HK1244510B - Rapid genotyping analysis and kits thereof - Google Patents

Description

Rapid and sensitive genotyping and nucleic acid detection

This application is a divisional application of the invention patent application with application number 201480027795.3 and invention name “Rapid and sensitive genotype identification and nucleic acid detection”, which was filed on March 17, 2014 and entered the Chinese national phase on November 3, 2015, with PCT international application PCT/CN2014/000275.

Related applications

This application claims priority based on U.S. Provisional Patent Application No. 61/791,933, filed on March 15, 2013, the entire contents of which are incorporated herein by reference.

Technical Field

The present invention relates to the field of identifying various genotypes associated with human diseases.

Background Art

The present invention relates to identifying different genotypes. Techniques such as sequence-specific primer-polymerase chain reaction (SSP-PCR), DNA sequencing, DNA fingerprinting, and single nucleotide polymorphism (SNP) genotyping have been applied to genotyping (PCT application publication number WO/2011/139750). Among these techniques, single nucleotide polymorphism (SNP) genotyping has a higher discrimination power. However, most genotyping tests involving DNA hybridization require high operating costs and long operation times. Therefore, it is necessary to develop more efficient genotyping methods that are faster, cheaper, and cover more genotypes.

Mycobacterium tuberculosis (MTB) testing

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (MTB). The emergence of drug-resistant Mycobacterium tuberculosis (DR-MTB) is a serious problem in TB control programs in both industrialized and developing countries. In addition to its clinical and epidemiological importance, drug-resistant TB (DR-MTB) has a significant economic impact due to its higher treatment costs than conventional MTB. DR-MTB is defined as resistance to at least two of the most effective first-line drugs, including rifampin (RIF) and isoniazid (INH). DR-MTB cases are increasing globally, resulting in significant morbidity and mortality. Resistance to first-line anti-TB drugs has been associated with mutations in several MTB genes: rpoB (resistance to RIF), katG, and inhA (resistance to INH).

Several different technologies are available for detecting DR-MTB. While traditional phenotypic drug susceptibility testing (DST) remains the "gold standard" for testing MTB for drug resistance, DST can take up to eight weeks to complete. Mycobacterium tuberculosis is a slow-growing bacterium. MTB divides only every 15-20 hours and typically requires four to six weeks to grow into colonies, depending on the medium used. Afterward, once MTB cultured during DST is identified, drug susceptibility testing typically takes another two weeks. This prolonged DST process can lead to delays in treating the disease, especially since treatment is often initiated before DST is completed. Alternative cultures under development may shorten testing times; however, some sample types are not suitable for these alternative cultures. Conventional DNA sequencing is not commonly used in commercial settings to detect MTB drug-resistance mutations due to its time-consuming nature and the specialized expertise required. Kits using different technologies are commercially available, including real-time PCR and conventional hybridization for detecting a single MTB type or drug-resistant MTB. Real-time PCR, in particular, often struggles to detect multiple drug-resistant MTB simultaneously due to its limited number of available channels. While conventional hybridization is also suitable for detecting multidrug-resistant MTB, it requires long incubation times (at least 4 hours). In contrast, the present invention, by utilizing polymerase chain reaction (PCR) and "flow-through" hybridization technology, provides an array test that can simultaneously detect both single-drug-resistant and multidrug-resistant MTB on a single platform, with a detection time shorter than conventional hybridization (~35 minutes). The entire test, from DNA extraction from the sample to analysis, takes less than 4 hours, and the test results are highly specific and sensitive.

Testing for beta-thalassemia

Beta-thalassemia is a hemoglobin disorder caused by autosomal mutations and can be further categorized based on the degree to which beta-globin production is affected. Some mutations result in a mild underproduction of beta-globin (denoted as β ⁺ ), while others result in absolute silencing of the beta-globin gene, resulting in either no production or production of only dysfunctional beta-globin (denoted as ^β0 ). An estimated 1.5% of the world's population is heterozygous for the beta-thalassemia genotype; offspring of heterozygous parents may be homozygous. Beta-thalassemia has three degrees of severity: mild (thalassemia minor, β ⁺ /β or ^β0 /β), moderate (thalassemia intermediate, β ⁺ /β or ^β0 /β), and severe (thalassemia major, β ⁺ /β ⁺ , ^β0 /β ⁺ , or ^β0 / ^β0 ). Individuals who inherit beta-thalassemia primarily experience hypochromic anemia and may require lifelong blood transfusions. The best way to manage β-thalassemia is to prevent the birth of a child with it. This requires prenatal testing and genetic counseling for prospective parents; this has been shown to be the most effective approach to managing β-thalassemia. β-thalassemia is prevalent in temperate regions, such as Mediterranean countries, the Middle East, and Southeast Asia. The molecular basis of β-thalassemia has been extensively studied, revealing that point mutations in the β-globin gene vary in their pattern and frequency across different regions.

