Kit for detecting silent sexually transmitted diseases (SSTDS) in a urine sample

Abstract

A diagnostic kit for simultaneously detecting at least seven silent sexually transmitted diseases (SSTDs) having a diagnostic strip which has detection zones with at least eight probes with SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24; at least 14 primers with SEQ ID NOs: 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23; an instruction manual; a nucleotide sequence set for simultaneously detecting at least seven silent sexually transmitted diseases (SSTDs); use of the kit to detect at least the following pathogens: Herpes Virus type 1, Herpes Virus type 2, Chlamydia trachomatis, Ureaplasma urealitycum, Mycoplasma hominis, Mycoplasma genitalium, and Human papillomavirus (HPV); a method for simultaneously detecting at least seven silent sexually transmitted diseases (SSTDs).

Claims

1. A diagnostic kit for simultaneously detecting at least 7 silent sexually transmitted diseases (STDs), comprising: a diagnostic strip, comprising detection zones with at least 8 probes of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24; at least 14 primers of SEQ ID NOs: 1, 2, 4, 5, 7, 8, 10, 1 1, 13, 14, 16, 17, 19, 20, 22, 23; instruction manual.

2. A method for simultaneously detecting at least 7 silent sexually transmitted diseases (STDs), comprising the steps of: i. extracting DNA from a body fluid sample by cell lysis, and centrifuging to obtain a pellet containing DNA samples to be amplified; ii. amplifying by Multiplex PCR, using the primers comprising SEQ ID NOs: 1, 2, 4, 5, 7, 8, 10, 1 1, 13, 14, 16, 17, 19, 20, 22, and 23; iii. hybridizing the amplified DNA in step ii), using the probes comprising SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, and 24; iv. detecting the presence or absence of the pathogens of interest in a nylon strip comprising the probes of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24.

3. The method of claim 2 wherein step i), body fluid samples are selected from urine, blood, saliva, urethral secretion, seminal fluid, vaginal secretion.

4. The method of claim 2, wherein in step iv) the pathogen detected is at least one of the pathogens selected from the group consisting of Herpes Virus type 1, Herpes Virus type 2, Chlamydia trachomatis, Ureaplasma urealitycum, Mycoplasma hominis, Mycoplasma genitalium, and Human papillomavirus (HPV).

5. A set of nucleic acid molecules for simultaneously detecting at least 7 silent sexually transmitted diseases (STDs), comprising the following sequences: TABLE-US-00002 Sequence (5′3′) SEQ ID NO: TGGCCAAGCTGACGGACATTT  1 5DigN/GAGAGCTTGATCTTGTCGGTT  2 /5AmMC6/TAAAGGTGAACGGCATGGTGAGCA  3 TGTTTCTGGGCAGCTGTATC  4 5DigN/CTATCGACGTTAGGGAAGGCAT  5 /5AmM6/CATAGATGCCAGCGCCGATACAGG  6 TCAAGGAGTGGAGTGTCTGCGTA  7 5DigN/TGTCGCTCCGATGCAGATGTTT  8 /5AmMC6/ATAGGGAGTGTTTCTCGCCAAGCT  9 GCAGGCGGGTTTGTAAGTTTGGTA 10 5DigN/AGCCTAAGCGTCAGTGATAGTCCA 11 /5AmMC6/ATAGGAAGAACACCGGTGGCGAA 12 TGGAGAATCACTGACGCAGCTAAC 13 5DigN/TGCGAAGGATGTCAAGAGTGGGTA 14 /5AmMC6/CCGCCTGAGTAGTATGCTCGCAAG 15 CGCAACGCTCAGGTGTCTAATGT 16 5DigN/AGAGCTTGGCGCATTGCTGA 17 /5AmMC6/TCGGCTCTCCGATGTTATCAAGTAGGA 18 CGTCCMARRGGAWACTGATC 19 5DigN/GCMCAGGGWCATAAYAATGG 20 /5AmMC6/TTTGTTACTGTTGTGAGAT ACCACTCGCAG 21 5DigN/GAAGAGCCAAGGACAGGTAC 22 CAACTTCATCCACGTTCACC 23 /5AmMC6/TAAGCAAATAGATGGCTCTGCCCT  24; and wherein the modification AmMC6 at the 5′ end of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21 and 24 adhere to a diagnostic strip.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1. Comparative graph of the Presence/Absence of DNA in urine samples. This graph shows that Kit-U detected the presence of DNA in 46 samples of the total of 46 samples tested, while the commercial laboratory kit only detected the presence of DNA in 8 samples. Using the same initial sample concentration in both cases. This shows that Kit-U sensitivity is higher than that of the commercial laboratory kit.

