WO2015114368A1 - Method of detecting trichomonas vaginalis - Google Patents

Abstract

A method of detecting the presence of Trichomonas vaginalis in a sample. The method involves detecting a target sequence taken from the TVAG_003780 gene in a sequence-specific manner. The method may involve a step of amplifying the target sequence, and may involve hybridising the target sequence to a nucleic acid probe and identifying hybridisation.

Description

METHOD OF DETECTING TRICHOMONAS VAGINALIS

TECHNICAL FIELD

This invention is in the field of detection methods and primers, probes and compositions used in those methods. More specifically, the invention relates to methods of detecting Trichomonas vaginalis and primers, probes and compositions used in those methods and kits for performing the methods.

BACKGROUND ART

Trichomonas vaginalis is an anaerobic, flagellated protozoan and is the cause of the disease trichomoniasis. Trichomoniasis is the most common pathogenic protozoan infection of humans in industrialized countries. Trichomoniasis is a sexually transmitted infection which affects both men and women, but is usually symptomatic in women and asymptomatic in men. Trichomoniasis can cause genital inflammation that makes it easier to get infected with the HIV virus, to pass the HIV virus on to a partner, or to contract other sexually transmitted infections.

Diagnosis of trichomoniasis is usually achieved via a laboratory test including an overnight culture. Such assays used to detect the presence of Trichomonas vaginalis have a sensitivity range of 75- 95%. Other more rapid testing is also available, e.g. the OSOM^® test which is a colour immunochromatographic test which is used to test urine samples from patients¹. The OSOM^® test has a sensitivity range of around 85-95% and a specificity of around 95%.

Molecular diagnostics for Trichomonas vaginalis are also available. For example, the Aptima assay is a nucleic acid amplification test that utilizes target capture, transcription-mediated amplification (TMA), chemiluminescent probe hybridization, and the automated Tigris DTS system to detect Trichomonas vaginalis 18S rRNA. Schwebke et al.² provides an evaluation of this test and shows sensitivity and specificity of the assay to be 100% and 99% respectively. However, the Aptima assay is performed in a central laboratory setting, and therefore the time required for a sample to be sent to a laboratory, the test to be performed and the results to be sent back to the clinic can be up to two weeks. Such long periods of time between testing and receipt of results can lead to patients not returning to the clinic for results, which leads to trichomoniasis being left untreated.

As disclosed by Pearce et al , there is a need for a point-of-care molecular diagnostic test for Trichomonas vaginalis in which results can rapidly be provided at the time of testing, without the patient having to leave the clinic. These rapid tests require molecular targets which are amenable to rapid amplification, but Pearce et al. did not disclose any such targets or the primers and probes which might be used for their detection.

It is an object of the invention to provide a reliable, sensitive and specific method of detecting Trichomonas vaginalis, which additionally allows for point of care diagnosis. Moreover, the methods should ideally be amenable to multiplex assays in which other pathogens can also be detected. DISCLOSURE OF THE INVENTION

The present invention is based on the identification of a particular target that is present in the genomic DNA of Trichomonas vaginalis and which allows for particularly rapid and effective detection of T. vaginalis. Methods of detection involving detecting the presence of this target are particularly useful for detecting the presence of T. vaginalis, as explained below.

The T. vaginalis genome sequence is extremely large (ca. 173 Mb) and contains many repeat regions. The genome size and the number of repeat regions is thought to be due to repeated uptake of non- Trichomonas DNA. These features of the Trichomonas vaginalis genome have caused its sequencing to be a long and difficult process. Furthermore, the fact that these features of the genome are thought to be due to uptake of non-Trichomonas DNA means that selecting regions to be used as a marker for detection faces the risk of cross-reactivity with the original source of the DNA sequence.

The inventors have identified a particular region of the Trichomonas vaginalis genome, TVAG 003780 (incorporated as SEQ ID NO: 1 and depicted in Figure 1). This region of the genome does not have a specific public annotation and is merely referred to as a "hypothetical protein". Neither DNA nor protein BLAST searches identify any putative conserved domains in the sequence.

This particular region of the genome is particularly suitable as a target for rapid detection of the presence of T. vaginalis in a sample because it is present in a high copy number (estimated to be in excess of 641 copies per genome). Therefore this sequence is easier to detect than sequences present only once or at low copy number in the genome because fewer rounds of amplification provide a large amount of amplified nucleic acid. In particular, the target lends itself to use in a rapid point-of- care test because its high copy number further increases the speed of the test. Furthermore, the target is found to be present in T. vaginalis isolates sourced from widely varying geographical areas. Also, it was not found to be present in any other species tested in exclusivity trials, and does not cross-react with human DNA, and therefore it is highly specific for T. vaginalis.

A skilled person would not have considered using TVAG_003780 as a target to detect the presence of T. vaginalis because it is considered to potentially be part of a transposable element. Transposable elements would be considered as sequences that could be easily removed from the genome, therefore rendering the T. vaginalis undetectable (false negative), or could have been inserted into the genome of another organism, therefore leading to false positives. Unexpectedly however, the sequence was found to be present in all T. vaginalis isolates tested, but not in the genomes of any other species.

Accordingly, the invention provides a method of detecting the presence of Trichomonas vaginalis in a sample, comprising a step of detecting a target which comprises the sequence of TVAG_003780 or a fragment thereof.

The invention also provides a pair of primers that is capable of amplifying a sequence within TVAG 003780 to provide an amplicon in a polymerase chain reaction.

The invention also provides a forward primer comprising a nucleic acid sequence comprising 12 or more contiguous nucleotides selected from SEQ ID NO: 4 wherein the sequence of SEQ ID NO: 4 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides.

The invention also provides a reverse primer comprising a nucleic acid sequence comprising 12 or more contiguous nucleotides selected from SEQ ID NO: 5; wherein the sequence of SEQ ID NO: 5 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides.

The invention also provides a nucleic acid probe which is capable of hybridising to the TVAG_003780 sequence or a fragment thereof.

The invention also provides a nucleic acid probe comprising a nucleic acid sequence which comprises 19 or more contiguous nucleotides selected from SEQ ID NO: 3 or its complement; wherein the sequence of SEQ ID NO: 3 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides.

The invention also provides a composition comprising a forward primer of the invention and a reverse primer of the invention.

The invention also provides a kit comprising a composition of the invention.

The invention also provides a method of detecting the presence of Trichomonas vaginalis in a sample comprising a step of amplifying the target sequence and a step of detecting a target sequence, wherein the target sequence consists of SEQ ID NO: 2, and said step of detecting comprises hybridising the target sequence to a nucleic acid probe comprising SEQ ID NO: 3 and a ferrocene label and identifying the occurrence of hybridisation by detecting the state of the labelled probe, and wherein said step of amplifying the target sequence uses the polymerase chain reaction and involves the use of a forward primer consisting of SEQ ID NO: 4 and a reverse primer consisting of SEQ ID NO: 5.

The invention also provides a method of diagnosis of trichomoniasis comprising a method or using a primer, probe, composition or kit of the invention.

Target sequence

The invention relates to methods, primers, probes, compositions and kits for detecting the presence of T. vaginalis in a sample by the detection of a target sequence. The target sequence is a nucleic acid sequence that is specific to T. vaginalis, but should not be present in any species other than T. vaginalis in order to reduce the occurrence of false positive results. The target sequence should be present in substantially all T. vaginalis isolates irrespective of geographical origin in order to reduce the occurrence of false negative results. The target sequence is a sequence that is particularly amenable to detection by rapid detection methods. The target sequence is such that it allows for multiplex detection, e.g. target sequences from other pathogens may be detected simultaneously with the T. vaginalis target sequence of the invention. The target sequence is the TVAG_003780 sequence or a fragment thereof. Where a fragment is used, the target sequence may be any length within the TVAG_003780 sequence. For example, the target sequence may be 50-1000, 100-500, 200-400, 250-350, 200-300, 100-200 or 100-150 nucleotides in length. In particular, the target sequence may be 50, 100, 105, 110, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 nucleotides in length.