Current detection technologies include microarrays, allele-specific PCR (AS-PCR), and direct sequencing. Microarrays can simultaneously detect multiple target DNA sequences on a chip, making them suitable for simultaneous testing of multiple samples. However, their application is limited by their high cost, and the results cannot be visually interpreted, requiring high-performance scanners and complex analysis software for interpretation. Allele-specific PCR uses allele-specific primers to detect polymorphisms or mutations. In this technique, only oligonucleotides that perfectly match the target DNA can act as primers to amplify the target DNA. This technique is limited by its limited throughput within a single reaction. Direct DNA sequencing involves an in vitro DNA replication process in which DNA polymerase selectively incorporates chain-terminating dideoxynucleotides. This technique has low throughput and requires lengthy operation times, resulting in low detection efficiency. The present invention, on the other hand, provides a DNA test that combines PCR and reverse dot-blot hybridization to provide highly sensitive and specific detection of β-globin gene mutations, assisting in the diagnosis of β-thalassemia.

Hepatitis B virus testing

Hepatitis B virus (HBV) is the smallest DNA virus, consisting of 3,000 nucleotides surrounded by a protein coat. HBV is transmitted through contact with the blood or body fluids of an infected individual. Approximately two billion people are infected with HBV; over 350 million of these individuals are carriers. Approximately one in five HBV infections will develop cirrhosis or liver cancer. HBV can be detected through a simple blood test for surface antigen or diagnosed using polymerase chain reaction (PCR) and measurement of HBV DNA. Furthermore, 10 HBV genotypes (A to J) and several subtypes have been identified through analysis of differences in HBV genome sequences. HBV genotypes and subtypes exhibit distinct geographical distributions. HBV genotypes differ in their pathogenicity and treatment. Determining HBV genotypes can provide information for the evaluation of chronic HBV infection and pre-treatment procedures. DNA sequencing is considered the gold standard for HBV genotyping. However, it is less efficient in detecting mixed genotypes. In addition, molecular DNA technology provides a specific method for highly sensitive detection of hepatitis B virus genotypes.

STD testing

Sexually transmitted infections (STDs) are common worldwide. According to a 2005 World Health Organization estimate, 4.48 million new cases of curable STDs (syphilis, gonorrhea, chlamydia, and trichomoniasis) occur annually, primarily among adults aged 15 to 49 worldwide. In Hong Kong, one in every 550 pregnant women was diagnosed with an STD (excluding HIV) in 2010. STDs can be transmitted to a pregnant woman and her child, either during pregnancy or after birth. Gonorrhea and chlamydia can cause infertility. Therefore, there is a potential need for STD testing among prospective couples and women before pregnancy. China also represents a potential market for STD testing, as the number of STD cases has increased dramatically over the past 20 years. Therefore, China needs rapid, low-cost, and accurate STD testing kits to help control STDs. Currently available STD testing kits can detect a single pathogen or three or four pathogens simultaneously, generally excluding human papillomavirus (HPV). Due to the increasing incidence of STDs, there is a need for simultaneous screening and detection of multiple STD pathogens.

Thrombophilia testing

Thrombophilia refers to an abnormal blood clotting condition that increases the risk of thrombosis. Research reports indicate that approximately 40% of patients with thrombophilia who develop thrombosis have genetic predispositions. This condition has been shown to increase the risk of cardiovascular diseases such as venous thrombosis and reproductive disorders such as recurrent miscarriage. There is also growing consensus that pregnant women with hereditary thrombophilia are more likely to experience adverse pregnancy outcomes, including recurrent miscarriage, intrauterine fetal death, intrauterine growth retardation, preeclampsia, and placental abruption.

Over the past two decades, several gene mutations have been identified as associated with inherited thrombophilia. The four most common mutations are Factor V Leiden (FVL) [1691G>A], Factor II (FII or Factor II) [20210G>A], Methylenetetrahydrofolate Reductase (MTHFR) [677C>T], and Methylenetetrahydrofolate Reductase (MTHFR) [1298A>C]. Testing for these gene mutations can help identify individuals at high risk for thrombophilia and recurrent miscarriage, thereby helping individuals prevent and reduce their risk and providing guidance for preventive treatment.

Summary of the Invention

SNP genotyping as a diagnostic tool

In addition to DNA fingerprinting, SNP genotyping can also be used to identify gene segments, polymorphic genes, genes with alterations, or genes with functional problems. The present invention provides methods and kits for rapidly and unambiguously identifying infectious pathogens or genetic diseases caused by the presence or absence of specific DNA sequences.