(2) FIG. 2. Graph showing the distribution of results obtained when analyzing the samples with Kit-U. Distribution of m.o. detected by Kit-U. This graph shows the distribution of the detections made using Kit-U, finding 34 detections for Ureaplasma urealiticum, 13 for Mycoplasma hominis, 3 Chlamidia trachomatis, 2 Mycoplasma genitalium, 1 HPV, and 10 negative samples.

(3) FIG. 3. Graph showing the distribution of results obtained when analyzing the samples by Commercial Laboratory. Distribution of m.o. detected by Commercial Laboratory. This graph shows the distribution of the detections made with the Commercial Laboratory Kit, finding that of the total of 8 samples detected by DNA, only 2 detections identify Mycoplasma hominis, while the remaining 6 samples were not identified.

(4) FIG. 4. This figure shows the spatial pattern showing the location of the detection probes for each pathogen. The detection strips are located parallel to this pattern to identify which pathogens are present in the sample.

DESCRIPTION OF THE INVENTION

(5) The present invention describes a Kit for simultaneously detecting pathogens causing Silent Sexually Transmitted Diseases (STDs) from a single non-invasive human urine sample, herein called “Kit-U”. The group of pathogens of interest detected are: Herpes Virus type 1, Herpes Virus type 2, Chlamydia trachomatis, Ureaplasma urealitycum, Mycoplasma hominis, Mycoplasma genitalium, and Human papillomavirus (HPV).

(6) The present invention describes a set of 24 sequences, wherein SEQ ID NOs: 1 to 18 are specifically designed for the detection of the first 6 pathogens of the group of pathogens of interest using 2 primers and 1 probe for each pathogen. Three already known sequences (2 primers, 1 probe) are used for HPV. Additionally, the present Kit-U requires the detection of the β-globin gene (internal control for DNA quality and amplification), for which another 3 known in the art sequences are used. This Kit-U allows to perform a detection method based on Multiplex PCR amplification of genome region sequences of different pathogens. The PCR product is hybridized on a diagnostic strip formed by a nylon membrane containing specific probes for different pathogens.

(7) The primers used in the Multiplex PCR step are previously labeled at their 5′ end with a digoxigenin molecule, which is used to detect the pathogen presence. Probes have a 5′ end modification with an Amino Modifier C6, (AmMC6) allowing the probe to adhere to the nylon membrane in the diagnostic strip.

(8) If the genome of any of the pathogens of interest to be detected is coupled to its complementary probe, it exposes the Digoxigenin molecule in one of the primers now located in the amplified DNA. This Digoxigenin molecule is detected through a reaction with anti-Digoxigenin antibody, this antibody is conjugated to alkaline phosphatase for its visualization with compound NB T/BCIP (5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium), which forms a purple precipitate on the membrane at the site of the probe that reacted with the pathogen DNA present in the sample.

(9) Hybridization between the pathogen DNA present in the urine sample and the specific probe is visualized by a color precipitate. The diagnostic strip includes a probe for the β-globin gene already known in the art, as amplification internal control. Detection zones are located in the diagnostic strip according to a spatial pattern (FIG. 4), established so that the diagnostic strip is compared with said pattern to determine the presence or absence of each specific pathogen.

