, all sequences were nanodropped. These oligonucleotides were tested at 50ng/ml, 25ng/ml, 12.5ng/ml, 5ng/ml, 2.5ng/ml and 1.25ng/ml. C. albicans target nucleic acid with TAMRA dye label was used in this experiment.

The results are shown in figure 5.

Discussion

This data indicates that the addition of a poly adenine string of l Omer, causes an increase in SERRS signal which is optimal at 12.5ng/ml with a 62.5% increase. Addition of 25ng/ml poly adenine string of 20mer causes a 77% SERRS enhancement. Addition of 12.5ng/ml poly adenine string of 30mer causes a 68% SERRS enhancement. Enhancement is compared to normal SERRS reactions where no extra DNA was added. Therefore, from this experiment it would appear that the addition of 25ng/ml poly adenine of 20mer is most effective at enhancing the SERRS signal.

Example 3 - Determining the effect of adding different lengths of strings of guanines to the SERRS reaction

The experiment of example 2 was repeated with poly guanine instead of poly adenine oligonucleotides. The results are shown in figure 6. Discussion

This data suggests that addition of extra DNA in the form of a string of 10 guanines results in an increase in the SERRS signal of 75%. Addition of a string of 20 guanines causes an 82% increase in SERRS signal whereas addition of a string of 30 guanines causes a 73% enhancement in SERRS signal compared to control reactions. Comparing this data to the previous example, overall additions of guanine oligonucleotides appears to be more effective than adenine oligonucleotides with best results from addition of 20mer guanine oligonucleotide.

Example 4 It was investigated whether additional length oligonucleotides of 50 and 100 bases could be capable of enhancing the SERRS signal. Figure 7 A shows the result of adding poly-A 50mer oligonucleotides to the SERRS reaction of C. albicans on TAMRA using two different addition methods. Figure 7B shows the result of adding poly-A l OOmer oligonucleotides to the SERRS reaction of C. glabrata on ATTO520 using the same two addition methods as follows.

Addition Method 1

2.5 μΐ of dye-labelled probe was pipetted into each of the wells. Water was then added to wells whereby the volume was dependent on the concentration of oligonucleotide being tested. 10 μΐ of spermine was then added. Oligonucleotide was then added to the reaction whereby the volume was again dependent on the concentration of oligonucleotide being tested. Finally, 120 μΐ of silver colloid was added to the wells of the detection plate . Addition Method 2

2.5 μΐ of dye-labelled probe was pipetted into each of the wells. Oligonucleotide and water were then added to wells whereby the volume of each constituent was determined by the concentration of the oligo being tested. Finally, 10 μΐ of spermine and 120 μΐ of silver colloid were added to the wells of the detection plate. The plate was then left for 5 minutes at room temperature before being analyzed on the SA- 1000 (Renishaw).

Discussion

The results show an enhancement of signal for concentrations of 5ng/ml of both 50mer and l OOmer oligonucleotides.

Example 5 - Adding an oligo chosen at random to the SERRS reaction As earlier work has indicated that a SERRS signal could be enhanced by the addition of all A, T, G or C oligomers, it was decided to test whether the addition of an oligonucleotide sequence chosen at random could drive similar enhancement in SERRS signal. A primer (ETEC hs l primer sequence: 5 '-GCA GTA AAA TGT GTT GTT CAT ATT TTC TG-3 ') from another experiment was chosen at random for this work. This primer was added to a SERRS reaction for the testing of C. glabrata target nucleic acid with an ATTO520 dye-labelled probe at 5ng/ml to 100 ng/ml using two methods of addition discussed in example 4. The concentration range was increased because it was observed for the all G's (20) oligo that the SERRS enhancement peaked at 25 ng/ml. It needed to be determined whether this SERRS enhancement would increase with increasing concentration of primer or if there would be a decrease in SERRS signal with increasing concentration.