As discussed above, commercially available hybridization formats require analysis using high-resolution image analyzers. Membrane-based allele-specific oligonucleotide probe-reverse dot hybridization (ASO-RDB) flow-through hybridization formats (e.g., U.S. Patent No. 5,741,647) can be applied to SNP genotyping. Microarray hybridization formats produce visible spots and can be analyzed visually and/or using less expensive image analyzers.

Flow-through hybridization

DNA hybridization is always the most important method that modern biological science studies and relates to gene molecular biology research.Various genomic nucleotide sequence complexity can be revealed and analyzed by hybridization in solution of specific complementary DNA sequence.In brief, target DNA is amplified or digested, and makes it hybridize with complementary DNA probe on solid support medium (such as nitrocellulose membrane).But traditional hybridization technique is limited to the area of film in principle, and usually needs longer incubation time.

Flow-through hybridization methods and equipment allow for precise control of hybridization conditions, eliminating the time constraints of traditional hybridization techniques. Flow-through DNA hybridization technology can reduce hybridization times from hours or days to minutes (the entire hybridization assay can be completed in 5-30 minutes, depending on the method used to generate the detection signal). Flow-through hybridization equipment is inexpensive to manufacture and uses 10 times less reagent volume than traditional hybridization equipment, making DNA detection technology more affordable than ever before. Flow-through hybridization technology provides more sensitive and accurate detection and identification results and is widely applicable to hybridization using a variety of different techniques, such as traditional Southern blotting, Northern blotting, dot blotting, slot blotting, and reverse dot blotting.

PCT application WO/2011/139750 describes a system of multiple lateral rapid flow-through detection devices connected to a central control unit. The hybridization device includes a central control unit connected to one or more lateral flow devices. The lateral flow devices perform hybridization and development procedures and are controlled and powered by the central control unit. A single lateral flow device or several devices can simultaneously test multiple reactions (or multiple samples and/or analytes) and perform procedures under different conditions under independent control. The lateral flow devices can be in the form of an "n x m" array (matrix) or a linear array. Further descriptions of methods and devices for performing flow-through hybridization can be found in U.S. Patents 5,741,647, 6,020,187, and PCT application WO/2011/139750. Those skilled in the art can practice the present invention using similar flow-through hybridization techniques described in U.S. Patents 5,741,647 or 6,020,187, or any novel method or device capable of performing flow-through hybridization techniques. Imaging systems, such as FT-Pro, attached to the hybridization devices can digitize signal detection. The hybridization device of the present invention can be an automated device, and its built-in digital function can be used to perform all analyses. The flow-through hybridization method has higher efficiency, accuracy and sensitivity than traditional hybridization techniques.

Drug-resistant Mycobacterium tuberculosis (MTB) detection

The present invention provides methods and kits for detecting Mycobacterium tuberculosis (MTB) resistant to rifampicin (RIF) and isoniazid (INH) using PCR and flow-through hybridization techniques. Mutations in the rpoB gene of the β subunit of MTB RNA polymerase are associated with resistance to rifampicin; mutations in the katG gene (catalase) and the INHA gene (enoyl ACP reductase) are associated with resistance to isoniazid. In the present invention, specific primers are used to amplify the INHA, rpoB, and katG genes; the specific primers have been shown to have no cross-reaction with the human genome. The primer RS-IAC serves as an internal control, which does not target any human or MTB genome, and is used to monitor the PCR amplification process. The probe set includes ten DNA probes for detecting drug resistance mutations in rpoB (D516V, D516G, H526D, H526Y, H526L1, S531L, and S531W), katG (S315T1 and S315T2), and inhA (-15TC/T). The probe set also includes five DNA probes for detecting the wild-type MTB genome, which helps verify that PCR amplification of the rpoB, katG, and inhA genes has been completed. Hybridization primers can be biotinylated for support.

Beta-thalassemia testing

The present invention provides a method and a kit for detecting beta-globin gene mutations, wherein the mutations include detection of TATA-28 (A>G), TATA-29 (A>G), start codon (G>A), codon 5 (-CT), codon 8/9 (+G), codon 15 (G>A), codon 16n (-C), codon 17 (A>T), codon 19 (A>G), codon 26 (G>A), codon 27/28 (+C), codon 30G>C, IVS1.1 (G>T), IVS1.1 (G>A), IVS1.5 (G>C), codon 41/42 (-TCTT), codon 43 (G>T), codon 71/72 (+A), IVS2.1 (G>A), IVS2.654 (C>T) and a 619 bp deletion.