(10) More specifically, the present application describes a diagnostic kit to simultaneously detect at least 7 silent Sexually Transmitted Diseases (STDs), which comprises a diagnostic strip, comprising detection zones with at least 8 probes of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24; at least 14 primers of SEQ ID NOs: 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23; instruction manual.

(11) The present specification also describes SEQ ID NOs: 1 to 18, each one separately, and sequences having at least 90% similarity thereof.

(12) The present application also describes a set of 24 nucleotide sequences described in detail in Table 1, the first 18 nucleotide sequences, SEQ ID NOs: 1 to 18, were designed specifically for this invention. Sequences 19 to 21 are known in the art for HPV detection, and sequences 22 to 24 are known in the art for β-globin detection. The nucleotide sequences of 2 primers and 1 probe, respectively, for each target pathogen to be detected are also described. The primers are useful to carry out an amplification of specific segments by Multiplex PCR amplification step, while the probe is useful to perform the detection of said amplified segments.

(13) TABLE-US-00001 TABLE 1 SEQ ID PATÓGEN Target Sequence (5′3′) NO Human DNA PRIMER TGGCCAAGCTGACGGACATTT  1 herpes virus polymerase PRIMER 5DigN/GAGAGCTTGATCTTGTCGGTT  2 1 gene PROBE /5AmMC6/TAAAGGTGAACGGCATGGTGAGCA  3 Human Virion PRIMER TGTTTCTGGGCAGCTGTATC  4 herpes virus glycoprotein PRIMER 5DigN/CTATCGACGTTAGGGAAGGCAT  5 2 I and virion PROBE /5AmMC6/CATAGATGCCAGCGCCGATACAGG  6 glycoprotein E gene Chlamydia Major outer PRIMER TCAAGGAGTGGAGTGTCTGCGTA  7 trachomatis membrane PRIMER 5DigN/TGTCGCTCCGA TGCAGATGTTT  8 protein Gene PROBE /5AmMC6/ATAGGGAGTGTTTCTCGCCAAGCT  9 Ureaplasma 16S RNA PRIMER GCAGGCGGGTTTGTAAGTTTGGTA 10 urealyticum ribosomal PRIMER 5DigN/AGCCTAAGCGTCAGTGATAGTCCA 11 gene PROBE /5AmMC6/ATAGGAAGAACACCGGTGGCGAA 12 Mycoplasma 16S RNA PRIMER TGGAGAATCACTGACGCAGCTAAC 13 hominis ribosomal PRIMER 5DigN/TGCGAAGGATGTCAAGAGTGGGTA 14 gene PROBE /5AmMC6/CCGCCTGAGTAGTATGCTCGCAAG 15 Mycoplasma 30S PRIMER CGCAACGCTCAGGTGTCTAATGT 16 genitalium ribosomal PRIMER 5DigN/AGAGCTTGG CGCATTGCTGA 17 protein S3 PROBE /5AmMC6/TCGGCTCTCCGATGTTATCAAGTAGGA 18 HPV Virus region PRIMER CGTCCMARRGGAWACTGATC 19 L1 PRIMER 5DigN/GCMCAGGGWCATAAYAATGG 20 PROBE /5AmMC6/TTTGTTACTGTTGTGAGATACCACTCGCAG 21 β-globine Constitutive PRIMER 5DigN/GAAGAGCCAAGGACAGGTAC 22 gene PRIMER CAACTTCATCCACGTTCACC 23 PRIMER /5AmMC6/TAAGCAAATAGATGGCTCTGCCCT 24

(14) The present application also describes a set consisting of 24 sequences that have at least 90% similarity to the sequences of the set described in Table 1.

(15) Sequences 19 to 24 are known in the art. They are herein described since HPV and β-globin detection is required as a control, together with the detection of the rest of the pathogens of the group: Herpes Virus type 1, Herpes Virus type 2, Chlamydia trachomatis, Ureaplasma urealitycum, Mycoplasma hominis, Mycoplasma genitalium.

(16) Primers having SEQ ID NOs: 2, 5, 8, 11, 14, 17, 20 and 22 are labeled at their 5′ end with Digoxigenin molecule. This marker molecule allows subsequently detect the hybridized sequences, thus identifying the corresponding pathogen.