The target sequence may be the TVAG 003780 sequence of SEQ ID NO: 1 or a fragment thereof, or its complement or a fragment thereof. As an alternative, the target sequence may be any naturally occurring allele or variant of TVAG 003780 or a fragment thereof, or its complement or a fragment thereof. The target sequence may be 90%, 95% or 100% identical to SEQ ID NO: 1 or a fragment thereof.

SEQ ID NO: 2 is a particular region within the TVAG 003780 sequence which has been found to be particularly useful as the target sequence. The target sequence may comprise 20 to 124 contiguous nucleotides of SEQ ID NO: 1 or its complement or SEQ ID NO: 2 or its complement. The target sequence may comprise at least 23, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, or at least 124 contiguous nucleotides of SEQ ID NO: 1 or its complement or of SEQ ID NO: 2 or its complement. Preferably, the target sequence to be detected comprises at least 24 contiguous nucleotides of SEQ ID NO: 1 or its complement. More preferably, the target sequence to be detected comprises at least 23 contiguous nucleotides of SEQ ID NO: 2 or its complement.

The target sequence may comprise the nucleic acid sequence of SEQ ID NO: 1 or its complement, or SEQ ID NO: 2 or its complement. Alternatively, the target sequence may consist of the nucleic acid sequence of SEQ ID NO: 1 or its complement, or SEQ ID NO: 2 or its complement.

The target sequence that is detected by the methods of the invention may be DNA or RNA. Usually it is DNA, in particular genomic DNA.

Usually, the target sequence will be double stranded and therefore the target sequence will comprise both SEQ ID NO: 1 and its complement (or fragments thereof).

Sample

The sample is a composition on which the method of the invention is performed in order to determine whether the target sequence is present. The sample may be a composition in which the target sequence is suspected to be present, or may be a composition in which the target sequence is potentially present. Methods of detecting the presence of T. vaginalis include methods that are performed on samples which are known or suspected to contain the target sequence, as well as compositions in which the target sequence is only potentially present. Even if a method is performed on a composition in which the target sequence is not actually present, the method is still considered to be a method of detecting the presence of T. vaginalis if the method is performed to detect any T. vaginalis that might be present.

The sample may be material obtained from a subject. The sample may be a cellular sample, i.e. a sample which contains cells. The sample may be obtained with minimal invasiveness or non- invasively, e.g., the sample may be a bodily fluid which may be obtained from a subject using a swab. Preferably, the sample is obtained from a genital swab, e.g. a vaginal swab.

The sample may have been treated since being obtained from the subject. For example, one skilled in the art will appreciate that samples can be purified, diluted, concentrated, centrifuged, frozen, etc. prior to target detection.

The subject is usually human, and may be male or female.

Methods of the invention

The methods of the invention for detecting the presence of T. vaginalis involve detecting the presence of the target sequence using any technique. The methods involve a step of detecting the target sequence that is capable of specifically detecting the target sequence rather than any other nucleic acid sequence. The methods of the invention are thus sequence-specific. Preferably, the methods of the invention are rapid detection methods which allow for point-of-care diagnosis.

Usually, the method involves a step of amplifying the target sequence in addition to the step of detecting the target sequence. The method may be sequence-specific by virtue of a sequence-specific step of amplifying the target sequence. Where a sequence-specific step of amplifying the target sequence is used, the step of detecting the target sequence does not need to be sequence-specific. As an alternative, the method may be sequence-specific by virtue of a sequence-specific step of detecting the target sequence. Where a sequence-specific step of detecting the target sequence is used, the step of amplifying the target sequence does not need to be sequence-specific. Usually, however, both the step of amplifying the target sequence and the step of detecting the target sequence are sequence-specific, as this can maximise overall specificity.

Therefore, the methods of the invention may involve a sequence-specific step of amplifying the target sequence and/or a sequence-specific step of detecting the target sequence.

Step of amplifying

In typical embodiments, methods of the invention comprise a step of amplifying the target sequence. This is particularly the case if the detection step requires a large number of copies of the target sequence to be present. As mentioned above, the step of amplifying the target sequence is preferably sequence-specific.

The amplifying step may involve any method of nucleic acid amplification known in the art, including but not limited to the polymerase chain reaction (PCR), the ligase chain reaction (LCR)⁴, strand displacement amplification (SDA)⁵, transcription mediated amplification⁶, nucleic acid sequence-based amplification (NASBA)⁷, helicase-dependent amplification⁸ and loop-mediated isothermal amplification⁹. Preferably, PCR is used. A standard amplification mixture for PCR comprises: a forward primer and a reverse primer wherein the two primers are complementary to the 3' ends of the sense and antisense strand of the target nucleic acid; a thermostable DNA polymerase; deoxynucleoside triphosphates (dNTPs); buffer; divalent cations (e.g. magnesium or manganese ions); and monovalent cations (e.g. potassium ions). The amplification mixture may further comprise dUTPs and optionally uracil-DNA glycosylase, e.g. for use in decontamination methods. UTPs may be incorporated into the amplified sequences, allowing decontamination to be performed using uracil-DNA glycolase prior to amplification in other potentially contaminated samples. Using such methods, contaminating sequences containing uracil may be removed without removing sequences naturally present in the sample (which will not contain uracil).

A typical thermostable polymerase is a Taq polymerase from the thermophilic bacterium Thermus aquaticus. An alternative is Pfu polymerase from Pyrococcus furiosus which has a proof reading activity absent from Taq polymerase and is therefore a higher fidelity enzyme.

In some embodiments, the step of amplifying the target sequence involves the use of one or more of the primers of the invention, which are described in more detail below.

The step of amplifying the target sequence may be performed prior to or simultaneously with the step of detecting the target sequence. Amplification will typically take place before detection, with amplification and detection being distinct steps in an overall method.

Step of detecting

As mentioned above, the step of detecting the target sequence will generally be sequence-specific. As an alternative, the step of detecting the target sequence may be non-sequence-specific, e.g. if the step of amplifying the target sequence is instead sequence-specific.

The detection step may involve hybridisation of the target sequence to a nucleic acid probe and subsequent identification of hybridisation. Further details of such hybridisation techniques are described below. As an alternative, the step of detecting may involve sequencing of the target.

As mentioned above, in some embodiments, the step of detecting involves hybridising the target sequence to a nucleic acid probe. The nucleic acid probe may be any probe that is capable of specifically hybridising to the target sequence, and so the probe will generally comprise a fragment of SEQ ID NO: 1 (or its complement) or SEQ ID NO: 2 (or its complement). Further details of nucleic acid probes are given below.

In some embodiments the detection step in a method of the invention involves identifying the occurrence of hybridisation after a probe has hybridised to the target sequence. Methods of identifying the occurrence of hybridisation include non-sequence-specific methods. Such methods include the use of a nucleic acid intercalating dye, for example, ethidium bromide or an asymmetrical cyanine dye, such as SYBR^®-Green. These dyes increase their fluorescent signal when bound to double-stranded nucleic acid and may be detected by a standard fluorescence detection system. Preferred methods of identifying the occurrence of hybridisation are sequence-specific methods, for example using a labelled nucleic acid probe. One or more labelled probes may be hybridised to the target sequence in a sequence-specific fashion. Nucleic acid probes may be labelled fluorescently, radioactively, enzymatically or electrochemically. Electrochemical labelling is preferred. As an alternative to using a labelled probe, the target sequence may be detected following hybridisation to an immobilised complementary sequence (for example, on a DNA array or "chip").

Methods of identifying the occurrence of hybridisation also include semi-specific detection of product. Such methods of identification include but are not limited to resolving the approximate molecular weight of the product, for example, by carrying out an electrophoresis of the reaction products prior to detection.