Hepatitis B virus testing

The present invention provides methods and kits for detecting whether eight hepatitis B virus genotypes (hepatitis B virus genotypes A, B, C, D, E, F, G and H) are present in a patient's serum sample.

Testing for STD pathogens

The present invention provides methods and kits for detecting the presence of protozoa, bacteria, and viruses in various specimens, such as urine, urogenital swabs (urethra, vagina, cervix and various lesions) and liquid-based cytology specimens (PreservCyt ^™ and SurePath ^™ ). Amplification controls (ACs) include those for detecting human DNA to check that the results have sufficient cell content and validity. ACs can be used to monitor the presence of PCR inhibitors and the adequacy of the amount of extracted DNA during the PCR amplification process. The absence of an AC signal indicates the presence of inhibitors or insufficient DNA amount (or absence of the specimen) during the PCR amplification process. The positive control (PC) contains a positive template nucleic acid molecule to monitor the performance of the PCR reagents.

DNA hybridization and PCR technologies can more quickly detect STD pathogens in urine, urogenital swabs (urethra, vagina, cervix, and various lesions), and liquid-based cytology specimens (PreservCyt™ and SurePath™). In one embodiment, the method and kit can detect 12 common STD pathogens: one protozoan (Trichomonas vaginalis), seven bacteria (Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealyticum, Sporozoites, and Treponema pallidum), and four viruses (Herpes simplex virus types 1 & 2, and human papillomavirus types 6 and 11). These pathogens are all associated with cervicitis, urethritis, trichomoniasis, and pelvic inflammatory disease.

The universal primers and pathogen-specific primers provided by the present invention are used to detect sexually transmitted disease pathogens. Universal primers are used to achieve simultaneous PCR balanced amplification of multiple different targets. The universal primer sequence has been tested for specificity by PCR and gel electrophoresis to prove that there is no cross-reaction with other pathogens and the human genome. Two universal primer sequences are added to the 5' end of the pathogen-specific primer and the AC-specific primer, respectively. The universal primer and pathogen-specific primer tags are further tested by PCR and gel electrophoresis to prove that there is no cross-reaction with other pathogens and the human genome (Figure 1). In one embodiment, the pathogen-specific primer and two universal sequence tags (primers) are included in a single PCR amplification. In the first round of PCR, the pathogen-specific primer binds to its specific target and creates a PCR amplification product carrying the universal sequence. Then, in the second round of PCR, the universal primer binds to the PCR product of the universal sequence tag (Figure 2).

Under optimized PCR, balanced amplification can be achieved to avoid biased results due to varying amplification efficiencies of different target pathogen DNA sequences. This embodiment also ensures that pathogen amplification products are generated exclusively by universal primers, enabling more accurate detection with DNA probes. The universal primer system described herein can also be applied to other methods and kits for detecting DR-MTB, beta-globulin, hepatitis B virus, thrombophilia, and other disease-related nucleic acids or genes not described in this invention.

Thrombophilia testing

The present invention provides a method for detecting gene mutations associated with hereditary thrombotic tendency, wherein the gene mutations include Factor V Leiden (FVL) [1691G>A], Factor II (FII or coagulation factor II) [20210G>A], Methylenetetrahydrofolate Reductase (MTHFR) [677C>T] and Methylenetetrahydrofolate Reductase (MTHFR) [1298A>C].

Although the validation data experiments of the present invention use PCR to amplify the sequence, any method that can produce sufficient amounts of a specific target sequence for identification and flow-through hybridization analysis can also be used. If the target sequence is obtained in sufficient quantities, PCR amplification may not be necessary. Detection can be achieved by detecting a label on the target DNA or the conjugate.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1a shows an electrophoretic image of PCR reactions of clinical samples amplified using universal primers (sequence numbers 285 and 286). Specific bands were observed only in positive samples containing pathogens (i.e., CT-positive, NG-positive, HPV-6, and HPV-11 positive), indicating that universal primers do not react with human DNA. Figure 1b shows an electrophoretic image of PCR reactions of human clinical samples amplified using pathogen-specific primers (sequence numbers 263-282) and universal primers (sequence numbers 285 and 286). Specific bands corresponding to specific pathogens were observed for each sample, indicating that the primers are highly specific to their corresponding pathogens. Similar situations can be observed in both single and multiple PCR reactions.

FIG2 shows an example of universal primers and gene-specific primers used in a single PCR amplification reaction.

Figure 3 shows a DR-MTB cassette (left) and the signal location of a DR-MTB assay according to an embodiment (right) (rules are for illustration purposes only). The IAC is an internal control that monitors the PCR amplification process. The HC is a hybridization control that monitors the hybridization process.

Figure 4 shows an example of the visual interpretation of different Mtb mutants.