(17) Probes having SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24 have a modification at the 5′ end (Amino Modifier C6, AmMC6) which allows their adhesion to the nylon membrane in the diagnostic strip.

(18) The present application describes the use of SEQ ID NOs: 1 to 24, for the preparation of a diagnostic kit comprising the same, useful for the detection of pathogens causing sexually transmitted diseases. Another embodiment of the present application is the use of the kit to detect at least pathogens of group: Herpes Virus type 1, Herpes Virus type 2, Chlamydia trachomatis, Ureaplasma urealitycum, Mycoplasma hominis, Mycoplasma genitalium, and Human papillomavirus (HPV).

(19) Sequences SEQ ID NOs: 1, 2, 3 are used for the detection of Human Herpes virus 1.

(20) Sequences SEQ ID NOs: 4, 5, 6 are used for the detection of Human Herpes virus 2.

(21) Sequences SEQ ID NOs: 7, 8, 9 are used for the detection of Chlamydia trachomatis.

(22) Sequences SEQ ID NOs: 10, 11, 12 are used for the detection of Ureaplasma urealyticum.

(23) Sequences SEQ ID NOs: 13, 14, 15 are used for the detection of Mycoplasma hominis.

(24) Sequences SEQ ID NOs: 16, 17, 18 are used for the detection of Mycoplasma genitalium.

(25) Sequences SEQ ID NOs: 19, 20, 21, known in the art, are used for the detection of Human papillomavirus (HPV).

(26) Sequences SEQ ID NOs: 22, 23, 24, known in the art, are used for the detection of β-globin gene, a constitutive gene that is used to control the correct extraction of DNA from the sample.

(27) The present application also describes a method for simultaneously detecting at least 7 Silent Sexually Transmitted Diseases (STDs), which comprises the steps of: i) extracting DNA from a body fluid sample by cell lysis, and centrifuging to obtain a pellet containing DNA samples to be amplified; wherein body fluids are selected from urine, blood, saliva, urethral secretion, seminal fluid, vaginal secretion; ii) amplifying by Multiplex PCR using the primers described in SEQ ID NOs: 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23; copies of DNA segments complementary to said primers are amplified; iii) hybridizing the amplified DNA in step ii), using the probes described in SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, by a hybridization reaction between the copies obtained in amplification of step ii) using said probes; iv) detecting the presence or absence of pathogens of interest on a nylon membrane comprising the probes of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, spatially arranged according to a spatial pattern previously determined.

Example

(28) Step 0:

(29) Preparation of urine sample for DNA extraction, transport medium removal.

(30) The sample was transferred to a 15 mL falcon tube, until a pellet from the total urine sample was obtained.

(31) It was centrifuged at 4,000 rpm for 15 min at room temperature (RT) at 20 to 22° C. The supernatant was removed, taking care not to detach the pellet.

(32) 1. 300 μL of Saline Phosphate Buffer (PBS) or Dubelcco Saline Phosphate Buffer (DPBS) was added. Between 300 and 600 μL of PBS can be added, depending on the pellet size.

(33) 2. The sample was then divided into two 1.5 mL Eppendorf tubes.

(34) 3. It was centrifuged at 12,000 rpm, for 5 min at RT.

(35) 4. The supernatant was removed carefully.

(36) 5. 500 μL of Lysis Solution was added.

(37) 6. It was left 12 hours at RT, and DNA extraction was continued.

(38) Step i

(39) DNA Extraction

(40) After removing the transport medium and adding 500 mL of lysis buffer to begin cell lysis, an extraction procedure was performed according to the steps described below:

(41) Note: Samples must have a minimum volume of 500 mL at the beginning of the extraction process.

(42) 1. Adding 250 mL of 100% ethanol. Mixing vigorously in vortex.

(43) 2. The extraction column was inserted into the 2 mL collection tubes.

(44) 3. The total of the sample from step 1 was transferred in the extraction column including any precipitate that may have been formed.