Discussion

It was observed that adding the primer to the SERRS reaction of C. glabrata on ATTO520 resulted in good SERRS enhancement (Figure 8). An increase in SERRS signal by 194 % and 173 % was obtained when adding concentrations of 5 ng/ml and 12.5 ng/ml of primer respectively using the first method of addition. Using 25 ng/ml also produces an enhancement in SERRS signal by 92 % but this is a decrease from the optimal enhancement observed. Addition of 50 ng/ml doesn't produce any considerable effect of the SERRS signal in comparison to the control whereas adding 100 ng/ml of primer results in a decrease in SERRS signa by 39 %.

An increase in SERRS signal by 161 % and 203 % was obtained when adding concentrations of 5 ng/ml and 12.5 ng/ml of primer respectively using the second method of addition. Using 25 ng/ml also produces an enhancement in SERRS signal by 96 % but this is a decrease from the optimal enhancement observed. Addition of 50 ng/ml doesn't produce any considerable effect of the SERRS signal in comparison to the control whereas adding 100 ng/ml of primer results in a decrease in SERRS signal by 46 %.

These results are quite significant as the SERRS enhancement obtained when using the all A's (20) and all G's (20) is similar to the enhancement obtained when a completely random primer is used.

Claims

1. A method for detection of a targeted nucleic acid sequence in a sample comprising, carrying out steps to:

a) hybridise dye-labelled oligonucleotide probes to targeted nucleic acid sequences, if present in the sample, to form hybridised dye-labelled oligonucleotide probes;

b) isolate hybridised dye-labelled oligonucleotide probes from unhybridised dye-labelled oligonucleotide probes;

c) add additional oligonucleotides to dye-labelled oligonucleotide probes isolated in step (b); and

2. The method according to claim 1 , wherein the additional oligonucleotides are : non-labelled;

not dye-labelled; or

dye-labelled with a different dye to the dye-labelled oligonucleotide probe.

3. The method according to claim 1 or 2, wherein the additional oligonucleotides are not arranged to hybridise with any target nucleic acid, and/or complementary sequences thereof, under stringent conditions.

4. The method according to any preceding claim, wherein, two or more, or a plurality of nucleic acid sequences are targeted for detection in a sample, wherein the additional oligonucleotides are dye-labelled with a different dye to all dye-labelled oligonucleotide probes in the SE(R)RS reaction.

5. The method according to any preceding claim, wherein the additional oligonucleotides comprise or consist of random nucleotide sequence; and/or

wherein the additional oligonucleotides comprise or consist of non-coding sequence; and/or

wherein the additional oligonucleotides are not complementary to the targeted nucleic acid sequence, or dye-labelled oligonucleotide probe.

6. The method according to any preceding claim, wherein the additional oligonucleotides are added to a concentration of up to 50ng/ml.

7. The method according to any preceding claim, wherein the dye-labelled oligonucleotide probe comprises a known sequence.

8. The method according to any preceding claim, wherein the dye-labelled oligonucleotide probe is complementary to the targeted nucleic acid sequence .

9. The method according to any preceding claim, wherein the dye label comprises any molecule which has surface enhanced Raman scattering (SERS) activity, or surface enhanced resonance Raman scattering (SERRS) activity.

10. The method according to any preceding claim, further comprising disassociating the hybridised dye-labelled oligonucleotide probe from the target nucleic acid sequence after removal of unhybridised oligonucleotide probe.

1 1. The method according to any preceding claim, wherein the hybridised dye-labelled oligonucleotide probe is isolated by washing.

12. The method according to claim 1 1 , wherein the washing comprises binding the targeted nucleic acid, having dye-labelled oligonucleotide probe hybridised thereon, to a solid support prior to washing away any unhybridised dye-labelled oligonucleotide probe .

13. The method according to claim 12, wherein the hybridised dye-labelled oligonucleotide probe may be released from the solid support after washing by disassociation from the targeted nucleic acid.