Figure 5 shows one embodiment of a β-thalassemia cassette and signal locations (gridlines are for illustrative purposes only).

Figure 6a shows an example of explaining different β-thalassemia genotypes. Figure 6b shows the test results of various clinical samples using the β-thalassemia cassette.

Figure 7 shows an example of a hepatitis B virus test kit that detects the presence of eight hepatitis B virus genotypes (hepatitis B virus A, B, C, D, E, F, G, and H). Universal primers IAC and HC are also included.

FIG8 shows an example to explain different HBV genotypes.

Figure 9a shows a comparison of the sensitivity of a STD test kit of the present invention and another manufacturer. Figure 9b shows a comparison of the performance of the STD test kit of the present invention and another manufacturer on clinical samples.

Figure 10a shows an embodiment of a STD reagent array, a cassette format and a signal position. The chip can detect the presence of 12 common pathogens: 1 protozoan (Trichomonas vaginalis), 7 bacteria (Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma, Mycoplasma hominis, Ureaplasma urealyticum, Ureaplasma urealyticum, Treponema pallidum) and 4 viruses (Herpes simplex virus type 1 & 2, human papillomavirus type 6 and type 11). Amplification control (AC) is used to monitor the presence of inhibitors and the adequacy of the amount of extracted DNA during PCR amplification. The absence of an amplification control signal indicates that there are inhibitors in the PCR amplification or the amount of DNA is insufficient (or the specimen is not present). Treponema pallidum (TP) can be provided as a positive control (PC), which contains a positive template nucleic acid molecule to monitor the performance of the PCR reagent. Figure 10b shows an example of a STD reagent kit array.

FIG. 11 shows an example of a thrombophilia test kit array.

FIG. 12 shows an example of a thrombophilia test kit array and the interpretation of its test results.

Summary of the Invention

The following terms are used to describe the present invention. Unless otherwise specified, the terms used to describe the present invention should be given the ordinary meanings understood by those skilled in the art.

As used herein, "allele-specific oligonucleotide probe-reverse dot hybridization (ASO-RDB)" refers to detection using ASO-RDB target molecules that can be captured in a solid by hybridization methods.

As used herein, the term "flow-through hybridization" refers to a hybridization process utilizing the technology described in US Patent No. 5,741,647.

As used herein, the term "flow-through hybridization device" or "flow-through apparatus" refers to the apparatus described in US Pat. No. 6,020,187 and/or the lateral flow apparatus described in PCT Application No. WO/2011/139,750 or any flow-through apparatus subsequently designed.

Detection of pathogens and target DNA

The methods, primers, probes, and kits provided herein are useful for detecting various nucleic acids, mutations, substances, and pathogens/or diseases. In one embodiment, the present invention is used to detect the presence (alone or multiple) of tuberculosis, hepatitis B, hepatitis C, SARS, respiratory viruses, venereal pathogens, β-globin, thrombotic tendencies, and/or related nucleic acids. The PCR method of the method uses specific primers to generate an amplification product, which hybridizes with an oligonucleotide probe. The resulting hybridization profile will reflect the presence of nucleic acids, gene mutations, substances, pathogens, and/or diseases.

In one embodiment, the hybridization of amplification product and oligonucleotide probe adopts diversion method, transverse diversion method or reverse diversion method.In one embodiment, the oligonucleotide probe used for hybridization is fixed on film.In the embodiment of diversion method, the sensitivity of hybridization depends on the total area of the film that comprises the probe array.In the embodiment of transverse diversion method, the sensitivity of hybridization depends on the total cross-sectional area that comprises the film that the probe spans in the flow direction.

In one embodiment, the present invention provides a method for detecting the presence of nucleic acids and/or exogenous substances, comprising the following steps: (a) amplifying a nucleic acid template nucleic acid molecule from a sample of a subject, wherein the primers are selected from the group consisting of sequence numbers 174-181, 201-205, 241-244, 263-286, and 299-306 to produce one or more amplification products; and (b) hybridizing the amplification products with oligonucleotide probes, the probes comprising the group consisting of sequence numbers 182-200, 206-240, 245-260, 287-298, and 307-314, to produce a hybridization profile with one or more oligonucleotide probes to show whether the target nucleic acid and/or exogenous substances are present in the sample of the subject. In one embodiment, the number of selected primers is 2-49; the number of selected probes is 2-92. In another embodiment, the number of selected primers is 2-24; the number of selected probes is 2-35.