(45) 4. Centrifuging at maximum speed (>10,000×g) for 1 minute.

(46) 5. The filtrate was removed and the collection tube was reused.

(47) 6. 500 mL of HBC Buffer (Wash Solution 1) was added.

(48) 7. HBC Buffer was diluted with isopropanol before use

(49) 8. Centrifuging at maximum speed for 30 seconds.

(50) 9. Filtrate and collection tube were removed.

(51) 10. The extraction column was inserted into a new 2 mL collection tube.

(52) 11. 700 mL of DNA Wash Buffer (Wash Solution 2) was added. DNA Wash Buffer was diluted with 100% ethanol before use.

(53) 12. Centrifuging at maximum speed for 30 seconds.

(54) 13. Filtrate was removed and the collection tube was reused.

(55) 14. Steps 10-12 were repeated for a second wash step with DNA Wash Buffer (Wash Solution 2).

(56) 15. The empty extraction column was centrifuged at maximum speed for 2 minutes to dry the column.

(57) 16. 100 mL of Elution Buffer (Elution Solution) preheated to 70° C. was added.

(58) 17. It was left at room temperature for 2 minutes.

(59) 18. It was centrifuged at 15,000 g for 1 minute.

(60) Stage ii.

(61) Amplification of the extracted DNA.

(62) This step was performed to increase the number of genome copies of pathogens from the group of interest to be detected. This is achieved with the use of primers of SEQ ID NOs: 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 23 and 24 described in Table 1. Which are labeled at their 5′ end with a Digoxigenin molecule that is reacted in the next step (hybridization).

(63) These primers are contained in the PCR Mix.

(64) 1. 20 μL of PCR Mix was distributed in each PCR tube.

(65) 2. Adding: 5 μL of sample to each tube 5 μL Positive control 5 μL Blank Control

(66) 3. Tubes were capped. Vortex for 3 sec. watching that there were no bubbles left.

(67) 4. Tubes were placed in a thermal cycler with the following program: Denaturation: 94° C./3 min. 34 cycles: 94° C./30 sec, 56° C./30 sec, 72° C./30 sec. Extension: 72° C./3 min.

(68) Step iii.

(69) Amplified DNA Hybridization.

(70) In this step, the amplified genome copies of step ii were reacted with the probes of SEQ ID NO 3, 6, 9, 12, 15, 18, 21 and 24 described in Table 1.

(71) Method:

(72) Hybridization

(73) (Method time: 3 h 30 min)

(74) Important: Before Starting the Method:

(75) a. Hybridization solution, Wash Solution 3, Wash Solution 4 and Wash Solution 5 were heated and maintained at 42° C. until used.

(76) A. PCR Product Denaturation: 1. 85 μL of Hybridization Solution was added to a 0.5 mL tube and then 15 μL of the PCR product was added. The tube was well capped. 2. Denaturing at 96° C. for 10 min. The tubes of the Thermocycler or thermal block were removed and immediately cooled on ice for 5 min.

(77) Extreme care was taken on maintaining the tubes closed to prevent amplicon contamination; especially when working with 0.2 mL tubes. 3. Centrifuging the tubes at 10,000×g for 10 sec before opening to avoid contamination.

(78) B. Diagnostic Strip Hydration While the tubes were cooled on ice (step 2, above), 200 μL of Hybridization Solution was added in an 8-channel tray, the diagnostic strips were added and hydrated at room temperature for 5 min. The diagnostic strips were removed with a tweezer from the tube containing the same.