Some methods of identifying the occurrence of hybridisation involve detection of an intact hybridised probe, but others involve digestion or hydrolysis of the probe. Thus the nucleic acid probe may be degraded following hybridisation to the target sequence. This may be achieved using a double-strand specific exonuclease. This allows for detection of a label attached to the probe following hydrolysis of the probe, e.g. by changing the label's environment in such a way that may be detected in order to determine the presence or absence of the target sequence. This sort of detection is useful with electrochemically-active labels, as disclosed by Pearce et al. ^w. The exonuclease may be a 5' to 3' exonuclease, such as a T7 exonuclease.

Identifying the occurrence of hybridisation may involve detecting the change in the environment of the label which occurs when the label is hydrolysed from the probe. With an electrochemical label, this change in environment of the probe may be detected by applying a potential difference to the sample and observing changes in the current flow.

Methods of identifying the occurrence of hybridisation may involve measurement of the hydrolysis of the probe concomitant with amplification of the target sequence. Such methods may make use of the nucleic acid exonuclease activity of a nucleic acid polymerase used in the step of amplifying the target sequence. Methods of amplification and detection using modified primers as described in United Kingdom patent application 1312995.2 (not yet published) may be used in the methods of the invention.

Amplification primers

The invention also provides primers that are capable of specifically hybridising to the target sequence. The primers are capable of being extended using one of the nucleic acid amplification methods described above. The primers of the invention may be used in the methods of the invention, where a step of amplifying the target sequence is used.

A primer of the invention will generally be at least 10 nucleotides long, e.g. the primer may be 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. The primer can be fully complementary to the target, but in some embodiments {e.g. in TMA) a primer can include a first region which is complementary to the target and a second region which is not. Shorter probe lengths are favoured if the GC content of the probe is high.

Target amplification will typically use two primers. These are usually referred to as "forward" and "reverse" primers, but the terms "forward" and "reverse" are used merely for convenience, and the directions specified in relation to each of the primer sequences are arbitrary. Therefore, primers described as forward may instead be reverse primers, and primers described as reverse may instead be forward primers. The primers of the invention are intended to be used in pairs, where the 3 ' ends of the two primers are directed towards one another so as to be useful in a step of amplifying.

The primers of the invention may comprise at least one modified nucleotide. The at least one modified nucleotide may protect the primer from exonuclease degradation. The presence of the at least one modified nucleotide in the primer may mean that the downstream amplified region cannot be hydrolysed by the exonuclease. Protection of the amplified product can be useful for increasing the signal produced during detection. Preferably, only one of the forward primer and the reverse primer comprises at least one modified nucleotide.

Modified nucleotide

A modified nucleotide may be any nucleotide which comprises at least one modified sugar moiety, at least one modified intemucleoside linkage and/or at least one modified nucleobase. The modification may prevent the nucleotide from being hydrolysed by an exonuclease. A modified nucleotide comprises at least one modification compared to a naturally occurring RNA or DNA nucleotide. The at least one modified nucleotide may comprise at least one modified sugar moiety. The modified sugar moiety may be a 2'-0-methyl sugar moiety. The modified sugar moiety may be a 2'-0- methoxyethyl sugar moiety. The modified sugar may be a 2'fluoro modified sugar. As an alternative, the modified sugar moiety may be a bicyclic sugar. Bicyclic sugars include 4'- (CH₂)n-0-2' bridges, wherein n is 1 or 2; and 4'-CH(CH₃)-0-2' bridges.

The at least one modified nucleotide may comprise at least one modified intemucleoside linkage. The at least one modified intemucleoside linkage may be at least one phosphoramidite linkage. The at least one modified intemucleoside linkage may be at least one phosphorothioate linkage.

The at least one modified nucleotide may comprise at least one modified nucleobase.

The at least one modified nucleotide may comprise more than one modification, e.g. a modified nucleotide may comprise a modified sugar moiety and a modified intemucleoside linkage. Alternatively, the modified nucleotide may comprise a modified sugar moiety and a modified nucleobase, or a modified intemucleoside linkage and a modified nucleobase. The modified nucleotide may comprise a modified sugar moiety, a modified intemucleoside linkage and a modified nucleobase.

The at least one modified nucleotide may be present at any position in the primer. For example the at least one modified nucleotide may be present at the 5' end of the primer, or may be present at the 3 ' end of the primer, or may be present in the central section of the primer, or may be interspersed throughout the primer. Usually, the at least one modified nucleotide is present at the 5' end of the primer. Where a modified nucleotide comprises a modified intemucleoside linkage at the 5' end of the primer, this means that the intemucleoside linkage between the first and second nucleotide is modified.

The modified primer may comprise multiple modified nucleotides. For example, the modified primer may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 modified nucleotides. Specifically, the modified primer may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 modified nucleotides. Each of the nucleotides of the modified primer may be modified nucleotides.

Where the modified primer comprises multiple modified nucleotides, the modified nucleotides are preferably contiguous nucleotides in the primer. As an alternative, the modified nucleotides may be spaced out along the primer, i.e. one or more unmodified nucleotides may be present in between the modified nucleotides. The spaces of unmodified nucleotides may comprise, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more unmodified nucleotides.

Preferably, the primer comprises 3 or 4 modified nucleotides ideally contiguous. These may be at the 5' end. Preferably, the 3 or 4 modified nucleotides comprise phosphorothioate linkages. In some embodiments, the primer comprises 3 contiguous phosphorothioate linkages at the 5' end, i.e. the linkages between each of the first to fourth nucleotides are phosphorothioate linkages. In some embodiments, the primer comprises four contiguous phosphorothioate linkages at the 5' end, i.e. the linkages between each of the first to fifth nucleotides are phosphorothioate linkages.

Amplification primers capable of specifically hyridising to the target sequence

A forward primer of the invention may comprise any nucleic acid sequence capable of specifically hybridising to SEQ ID NO: 1 or its complement, or SEQ ID NO: 2 or its complement.

A forward primer of the invention may comprise a nucleic acid sequence comprising at least 10 contiguous nucleotides of SEQ ID NO: 4. This sequence may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides relative to SEQ ID NO: 4, provided that the primer is still capable of specifically hybridising to the target sequence. In some embodiments there are 1, 2, 3, 4 or 5 additions, deletions and/or substitutions of single nucleotides relative to SEQ ID NO: 4. However, in other embodiments there are no additions, deletions and/or substitutions of single nucleotides made to SEQ ID NO: 4. Preferably, the forward primer comprises a nucleic acid sequence comprising at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26 or at least 27 contiguous nucleotides of SEQ ID NO: 4. Even more preferably, the forward primer comprises all of the nucleic acid sequence of SEQ ID NO: 4. The forward primer may consist of the nucleic acid sequence of SEQ ID NO: 4. In one embodiment, the forward primer may comprise or consist of the nucleotide sequence of A*A*C*A*CAAAGCTTCAAACGGTAGTGAGAG (SEQ ID NO: 4) or

A*A*C*ACAAAGCTTCAAACGGTAGTGAGAG.

N* indicates that the nucleotide at the specified position is a modified nucleotide. Where the modified nucleotide N*comprises a modified internucleoside linkage the modified internucleoside linkage is between the specified nucleotide and the next nucleotide in the 3' direction.

Therefore, the forward primer may comprise the nucleotide sequence of SEQ ID NO: 4, wherein nucleotides 1-3 or 1-4 are modified nucleotides. The nucleotides of the primer are numbered from the 5' end. Therefore nucleotides 1-3 or 1-4 are the 3 or 4 nucleotides at the 5' end of the primer. The modified nucleotides may comprise phosphorothioate linkages.

A reverse primer of the invention may comprise any nucleic acid sequence capable of specifically hybridising to SEQ ID NO: 1 or its complement, or SEQ ID NO: 2 or its complement. A reverse primer of the invention may be used in combination with a forward primer of the invention to amplify the target sequence. Preferably, the reverse primer of the invention is capable of hybridising to the strand of the target sequence that is complementary to the strand of the target sequence to which the forward primer of the invention hybridises.