In another embodiment, the present invention provides a method for detecting the presence of nucleic acids and/or exogenous substances in a sample, the method comprising the following steps: (a) amplifying a template nucleic acid molecule from a sample from a subject, wherein the primers used are selected from sequence numbers: 174-181, 201-205, 241-244, 263-286 and 299-306 to produce sufficient single one or more amplification products; and (b) hybridizing the amplification products with oligonucleotide probes, the probes comprising single one or more oligonucleotide probes selected from sequence numbers 182-200, 206-240, 245-260, 287-298, and 307-314 to produce a hybridization spectrum showing whether the target nucleic acid and/or exogenous substances are present in the sample from the subject.

In one embodiment, the present invention provides a method for detecting multidrug-resistant Mycobacterium tuberculosis (DR-MTB) or its nucleic acid, the method comprising the following steps: (a) obtaining a sample containing a template nucleic acid molecule; (b) amplifying the template nucleic acid molecule using primers selected from primer sequence numbers 174-181 to obtain an amplification product; and (c) hybridizing the obtained amplification product with an oligonucleotide probe selected from sequence numbers 182-200, the hybridization result obtained indicating the presence or absence of multidrug-resistant Mycobacterium tuberculosis (DR-MTB) or its nucleic acid. In one embodiment, the present invention provides a method for detecting multidrug-resistant Mycobacterium tuberculosis (DR-MTB) or its nucleic acid, the method comprising the following steps: (a) obtaining a sample containing a template nucleic acid molecule; (b) amplifying the template nucleic acid molecule using primers selected from primer sequence numbers 174-181 to obtain an amplification product; and (c) hybridizing the obtained amplification product with an oligonucleotide probe selected from sequence numbers 182-200, the hybridization result obtained indicating the presence or absence of multidrug-resistant Mycobacterium tuberculosis (DR-MTB) or its nucleic acid. In one embodiment, the primer comprises a tag that generates a signal. In one embodiment, the amplification product is hybridized with a plurality of oligonucleotide probes by a flow-through method, a lateral flow-through method or a reverse flow-through method and/or related equipment.

In one embodiment, the above method can detect the presence of multidrug-resistant Mycobacterium tuberculosis (DR-MTB), or a nucleic acid having one or more of the following DR-MTB mutations: one of seven mutations in the rpoB gene; one of two mutations in the katG gene; and a mutation in the inhA gene. The present invention also provides a kit for detecting the presence of multidrug-resistant Mycobacterium tuberculosis (DR-MTB) or its nucleic acid, the kit comprising primers selected from sequence numbers 182-200 and oligonucleotide probes selected from sequence numbers 174-181. In one embodiment, the kit is used to detect the presence of multidrug-resistant mycobacteria (DR-MTB), or the nucleic acid of DR-MTB, and the kit comprises primers with sequence numbers 174-181 and oligonucleotide probes with sequence numbers 182-200.

The present invention also provides a method for detecting a β-globin gene mutation in a subject with β-thalassemia, comprising the following steps: (a) obtaining a sample comprising a template nucleic acid molecule from the subject; (b) amplifying the template nucleic acid molecule using primers with sequence numbers 201-205 to obtain an amplified product; and (c) hybridizing the obtained amplified product with an oligonucleotide probe selected from sequence numbers 206-240, wherein the hybridization result indicates the β-thalassemia genotype. In one embodiment, the method for detecting a β-globin gene mutation in a subject with β-thalassemia comprises the following steps: (a) obtaining a sample comprising a template nucleic acid molecule from the subject; (b) amplifying the template nucleic acid molecule using primers with sequence numbers 201-205 to obtain an amplified product; and (c) hybridizing the obtained amplified product with an oligonucleotide probe selected from sequence numbers 206-240, wherein the hybridization result indicates the β-thalassemia genotype. In one embodiment, the primer comprises a tag that generates a signal. In one embodiment, the amplified product is hybridized with a plurality of oligonucleotide probes using a flow-through method, a lateral flow-through method, or a reverse flow-through method and/or related equipment. In one embodiment, the above-mentioned method for detecting a β-globin mutation can detect one of 21 β-globin mutations.

The present invention also provides a kit for detecting a β-globin gene mutation, the kit comprising primers selected from SEQ ID NOs. 201-205 and oligonucleotide probes selected from SEQ ID NOs. 206-240. In one embodiment, the kit for detecting a β-globin mutation comprises primers selected from SEQ ID NOs. 201-205 and oligonucleotide probes selected from SEQ ID NOs. 206-240.