14. The method according to claim 13, wherein SE(R)RS reagents are added to the disassociated dye-labelled oligonucleotide probe.

15. The method according to any preceding claim, wherein the sample comprises a bodily fluid sample; or wherein the sample comprises an environmental sample; or wherein the sample comprises a food or beverage sample; or

wherein the sample comprises a cell culture sample; or

wherein the sample comprises a sample of pre-extracted nucleic acid.

16. The method according to any preceding claim, wherein the nucleic acid in the sample comprises a mixture of mammalian and non-mammalian nucleic acid.

17. The method according to any preceding claim, wherein the targeted nucleic acid sequence comprises a species and/or strain specific sequence.

18. The method according to any preceding claim, wherein SERS or SERRS detection comprises the use of a reagent comprising metallic nanoparticles.

19. The method according to claim 18, wherein the SERS or SERRS reagent comprises silver and/or gold.

20. The method according to any preceding claim, wherein the SE(R)RS detection comprises a reagent comprising a polyamine; and optionally wherein the polyamine is spermine.

21. The method according to any preceding claim, wherein the nucleic acid comprises RNA, and the RNA is transcribed to cDNA by reverse transcriptase .

22. The method according to any preceding claim, wherein the method comprises detecting, or attempting to detect, two or more different target nucleic acid sequences in the sample.

23. A method of enhancing SERRS or SERS detection of dye-labelled oligonucleotide probe, comprising addition of oligonucleotide to the dye-labelled oligonucleotide probe prior to detection, wherein the oligonucleotide is:

non-labelled;

not dye-labelled; or

dye-labelled with a different dye to the dye-labelled oligonucleotide probe.

24. Use of oligonucleotides in a solution or suspension comprising dye-labelled oligonucleotide probe to increase intensity, signal to noise ratio and/or longevity of the SERRS or SERS signal from the dye-labelled oligonucleotide probe,

wherein the oligonucleotides are:

non-labelled;

not dye-labelled; or

dye-labelled with a different dye to the dye-labelled oligonucleotide probe.

25. A SERRS or SERS reagent composition for detection of dye-labelled oligonucleotide probe, the composition comprising:

a metal colloid; and

oligonucleotides, wherein the oligonucleotides are:

non-labelled;

not dye-labelled; or

dye-labelled with a different dye to the dye-labelled oligonucleotide probe.

26. The SERRS or SERS reagent composition according to claim 25, wherein the SE(R)RS reagent composition further comprises a polyamine.

27. A kit for SERS or SERRS detection of a target nucleotide sequence in a sample comprising:

a dye-labelled oligonucleotide probe;

a metal colloid suspension; and

oligonucleotides, wherein the oligonucleotides are non-labelled; not dye- labelled; or dye-labelled with a different dye to the dye-labelled oligonucleotide probe.

28. A method of diagnosis of a disease comprising the use of the method according to any of claims 1 to 22, the SERRS or SERS reagent composition according to claims 25 or 26, or the kit according to claim 26, to detect a diseased state .

29. A method of analysing the status, progress or severity of a disease comprising the use of the method according to any of claims 1 to 23, the SERRS or SERS reagent composition according to claims 25 or 26, or the kit according to claim 27.

30. The method according to claim 28 or claim 29, wherein the disease is an infection or cancer.

3 1. Use of additional oligonucleotides in a nanoparticle suspension containing dye- labelled oligonucleotide probe to alter an aggregate size distribution of clusters of the nanoparticles.

32. A method of altering an aggregate size distribution of clusters of SERRS or SERS active nanoparticles in a suspension containing dye-labelled oligonucleotide probe, comprising adding oligonucleotides to the suspension.

33. The use according to claim 3 1 , or method according to claim 32, wherein the oligonucleotides are non-labelled; not dye-labelled; or dye-labelled with a different dye to the dye-labelled oligonucleotide probe.

34. The method, use, composition, or kit substantially as described herein, optionally with reference to the accompanying drawings.