The reverse primer may comprise a nucleic acid sequence comprising at least 10 contiguous nucleotides of SEQ ID NO: 5. This sequence may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides relative to SEQ ID NO: 5. In some embodiments there are 1, 2, 3, 4 or 5 additions, deletions and/or substitutions of single nucleotides relative to SEQ ID NO: 5. However, in other embodiments there are no additions, deletions and/or substitutions of single nucleotides made to SEQ ID NO: 5. Preferably, the reverse primer comprises a nucleic acid sequence comprising at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24 or at least 25 contiguous nucleotides of SEQ ID NO: 5. Even more preferably, the reverse primer comprises all of the nucleic acid sequence of SEQ ID NO: 5. The reverse primer may consist of the nucleic acid sequence of SEQ ID NO: 5.

The reverse primer may comprise or consist of the nucleotide sequence of

A*A*A*C*CTGACTTCTTTGCAGAGATGAC (SEQ ID NO: 5) or

A*A*A*CCTGACTTCTTTGCAGAGATGAC, i. e. the first primer may comprise the nucleotide sequence of SEQ ID NO: 5, wherein nucleotides 1-3 or 1-4 are modified nucleotides. The modified nucleotides may comprise phosphorothioate linkages.

The invention provides a pair of primers that is capable of amplifying a target sequence within TVAG 003780 to provide an amplicon. Primer pairs of the invention of particular interest are those useful in PCR, and one such pair is embodied as SEQ ID NOs: 4 and 5. The invention also provides pairs of primers comprising a primer comprising SEQ ID NO: 4, wherein nucleotides 1-3 or 1-4 are modified nucleotides optionally comprising phosphorothioate linkages, and a primer comprising SEQ ID NO: 5, wherein SEQ ID NO: 5 is unmodified, i.e. comprises no modified nucleotides. The invention also provides pairs of primers comprising a primer comprising SEQ ID NO: 5, wherein nucleotides 1-3 or 1-4 are modified nucleotides optionally comprising phosphorothioate linkages, and a primer comprising SEQ ID NO: 4, wherein SEQ ID NO: 4 is unmodified, i.e. comprises no modified nucleotides.

Nucleic acid probes

The invention provides nucleic acid probes that are capable of specifically hybridising to the target sequence. The nucleic acid probes may be used in the step of detecting the target sequence of the methods of the invention. The probes are normally labelled.

Nucleic acid probes of the invention are typically 15 to 45 nucleotides in length, i.e. the nucleic acid probe may be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 nucleotides in length.

As mentioned above, a probe will generally comprise a fragment of SEQ ID NO: 1 (or its complement) or a fragment of SEQ ID NO: 2 or its complement. One such fragment is SEQ ID NO: 3. The nucleic acid probe may comprise a sequence comprising at least 19, at least 20, at least 21 or at least 22 contiguous nucleotides from SEQ ID NO: 3 or its complement; wherein the sequence may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides relative to SEQ ID NO: 3 or its complement, provided that the sequence is still capable of specifically hybridising to the target and that the occurrence of the hybridisation may be identified. In some embodiments there are 1, 2, 3, 4 or 5 additions, deletions and/or substitutions of single nucleotides relative to SEQ ID NO: 3 or its complement. However, in other embodiments there are no additions, deletions or substitutions relative to SEQ ID NO: 3 or its complement.

The nucleic acid probes may include one or more additional moieties other than the region capable of hybridising to the target sequence. The additional moieties may be additional nucleic acid sequences or be non-nucleic acid moieties. For example, the probe may include a linker region which attaches it to an array. The additional moiety may be a label moiety. Particular types of labels that may be used are described in more detail below.

Labels

The nucleic acid probes and/or primers described above may be linked to a label to assist their detection. That label may be radioactive, enzymatically active, fluorescently active, luminescently active, or electrochemically active.

Where detection of multiple nucleic acid sequences are undertaken simultaneously, for example where detection of the target sequence and detection of an internal control nucleic acid sequence are both undertaken simultaneously, or where detection of the T. vaginalis and detection of a nucleic acid sequence from one or more additional pathogens are undertaken simultaneously, the labels used to assist in the detection of the multiple nucleic acid sequences are preferably distinguishable from each other, for example, they may be different fluorophores or they may be different electrochemically active agents or electrochemically active labels providing electrochemically distinguishable activity.

The present invention is especially suitable for use with electrochemically labelled probes and/or primers. In particular, the electrochemical label may include those comprising metallo-carbocyclic pi complexes, that is organic complexes with partially or fully delocalized pi electrons. Suitable labels include those comprising sandwich compounds in which two carbocyclic rings are parallel, and also bent sandwiches (angular compounds) and monocyclopentadienyls. Preferably, the electrochemically active markers are metallocene labels. More preferably they are ferrocene labels. These can be used as disclosed by Pearce et al.¹⁰.

Examples of ferrocene labels which may be incorporated into the primers and/or probes of the invention can be found in WO03/074731, WO2012/085591 and WO 2013/190328 . For example, the ferrocene label may have the structure of formula I in WO2012/085591. As an alternative, the ferrocene label may have the structure of formula I in WO 2013/190328.

Internal controls

Methods of the invention will generally include an internal control nucleic acid. This is used to provide confirmation that the step of amplifying the target sequence and/or the step of detecting the target sequence works correctly.

The internal control comprises a nucleic acid sequence that will not be present in the sample. The internal control nucleic acid sequence may be taken from a plant or bacterium, wherein the nucleic acid sequence is not present in animals and is highly specific to this plant or bacterium. One example of a possible bacterium from which the control nucleic acid may be taken for an animal sample is Pectobacterium atrosepticum, although any control nucleic acid may be used that will not be present in the sample.

The internal control nucleic acid sequence may be DNA or RNA, but is preferably DNA.

The techniques described above for detecting and amplifying the T. vaginalis target can also be used for detection and amplification of an internal control nucleic acid sequence. Similarly, the characteristics of primers and probes can be the same as described above.

Thus the internal control nucleic acid may be any length provided that it is capable of being identified as the internal control nucleic acid. For example, the internal control nucleic acid may be 50-1000, 100-500, 100-200 or 100-150 nucleotides in length. In particular, the internal control nucleic acid sequence may be 50, 100, 105, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 nucleotides in length. Ideally, the length of the internal control nucleic acid is similar to the length of the target sequence.

The internal control nucleic acid may comprise the nucleic acid sequence of SEQ ID NO: 6, or its complement, or a fragment of SEQ ID NO: 6 or its complement; wherein the sequence is mutated by up to 5 additions, deletions or substitutions of single nucleotides. The internal control nucleic acid sequence may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides relative to SEQ ID NO: 6 or its complement.

The step of detecting the internal control nucleic acid sequence may be conducted using the same technique(s) as that used to detect the target sequence. Alternatively, the step of detecting the internal control nucleic acid sequence may be conducted using a different technique(s) to that used to detect the target sequence. Preferably, the step of detecting the internal control nucleic acid sequence is conducted using the same technique as that used to detect the target sequence.

Where the step of detecting the internal control nucleic acid sequence involves hybridisation of the internal control nucleic acid sequence to a nucleic acid probe, the nucleic acid probe may comprise a nucleic acid sequence comprising at least 20 contiguous nucleotides containing SEQ ID NO: 7 or its complement; wherein the sequence may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides relative to SEQ ID NO: 7 provided that the sequence is still capable of specifically hybridising to the internal control nucleic acid sequence. In some embodiments there are 1, 2, 3, 4 or 5 additions, deletions and/or substitutions of single nucleotides relative to SEQ ID NO: 7. However, in other embodiments there are no additions, deletions and/or substitutions of single nucleotides made to SEQ ID NO: 7.