The present invention also provides a method for detecting hepatitis B virus or its nucleic acid, comprising the following steps: (a) obtaining a sample containing a template nucleic acid molecule; (b) amplifying the template nucleic acid molecule using primers selected from sequence numbers 241-244 to obtain an amplified product of hepatitis B virus; and (c) hybridizing the obtained amplified product with an oligonucleotide probe selected from sequence numbers 245-260, wherein the hybridization result indicates the presence or absence of hepatitis B virus or its nucleic acid. In one embodiment, the method for detecting hepatitis B virus or its nucleic acid comprises the following steps: (a) obtaining a sample containing a template nucleic acid molecule; (b) amplifying the template nucleic acid molecule using primers selected from sequence numbers 241-244 to obtain an amplified product; and (c) hybridizing the obtained amplified product with an oligonucleotide probe selected from sequence numbers 245-260, wherein the hybridization result indicates the presence or absence of hepatitis B virus or its nucleic acid. In one embodiment, the primer comprises a tag that generates a signal. In one embodiment, the amplified product is hybridized with multiple oligonucleotide probes using a flow-through method, a lateral flow-through method, or a reverse flow-through method and/or related equipment.

In one embodiment, the above method can detect hepatitis B virus or its nucleic acid, wherein the hepatitis B virus is selected from genotypes A to H. The present invention also provides a kit for detecting hepatitis B virus or its nucleic acid, comprising primers selected from sequence numbers 241-244 and oligonucleotide probes 245-260. In one embodiment, the kit for detecting hepatitis B virus or its nucleic acid comprises primers 241-244 and oligonucleotide probes 245-260.

The present invention also provides a method for detecting the presence of a sexually transmitted disease pathogen or its nucleic acid in a subject, the method comprising the following steps: (a) obtaining a sample comprising a template nucleic acid molecule from the subject; (b) amplifying the template nucleic acid molecule using primers selected from sequence numbers 263-286 to obtain an amplification product; and (c) hybridizing the obtained amplification product with an oligonucleotide probe selected from sequence numbers 287-298, the hybridization result indicating the presence of a nucleic acid from or that causes a sexually transmitted disease in the sample. In one embodiment, the present invention provides a method for detecting the presence of a sexually transmitted disease pathogen or its nucleic acid in a subject, the method comprising the following steps: (a) obtaining a sample comprising a template nucleic acid molecule from the subject; (b) amplifying the template nucleic acid molecule using primers selected from sequence numbers 263-286 to obtain an amplification product; and (c) hybridizing the obtained amplification product with an oligonucleotide probe selected from sequence numbers 287-298, the hybridization result indicating the presence of a nucleic acid from or that that causes a sexually transmitted disease in the sample. In one embodiment, the primer comprises a tag that generates a signal. In one embodiment, the amplified product is hybridized with a plurality of oligonucleotide probes by a flow-through method, a lateral flow-through method or a reverse flow-through method and/or related equipment.

In one embodiment, the method for detecting STD pathogens in a subject can detect the following STD pathogens: Trichomonas vaginalis, Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealyticum, Sporozoites, Treponema pallidum, Herpes simplex virus type 1, Herpes simplex virus type 2, Human papillomavirus type 6, and Human papillomavirus type 11. The present invention also provides a kit for detecting the presence of STD pathogens or nucleic acids thereof in a subject, the kit comprising primers selected from sequence numbers 263-286 and oligonucleotide probes selected from sequence numbers 287-298. In one embodiment, the kit for detecting STD pathogens or nucleic acids thereof in a subject sample comprises primers selected from sequence numbers 263-286 and oligonucleotide probes selected from sequence numbers 287-298.

The present invention provides a method for detecting a gene mutation associated with a thrombotic tendency, the method comprising the following steps: (a) obtaining a sample comprising a template nucleic acid molecule; (b) amplifying the template nucleic acid molecule using primers selected from SEQ ID NOs: 299-306 to obtain an amplified product; and (c) hybridizing the obtained amplified product with an oligonucleotide probe selected from SEQ ID NOs: 307-314, the hybridization result obtained indicating the presence or absence of a gene mutation associated with a thrombotic tendency. In one embodiment, the primers comprise a tag that generates a signal. In another embodiment, the method is used to detect a gene mutation associated with a thrombotic tendency, the method comprising the following steps: (a) obtaining a sample comprising a template nucleic acid molecule; (b) amplifying the template nucleic acid molecule using primers selected from SEQ ID NOs: 299-306 to obtain an amplified product; and (c) hybridizing the obtained amplified product with an oligonucleotide probe selected from SEQ ID NOs: 307-314, the hybridization result obtained indicating the presence or absence of a gene mutation associated with a thrombotic tendency. In one embodiment, the primers comprise a tag that generates a signal. In one embodiment, the amplified product is hybridized with a plurality of oligonucleotide probes by a flow-through method, a lateral flow-through method, a reverse flow-through method and/or related equipment.