(79) C. Hybridization of PCR Products on Diagnostic Strips 1. Denaturated PCR products were added (100 μL) on each diagnostic strip, and incubated at 42° C. for 60 min gently shaking, avoiding bubbles. 2. Hybridization Solution was removed with a sterile plastic Pasteur pipette 3. 1 mL of Wash Solution 3 was added. Incubated at 42° C. for 10 min with stirring. Step 3 was repeated. 4. Wash Solution 3 was removed with a sterile plastic Pasteur pipette and 1 mL of Wash Solution 4 was added. It was incubated at 42° C. for 2 min with shaking. 5. Conjugate Solution was prepared by adding 1.3 μL of Conjugate to the Conjugate Solution. It was mixed inverting (NO Vortex). 6. Wash Solution 4 was removed with a sterile plastic Pasteur pipette and 0.5 mL of freshly prepared Conjugate Solution was added. It was incubated at 42° C. for 30 min with shaking. Conjugate Solution was removed. 7. 1 mL of Wash Solution 5 was added. Incubated at 42° C. for 5 min with shaking. This step was repeated. 8. Wash Solution 5 was removed.

(80) D. Development of the Diagnostic Strip 1. 0.5 mL of Substrate Solution was added. It was incubated at 42° C. for 30 min in darkness without shaking. 2. Reaction was stopped by adding approximately 1 mL of distilled water with a pipette. This step was repeated at least twice. 3. Water was removed by inversion taking care not to sweep along the diagnostic strips. 2 mL of distilled water was added, left to stand for at least 30 min. 4. The diagnostic strips were removed with tweezers and placed on a paper sheet to dry. Once dried they were covered with a scotch tape to store them. Optional: can be digitized. 5. The results are obtained immediately. The diagnostic strip was placed parallel to the pattern (FIG. 4) included in the kit.

(81) Step iv

(82) Results and Interpretation of the Diagnostic Strip

(83) The diagnostic strip contains specific probes to detect pathogens: Herpes Virus type 1, Herpes Virus type 2, Chlamydia trachomatis, Ureaplasma urealitycum, Mycoplasma hominis, Mycoplasma genitalium and Human papillomavirus (HPV). These probes are immobilized in the diagnostic strip, allowing a simple reading from visualizing a mark in a specific spatial zone in the strip.

(84) Controls POSITIVE: there must be presence of bands for all the pathogens contained in the kit, including β-globin band. BLANK: absence of bands.

(85) This example was performed twice, a first time using the primers and probes described in Table 1 of this Kit-U, and a second time using the primers and probes contained in a commercial laboratory kit. It allowed to obtain comparative results (FIGS. 1, 2, 3) regarding he detection sensitivity obtained with the sequences described in the present application compared to the sensitivity obtained using the sequences of a commercial laboratory kit.

(86) The results can be read by facing each diagnostic strip to the pattern containing the ordered probes shown in FIG. 4, trying to match the baseline to ensure the correct interpretation of the results.

(87) A comparative analysis versus the Commercial Laboratory Kit was carried out to validate this Kit-U, samples were taken from 47 patients who donated two urine samples each. Both samples were taken at the same time.

(88) FIG. 1 shows a comparison of sensitivity between KIT-U vs. Commercial Laboratory Kit. Using the Kit-U it was possible to obtain enough DNA from 100% of the urine samples to obtain a result. In contrast, the analysis performed using the commercial laboratory kit did not detect the presence of DNA in 82.6% (n=38) of the samples.

(89) FIG. 2 shows the pathogens detected using the Kit-U. It is observed that Kit-U was able to detect the presence of at least one pathogen in 78.2% (n=36) of the analyzed samples, with only 21.8% (n=10) of negative cases.

(90) In contrast, when using the commercial laboratory kit—see results in FIG. 3A—from the total samples (n=8) where DNA was detected, 75% (n=6) were negative for the pathogens evaluated and only 25% (n=2) were positive for Mycoplasma hominis.

(91) This shows that the primers and probes described in SEQ ID NOs: 1 to 18 comprised by the Kit-U, provide improved sensitivity compared to the primers and probes of the Commercial Laboratory Kit.

(92) It is important to mention that these sequences of primers and probes designed for this Kit-U, SEQ ID NOs: 1 to 18, together with the additional sequences 19 to 24 known from the art and using the detection method, can be used with any biological sample from which amplifiable DNA can be obtained, preferably fluids, such as blood, saliva, urethral secretion, seminal fluid, vaginal secretion.