Where used, a step of amplifying the internal control nucleic acid sequence may involve the same technique(s) as the step of amplifying the target sequence, or may involve different techniques that the step of amplifying the target sequence. Preferably, the step of amplifying the internal control nucleic acid sequence is conducted using the same technique as that used to amplify the target sequence.

Where the intemal control nucleic acid sequence is that of SEQ ID NO: 6, a forward control primer having the sequence of SEQ ID NO: 8 and a reverse control primer having a sequence of SEQ ID NO: 9 may be used as the primer pair used in a step of amplifying the internal control nucleic acid sequence. The forward and reverse control primers may, however, be longer or shorter in length than SEQ ID NOs: 8 and 9, respectively. The forward and reverse control primers may be 12 to 60 nucleotides in length, i.e. the primers may be 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 nucleotides in length. Shorter probe lengths are favoured if the GC content of the probe is high.

Use of shorter primers and probes

In some embodiments, primers and probes may be shorter than those specified above particularly if steps are taken to increase the melting temperature of the primer, for example by increasing its GC content. In particular, lengths of 1, 2, 3, 4 or 5 nucleotides shorter than the lengths and ranges specifically disclosed above are contemplated.

The use of shorter primer and probes may be facilitated by the use of minor groove binder moieties and also locked nucleic acids (LNAs) which increase thermal stability of primers and probes and increase the melting temperature of the primer or probe. The use of such modifications is contemplated as part of the present invention in conjunction with probes and primers as disclosed above, but with oligomeric lengths and ranges shortened by 5 nucleotides from those specified above. The present invention contemplates use of shorter primer and probes wherein increased thermal stability is facilitated by the use of minor groove binder moieties and/or LNAs in combination with primers and probes described herein which do not have increased thermal stability as facilitated by the use of minor groove binder moieties and/or LNAs as well as exclusive use of primers and probes having increased thermal stability as facilitated by the use of minor groove binder moieties and/or lock nucleic acids.

Locked nucleic acids

Locked nucleic acids (LNAs) are well known in the art¹¹ An LNA is a nucleic acid incorporating one or more modified RNA or DNA nucleotides (optionally in combination with ordinary DNA or RNA nucleotides). In the modified residue an extra covalent bridge connects the 2' and 3' carbons and "locks" the ribose sugar in the 3'- endo structural conformation as normally found in the A- form of RNA and DNA. LNAs include all nucleic acids incorporating locked nucleotides at some or all residue positions. The lock may be achieved by any chemical bridge connecting the 2' and 3' carbons of the sugar moiety. Preferably the lock is achieved in a 2'-0, 4'-C methylene linkage.

LNAs display increased thermal stability, with melting temperature rising by about 5°C compared to corresponding DNA or RNA oligomers. Because of the elevated melting temperature, the risk of LNA primers and probes forming hairpin structures detrimental to efficient PCR reactions is increased. Good primer and probe design therefore becomes even more essential and in relation to the present application LNA primers and probes corresponding to those disclosed in SEQ ID NOS: 3, 4, 5, 7, 8 and 9 optionally shortened by 1, 2, 3, 4 or 5 nucleotides from either end, or from both ends, are especially preferred.

LNAs can be readily prepared and are commercially available from a number of suppliers.

Minor groove binding moieties

The nucleic acid probes and primers of the invention (including LNA probes and primers) may be conjugated to minor groove binder (MGB) moieties¹². MGB moieties are isometrical-shaped groups which bind in the minor groove of a double helix forming between the probe or primer and target. They stabilize the double stranded region and increase the melting temperature and specificity of the probe/primer, allowing shorter probes/primers to be used. Examples of minor groove binding moieties include distamycin and netropsin analogues, oligoamides built from heterocyclic and aromatic amino acids, and bis-amidines separated by aromatic and heterocyclic rings. Minor groove binding moieties may be readily prepared and attached to primers and probes and are commercially available from a number of suppliers.

Additional target sequences

The methods of the invention for detecting the presence of T. vaginalis may further comprise a step of detecting one or more additional target sequence(s). The methods of the invention may further comprise amplifying one or more additional target sequence(s). The methods of the invention may further comprise a step of detecting at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten additional target sequences. The methods of the invention may further comprise a step of amplifying at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten additional target sequences.

The techniques described above for detecting and amplifying the target sequence can also be used for detection and amplification of any additional target sequences. Similarly, the characteristics of primers and probes can be the same as described above.

One or more of the additional target sequence(s) may be further T. vaginalis target sequences. One or more of the additional target sequence(s) or may be target sequences from other pathogens. Using one or more additional target sequence(s) from other pathogens allows for simultaneous detection of multiple pathogens.

Detection of multiple pathogens

The methods of the invention may be used to detect multiple pathogens simultaneously. For example, the methods may involve detecting T. vaginalis and Chlamydia trachomatis (C. trachomatis). The methods may involve detecting, in addition to T. vaginalis, one or both of C. trachomatis and Neisseria gonorrhoeae (N. gonorrhoeae) in the sample. The methods may also involving detecting other pathogens, particularly pathogens which cause sexually transmitted infections.

The techniques described above for detecting and amplifying the first target sequence and/or the second target sequence can also be used for detection and amplification of target sequences from C. trachomatis and N. gonorrhoeae. Similarly, the characteristics of primers and probes can be the same as described above.

Where C. trachomatis is additionally detected, the method further comprises a step of detecting a C. trachomatis specific target sequence. This step may be performed as described in WO 2011/073675. The C. trachomatis specific target sequence may comprise at least 10, at least 15, at least 20, at least 25 or at least 30, at least 40, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 contiguous nucleotides of SEQ ID NO: 10 or a fragment thereof or SEQ ID NO: 11 or a fragment thereof. The step of detecting the C. trachomatis specific target sequence may comprise a step of hybridising the C. trachomatis specific target sequence to a C. trachomatis specific nucleic acid probe and identifying the occurrence of hybridisation. Any probe which is capable of hybridising to the C. trachomatis specific target sequence may be used. The C. trachomatis specific nucleic acid probe may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, at least 30 or at least 32 contiguous nucleotides of SEQ ID NO: 12. The C. trachomatis specific nucleic acid probe may comprise SEQ ID NO: 12. The C. trachomatis specific nucleic acid probe may consist of SEQ ID NO: 12. The method may further comprise a step of amplifying the C. trachomatis specific target sequence. This amplification may be performed using any technique for amplification mentioned above. Preferably PCR is used. The step of amplifying the C. trachomatis specific target sequence may involve any primer pair that is capable of amplifying the C. trachomatis specific target sequence. Preferably, a forward primer comprising 12 or more contiguous nucleotides selected from SEQ ID NO: 13 wherein the sequence of SEQ ID NO: 13 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides and/or a reverse primer comprising 12 or more contiguous nucleotides selected from SEQ ID NO: 14 wherein the sequence of SEQ ID NO: 14 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides, are used.

Where N. gonorrhoeae is additionally detected, the method further comprises a step of detecting a first N. gonorrhoeae specific target sequence and/or a second N. gonorrhoeae specific target sequence. This step may be performed as described in United Kingdom patent application 1416459.4 (not yet published). The first N. gonorrhoeae specific target sequence may comprise at least 10, at least 15, at least 20, at least 25 or at least 30, at least 40, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 contiguous nucleotides of SEQ ID NO: 15 or a fragment thereof or SEQ ID NO: 16 or a fragment thereof. As for the T. vaginalis target sequences mentioned above, the first N. gonorrhoeae specific target sequence specific target sequence may be of any length.

The second N. gonorrhoeae specific target sequence may comprise at least 10, at least 15, at least 20, at least 25 or at least 30, at least 40, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 contiguous nucleotides of SEQ ID NO: 17 or a fragment thereof or SEQ ID NO: 18 or a fragment thereof. As for the T. vaginalis target sequences mentioned above, the second N. gonorrhoeae specific target sequence specific target sequence may be of any length.

The step of detecting the first N. gonorrhoeae specific target sequence and/or the second N. gonorrhoeae specific target sequence may comprise a step of hybridising the N. gonorrhoeae specific target sequence to a N. gonorrhoeae specific nucleic acid probe and identifying the occurrence of hybridisation. Any probe which is capable of hybridising to the N. gonorrhoeae specific target sequences may be used. The first N. gonorrhoeae specific nucleic acid probe which is capable of hybridising to the first N. gonorrhoeae specific target sequence may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20 or at least 22 contiguous nucleotides of SEQ ID NO: 19. The first N. gonorrhoeae specific nucleic acid probe may comprise SEQ ID NO: 19. The first N. gonorrhoeae specific nucleic acid probe may consist of SEQ ID NO: 19. The second N. gonorrhoeae specific nucleic acid probe which is capable of hybridising to the second N. gonorrhoeae specific target sequence may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20 or at least 22 contiguous nucleotides of SEQ ID NO: 20. The second N. gonorrhoeae specific nucleic acid probe may comprise SEQ ID NO: 20. The second N. gonorrhoeae specific nucleic acid probe may consist of SEQ ID NO: 20. The method may further comprise a step of amplifying the first N. gonorrhoeae specific target sequence and/or the second N. gonorrhoeae specific target sequence. This amplification may be performed using any technique for amplification mentioned above. Preferably PCR is used. The step of amplifying the first N. gonorrhoeae specific target sequence may involve any primer pair that is capable of amplifying the first N. gonorrhoeae specific target sequence. Preferably, a forward primer comprising 12 or more contiguous nucleotides selected from SEQ ID NO: 21 wherein the sequence of SEQ ID NO: 21 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides and/or a reverse primer comprising 12 or more contiguous nucleotides selected from SEQ ID NO: 22 wherein the sequence of SEQ ID NO: 22 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides, are used. The step of amplifying the second N. gonorrhoeae specific target sequence may involve any primer pair that is capable of amplifying the second N. gonorrhoeae specific target sequence. Preferably, a forward primer comprising 12 or more contiguous nucleotides selected from SEQ ID NO: 23 wherein the sequence of SEQ ID NO: 23 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides and/or a reverse primer comprising 12 or more contiguous nucleotides selected from SEQ ID NO: 24 wherein the sequence of SEQ ID NO: 24 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides, are used.

As mentioned above, the labels included in the nucleic acid probes used to assist in the detection of nucleic acid sequences from different pathogens are preferably distinguishable from each other. For example, they may be different fluorophores or they may be different electrochemically active agents or electrochemically active labels providing electrochemically distinguishable activity.

Accordingly, the invention also provides methods for detecting T. vaginalis, C. trachomatis and N. gonorrhoeae in a sample. The method may comprise a step of detecting at least one target sequence from T. vaginalis, at least one C. trachomatis specific target sequence and at least one N. gonorrhoeae specific target sequence. Preferably, the T. vaginalis specific target sequence is

TVAG 003780 (SEQ ID NO: 1) or a fragment thereof or SEQ ID NO: 2 or a fragment thereof.

Preferably the C. trachomatis specific target sequence is SEQ ID NO: 10 or a fragment thereof or

SEQ ID NO: 11 or a fragment thereof. Preferably, the at least one target sequence from N. gonorrhoeae is selected from SEQ ID NO: 15 or a fragment thereof or SEQ ID NO: 16 or a fragment thereof; and SEQ ID NO: 17 or a fragment thereof or SEQ ID NO: 18 or a fragment thereof.

Compositions

The invention also provides compositions comprising one or more of the primers and/or nucleic acid probes described above.

The compositions of the invention may comprise a forward primer and a reverse primer capable of specifically hybridising to the target sequence. The composition may further comprise a nucleic acid probe capable of specifically hybridising to the target sequence. The composition may further comprise a DNA polymerase. These compositions are suitable for performing amplification of the target e.g. by PCR.

The compositions may further comprise an internal control nucleic acid sequence. The compositions may further comprise a forward control primer and a reverse control primer capable of specifically hybridising to the internal control nucleic acid sequence. The composition may further comprise a control nucleic acid probe capable of specifically hybridising to the internal control nucleic acid sequence.

These compositions may be added to a sample and the conditions required for detection and where used amplification applied to the composition in order to detect the presence of T. vaginalis in the sample.

The invention also provides kits comprising a composition of the invention. A kit may further comprise instructions for use.

Cartridge

The invention also provides a cartridge for use in a method of detecting the presence of T. vaginalis in a sample. The cartridge allows for rapid point-of-care detection of T. vaginalis when used in combination with a suitable detection apparatus (cartridge reader).

The cartridge comprises a sample inlet for receiving a sample, a portion in which a step of amplifying the target can take place, and a portion in which a step of detecting the target sequence can take place.

The cartridge may also comprise a portion in which a step of extracting nucleic acids from the sample can take place. The cartridge may be a pneumatically controlled cartridge.

General

The term "comprising" encompasses "including" as well as "consisting" e.g. a composition "comprising" X may consist exclusively of X or may include something additional e.g. X + Y.

The term "about" in relation to a numerical value x is optional and means, for example, x+10%.

The term "specifically hybridises" means capable of hybridising to the sequence of the intended target and not to sequences that are not present in intended target.

Unless specifically stated otherwise, a process comprising a step of mixing two or more components does not require any specific order of mixing. Thus components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc.

The text above refers in several places to addition, deletion or a substitution of a single nucleotide within a particular sequence. The sequence may be mutated by a number of additions and a number of deletions within the specified sequence. Alternatively, the sequence may be mutated by a number of additions and a number of substitutions within the specified sequence. Alternatively, the sequence may be mutated by a number of substitutions and a number of deletions within the specified sequence. Alternatively, the sequence may be mutated by a number of additions, a number of deletions and a number of substitutions within the specified sequence. In all cases, the number of mutated nucleotides may be 1, 2, 3, 4 or 5.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. The sequence of TVAG 003780 (the coding strand of which is incorporated as SEQ ID NO: 1), showing the location of hybridisation of two specific primers (SEQ ID NOs: 4 and 5) which amplify the target sequence SEQ ID NO: 2, and a specific nucleic acid probe (SEQ ID NO: 3).

Figure 2. Sensitivity of the Trichomonas vaginalis assay following extraction, nucleic acid amplification and detection. Error bars show the SD (n=5).

Figure 3. Inclusivity test of the Trichomonas vaginalis assay using thirteen isolates from a range of geographical locations. Error bars show the SD (n=5).

Figure 4. Exclusivity assay using DNA molecules from a range of organisms. Error bars show the SD (n=3).

Figure 5. Frequency distribution of clinical samples tested using the Trichomonas vaginalis assay. MODES FOR CARRYING OUT THE INVENTION

Trichomonas vaginalis assay

A sub-circuit assay which mimics a point-of-care cartridge was produced in which three steps take place sequentially: extraction of the nucleic acids from T. vaginalis cells; amplifying the target sequence SEQ ID NO: 2 by PCR; and detecting the target sequence by hybridisation of the target sequence to a nucleic acid probe consisting of SEQ ID NO: 3 with a conjugated diferrocene label (di275). The assay was performed as disclosed by Pearce et al.¹⁰.

Primers consisting of the nucleic acid sequences of SEQ ID NOs: 4 and 5 were included in the PCR reagents in the cartridge in order to amplify the sequence of SEQ ID NO: 2 using semi-rapid amplification which is completed in less than one hour.

The nucleic acid probe consisting of SEQ ID NO: 3 was labelled with a ferrocene label. Following hybridisation of the probe to the target, the double-stranded product was specifically hydrolysed by T7 exonuclease causing the diferrocene label to be cleaved from the rest of the probe. The change in environment of the label which occurs when the label is cleaved from the probe was detected by applying a potential difference to a detection region of the detection sub-circuit and observing changes in the current flowing through the detection region.

The cartridge also allowed for a step of amplifying a Pectobacterium atrosepticum internal control nucleic acid sequence and a step of detecting the P. atrosepticum internal control nucleic acid sequence via hybridisation of the internal control nucleic acid to a control nucleic acid probe and identification of hybridisation. The extraction and amplification of the internal control nucleic acid sequence took place simultaneously with the extraction and amplification of the target sequence. Primers having the nucleic acid sequences of SEQ ID NOs: 8 and 9 were included in the PCR reagents in the cartridge in order to amplify the sequence of SEQ ID NO: 6. Amplification of the internal control DNA was performed in the same amplification chamber as the target sequence amplification.

A nucleic acid probe consisting of the nucleic acid sequence of SEQ ID NO: 7 was included in the sub-circuit in order to perform the step of hybridisation of the internal control nucleic acid sequence to a nucleic acid probe. The nucleic acid probe was labelled with the same diferrocene label as was used to label the target nucleic acid probe. Following hybridisation of the internal control nucleic acid sequence to the control nucleic acid probe, the double-stranded product was specifically hydrolysed and detected in the same way as for the target sequence (see above). Detection of the amplified internal control DNA occurred in a separate chamber to the detection of the target sequence. The use of separate chambers for the detection of the internal control DNA and the target sequence allows for the same label to be attached to both the control nucleic acid probe and the target nucleic acid probe. An alternative arrangement would be to detect the target and internal control DNA in the same chamber, but for distinguishable labels to be attached to the control nucleic acid probe and the target nucleic acid probe.

Determining assay sensitivity

The T. vaginalis assay was performed on samples which contained different copy numbers of the T. vaginalis genome in the reaction mixture. The target sequence was detected in all samples containing 50000, 5000, 500, 50 and 5 genomic copies (see Fig 2). Target sequence was not detected in the negative control (see Fig 2). Therefore the preliminary sensitivity of the T. vaginalis assay was found to be 5 genome copies of T. vaginalis genomic DNA. The fact that such high copy numbers of the target sequence are present in each genome copy means that sensitivity is increased because even with a very low genome copy present, a high level of target sequence is present allowing for detection.

The high copy number of the target sequence on the Trichomonas vaginalis genome also means that fewer rounds of amplification are required without losing the sensitivity of the test, because even very few rounds of amplification of the target sequence will result in a large amount of amplified target sequence. In order to achieve a point-of-care test it is necessary to use rapid amplification techniques. However, such techniques can lead to false negative results if the sequence is missed because not enough time is taken for adequate hybridisation of primers etc to take place. However, the presence of a high copy number of the target sequence in the genome allows for rapid amplification to be used without the possibility of the target sequence being missed.

Determining assay inclusivity

It is important that the assay is capable of detecting T. vaginalis isolated from different geographical locations. Therefore the thirteen different strains of T. vaginalis listed in Table 1 were tested using the T. vaginalis assay to ensure that all strains of T. vaginalis were detected by the assay irrespective of source. Table 1

The results of the inclusivity testing are provided in Figure 3. All strains were detected using the assay. T. vaginalis was not detected in the negative control sample. The presence of the target sequence in many T. vaginalis isolates obtained from a variety of geographical locations confirms the ubiquitous nature of this sequence in T. vaginalis and thus may reduce the number of false negative results.

Determining assay exclusivity

It is important that the assay is capable of distinguishing the presence of T. vaginalis from the presence of other sexually transmitted pathogens, as well as any other pathogens. Therefore the assay was performed on samples containing a range of organisms to check exclusivity. The results of the exclusivity testing are provided in Figure 4. No cross-reactivity was demonstrated when the assay was tested against the panel of organisms, i. e. no organism other than T. vaginalis was detected at levels higher than those present in the negative control.

Testing assay performance in clinical samples

One hundred and forty eight vaginal swab samples were obtained for testing. These had been pre- typed using the Gen-Probe APTIMA T. vaginalis assay, as 53 positive and 95 negative samples.

The T. vaginalis sub-circuit assay was performed on each sample. The internal control DNA was added to the sample prior to extraction. The testing of these samples, but not the particular sequences amplified has been published'. A preliminary assay "cut-off was assigned based on the negative mean plus 3 x standard deviation of the mean. Using this preliminary cut-off the assay produced one false positive result (at peak height 1051-2000nA) and two false negative results (at peak heights of 0-5 and 16-20nA). Using these data the sensitivity and specificity of the assay was determined and these data are provided in Table 2. The sensitivity of the assay was found to be 96.2% and the specificity of the assay was found to be 99.0%. Therefore, the assay is shown to be highly sensitive and specific, as well as being highly amenable for use in a rapid point-of-care diagnostic test. The high degree of sensitivity and specificity is not compromised in order to achieve a rapid point-of-care test.

Table 2

REFERENCES

1. Zaki et al. PUJ, 2011, 4(2): 177-184

2. Schwebke et al. Journal of Clinical Microbiology, 2011, 49(12) :4106-4111

3. Pearce et al. (2013) Sex Transm Infect. 89:495-497.

4. Wiedmann M. et al. PCR Methods and Applications 1994 3(4)S51-64

5. Walker et al. Nucleic Acids Res.1992. 20(7) 1691-1696

6. Wroblewski J. et al. J. Clin. Microbiol. 2006:44(9):3306-3312

7. Compton J. Nature 1991 :350(6313):91-2

8. Vincent M. et al. EMBO Rep. 2004 5(8) 795-800

9. Notomi et al. Res. 2000 23 (12):E63.

10. Pearce et al. (2011) IEEE Trans Biomed Eng. 58(3): 755-758.

11. Devor (2005) Integrated DNA technologies technical bulletin

12. Katyavin et al. (2000) Nucleic Acid Res. 28(2):655-661

Claims

1. A method of detecting the presence of Trichomonas vaginalis in a sample, comprising a step of detecting a target which comprises the sequence of TVAG_003780 or a fragment thereof.

2. The method of claim 1, wherein said step of detecting comprises hybridising the target to a nucleic acid probe and identifying the occurrence of hybridisation.

3. The method of claim 1 or claim 2, wherein said target comprises at least 23 contiguous nucleotides contained in SEQ ID NO: 1 or SEQ ID NO: 2.

4. The method of any one of claims 1-3, wherein the target comprises at least 30 contiguous nucleotides contained in SEQ ID NO: 2.

5. The method of any one of claims 2-4, wherein the nucleic acid probe is capable of specifically hybridising to the target.

6. The method of any one of claims 2-5, wherein the nucleic acid probe comprises at least 19 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 2 or its complement; wherein the sequence of SEQ ID NO: 2 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides.

7. The method of claim 6, wherein the nucleic acid probe comprises or consists of the nucleotide sequence of SEQ ID NO: 3.

8. The method of any one of claims 5-7, wherein the nucleic acid probe comprises a minor groove binder moiety and/or wherein said nucleic acid probe is a locked nucleic acid (LNA).

9. The method of any preceding claim, comprising a step of amplifying the target using the polymerase chain reaction, transcription mediated amplification, nucleic acid sequence based amplification (NASBA), helicase-dependent amplification, recombinase polymerase amplification, strand displacement amplification or loop-mediated isothermal amplification.

10. The method of claim 9, wherein the step of amplifying the target uses the polymerase chain reaction.

11. A pair of primers that is capable of amplifying a sequence within TVAG 003780 to provide an amplicon in a polymerase chain reaction.

12. A forward primer comprising a nucleic acid sequence comprising 12 or more contiguous nucleotides selected from SEQ ID NO: 4 wherein the sequence of SEQ ID NO: 4 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides.

13. The forward primer of claim 12, comprising a nucleic acid sequence consisting of the nucleotide sequence of SEQ ID NO: 4.

14. A reverse primer comprising a nucleic acid sequence comprising 12 or more contiguous nucleotides selected from SEQ ID NO: 5; wherein the sequence of SEQ ID NO: 5 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides.

15. The reverse primer of claim 14, comprising a nucleic acid sequence consisting of the nucleotide sequence of SEQ ID NO: 5.

16. A nucleic acid probe which is capable of hybridising to the TVAG 003780 sequence or a fragment thereof.

17. A nucleic acid probe comprising a nucleic acid sequence which comprises 19 or more contiguous nucleotides selected from SEQ ID NO: 3 or its complement; wherein the sequence of SEQ ID NO: 3 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides.

18. The nucleic acid probe of claim 17, comprising a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 3.

19. The nucleic acid probe of claim 18, comprising a nucleic acid sequence consisting of the nucleotide sequence of SEQ ID NO: 3.

20. The forward primer, reverse primer or nucleic acid probe of any one of claims 12-19, comprising a minor groove binder moiety or wherein said primer or said nucleic acid probe are locked nucleic acids (LNAs).

21. The forward primer, reverse primer or probe of any one of claims 12-20, wherein the primer or nucleic acid probe is conjugated to an electrochemically active label.

22. The method of claim 9 or claim 10, wherein said step of amplifying the target sequence involves the use of a forward primer as defined in any one of claims 12, 13, 20 or 21.

23. The method of any one of claims 9, 10 or 22, wherein said step of amplifying the target sequence involves the use of a reverse primer as defined in any one of claims 14, 15, 20 or 21.

24. The method of any one of claims 1-10 or 22-23, wherein said method involves the use of a nucleic acid probe as defined in any one of claims 16-21.

25. The method of any one of claims 1-10 or 22-24, wherein said method further includes a step of detecting the presence of an internal control nucleic acid sequence.

26. The method of claim 25, wherein said internal control nucleic acid sequence is a Pectobacterium atrosepticium nucleic acid sequence.

27. The method of claim 25 or claim 26, wherein said step of detecting the presence of a nucleic acid sequence for use as an internal positive control comprises: a) a step of amplifying said nucleic acid sequence for use as an internal positive control, and/or b) a step of hybridising the nucleic acid sequence for use as an internal positive control to a probe and a step of identifying the occurrence of hybridisiation.

28. A composition comprising a forward primer as defined by any one of claims 12, 13, 20 or 21 and a reverse primer as defined by any one of claims 14, 15, 20 or 21.

29. The composition of claim 28, further comprising a nucleic acid probe as defined by any one of claims 16-21.

30. The composition of claim 28 or claim 29, further comprising a Pectobacterium atrosepticium nucleic acid sequence for use as an internal positive control.

31. The method of claim 26 or the composition of claim 30, wherein said Pectobacterium atrosepticium is of the strain ATCC BAA-672.

32. The composition of claim 30 or claim 31, further comprising a forward control primer and reverse control primer which are capable of amplifying the Pectobacterium atrosepticium nucleic acid sequence.

33. The composition of any one of claims 30-32, further comprising a control nucleic acid probe which is capable of hybridising to the Pectobacterium atrosepticium nucleic acid sequence.

34. A kit comprising a composition as claimed in any of claims 28 to 33.

35. A method of detecting the presence of Trichomonas vaginalis in a sample comprising a step of amplifying the target sequence and a step of detecting a target sequence, wherein the target sequence consists of SEQ ID NO: 2, and said step of detecting comprises hybridising the target sequence to a nucleic acid probe comprising SEQ ID NO: 3 and a ferrocene label and identifying the occurrence of hybridisation by detecting the state of the labelled probe, and wherein said step of amplifying the target sequence uses the polymerase chain reaction and involves the use of a forward primer consisting of SEQ ID NO: 4 and a reverse primer consisting of SEQ ID NO: 5.

36. A method of diagnosis of trichomoniasis comprising a method or using a primer, probe, composition or kit of any one of the preceding claims.

37. The method of any one of claims 1-10, 22-27, 31, 35 or 36, further comprising detecting the presence of Chlamydia trachomatis in the sample, the method further comprising a step of detecting a Chlamydia trachomatis specific target sequence.

38. The method of claim 37, wherein the Chlamydia trachomatis specific target sequence comprises at least 10, at least 15, at least 20, at least 25 or at least 30 contiguous nucleotides of SEQ ID NO: 10.

39. The method of claim 37 or claim 38, wherein the step of detecting a Chlamydia trachomatis specific target sequence comprises a step of hybridising the Chlamydia trachomatis specific target sequence to a Chlamydia trachomatis specific nucleic acid probe and identifying the occurrence of hybridisation, wherein optionally the Chlamydia trachomatis specific nucleic acid probe comprises at least 10 contiguous nucleotides of SEQ ID NO: 12.

40. The method of any one of claims 37-39, further comprising a step of amplifying the Chlamydia trachomatis specific target sequence using PCR, wherein optionally the step of amplifying involves the use of a forward primer comprising 12 or more contiguous nucleotides selected from SEQ ID NO: 13 wherein the sequence of SEQ ID NO: 13 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides and the use of a reverse primer comprising 12 or more contiguous nucleotides selected from SEQ ID NO: 14 wherein the sequence of SEQ ID NO: 14 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides.

41. The method of any one of claims 1-10, 22-27, 31, or 35-40 further comprising detecting the presence of Neisseria gonorrhoeae in the sample, the method further comprising a step of detecting a first Neisseria gonorrhoeae specific target sequence and/or a second Neisseria gonorrhoeae specific target sequence.

42. The method of claim 41, wherein the first Neisseria gonorrhoeae specific target sequence comprises SEQ ID NO: 15 or a fragment thereof or SEQ ID NO: 16 or a fragment thereof.

43. The method of claim 41 or claim 42, wherein the step of detecting comprises a step of hybridising the first Neisseria gonorrhoeae specific target sequence to a first Neisseria gonorrhoeae specific nucleic acid probe and identifying the occurrence of hybridisation, wherein optionally the first Neisseria gonorrhoeae specific nucleic acid probe comprises at least 10 contiguous nucleotides of SEQ ID NO: 19.

44. The method of any one of claims 41-43, further comprising a step of amplifying the first Neisseria gonorrhoeae specific target sequence using PCR, wherein optionally the step of amplifying involves the use of a forward primer comprising 12 or more contiguous nucleotides selected from SEQ ID NO: 21 wherein the sequence of SEQ ID NO: 21 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides and the use of a reverse primer comprising 12 or more contiguous nucleotides selected from SEQ ID NO: 22 wherein the sequence of SEQ ID NO: 22 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides.

45. The method of any one of claims 41-44, wherein the second Neisseria gonorrhoeae specific target sequence comprises SEQ ID NO: 17 or a fragment thereof or SEQ ID NO: 18 or a fragment thereof.

46. The method of any one of claims 41-45, wherein the step of detecting comprises a step of hybridising the second Neisseria gonorrhoeae specific target sequence to a second Neisseria gonorrhoeae specific nucleic acid probe and identifying the occurrence of hybridisation, wherein optionally the second Neisseria gonorrhoeae specific nucleic acid probe comprises at least 10 contiguous nucleotides of SEQ ID NO: 20.

47. The method of any one of claims 41-46, further comprising a step of amplifying the second Neisseria gonorrhoeae specific target sequence using PCR, wherein optionally the step of amplifying involves the use of a forward primer comprising 12 or more contiguous nucleotides selected from SEQ ID NO: 23 wherein the sequence of SEQ ID NO: 23 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides and the use of a reverse primer comprising 12 or more contiguous nucleotides selected from SEQ ID NO: 24 wherein the sequence of SEQ ID NO: 24 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides.

48. A method for detecting N. gonorrhoeae, C. trachomatis and T. vaginalis in a sample.