In one embodiment, the method is used to detect the gene mutation relevant to thrombosis tendency, and the gene is selected from Factor V Leiden (FVL), prothrombin Factor II (FII or coagulation factor II), and Methylenetetrahydrofolate Reductase (MTHFR). The present invention also provides a test kit for detecting the gene mutation relevant to thrombosis tendency, the test kit comprising a primer selected from sequence numbers 299-306 and an oligonucleotide probe selected from sequence numbers 307-314. In one embodiment, the test kit comprises a primer selected from sequence numbers 299-306 and an oligonucleotide probe selected from sequence numbers 307-314.

By quoting the following experimental details, the present invention can be better understood. However, it should be understood by those skilled in the art that the examples provided are only for illustration and are not intended to limit the scope of the present invention. The scope of the present invention will be defined by the following claims.

Throughout this application, various references or publications are cited. The entire text and disclosures of these references or publications are incorporated into this application to more fully describe the present invention. It should be noted that the transitional term "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended, not excluding the presence of unrecited elements or method steps.

Example 1

Test Procedure

Separation steps for DNA, PCR amplification, hybridization, sample detection, and result analysis, as described in WO/2011/139750 or known to those skilled in the art, can be applied to the present invention. Various internal controls (ICs) can be included in the test assays to verify DNA samples, PCR amplification, and result visualization. The size and format of each flow-guiding membrane array can be customized to individual needs or to optimize reaction chamber usage for maximum cost savings.

Example 2

Genotyping multidrug-resistant tuberculosis

In one embodiment, the present invention provides a method and kit for genotyping DR-MTB. The method and kit utilize polymerase chain reaction (PCR) and flow-through hybridization techniques to detect Mycobacterium tuberculosis (MTB) resistant to rifampicin (RIF) and isoniazid (INH). The corresponding primers used to amplify the rpoB gene, katG gene, and INHA gene have been shown to have no cross-reaction with the human genome. RS-IAC is an internal control primer that does not target the human or MTB genome and is used to monitor the PCR amplification process. The probes used to detect drug-resistant gene mutations include the rpoB gene (D516V, D516G, H526D, H526Y, H526L1, S531L, and S531W), the katG gene (S315T1 and S315T2), and the inhA gene (-15TC/T). There are also five probes for detecting mutations in wild-type MTB, which helps verify whether the PCR amplification reaction of the rpoB gene, katG gene and inhA gene has been completed. In one embodiment, the primers are biotinylated to facilitate hybridization.

In one embodiment, the present invention provides a method for detecting multidrug-resistant Mycobacterium tuberculosis (DR-MTB), comprising the following steps: (a) obtaining a sample comprising a template nucleic acid molecule; (b) amplifying the template nucleic acid molecule with a primer selected from the group consisting of sequence numbers 174-181, thereby producing an amplification product; and (c) hybridizing the amplification product with a group consisting of oligonucleotide probes selected from the group consisting of sequence numbers 182-200, the resulting hybridization spectrum indicating the presence of multidrug-resistant Mycobacterium tuberculosis. In one embodiment, the present invention can detect multidrug-resistant Mycobacterium tuberculosis having one or more of the following mutations: seven mutations in the rpoB gene; two mutations in the katG gene; and one mutation in the inhA gene. In one embodiment, the amplification product is hybridized to a plurality of oligonucleotide probes by a flow-through method, a lateral flow-through method, or a reverse flow-through method.

Figure 3 shows a DR-MTB box (left) and the various positions of the probe and signal (right). IAC is the internal amplification control to monitor the PCR amplification process. HC is the hybridization control to monitor the hybridization process. Figure 4 shows an embodiment of an array configuration for detecting DR-MTB. In one embodiment, MTB strains that are resistant to RIF and isoniazid are detected. In another embodiment, MTB strains that are resistant to RIF and INH (multi-drug resistance) are detected. In one embodiment, MTB strains that are neither resistant to rifampicin (RIF) nor isoniazid (INH) are detected. The test includes various controls.

Primers

Primer ID Primer sequence (5'-3') 5' modification Serial number MTB-DR-Rpob-F TCAACATCCGGCCGGTGGTC Biotin 174 MTB-DR-Rpob-R CCGGCACGCTCACGTGACAGA Biotin 175 MTB-DR-KatG-F TGGCACCGGAACCGGTAAGGA Biotin 176 MTB-DR-KatG-R CGCCAGCAGGGCTCTTCGT 177 MTB-DR-inhA-F AAGTTCCCGCCGGAAATCGCAG Biotin 178 MTB-DR-inhA-R CCGGTAACCAGGACTGAACGGGAT 179 RS-IAC-F CGAGTTCCCGCCCATAACC 180 RS-IAC-R GGAAGTTGCAACGCCGTCC Biotin 181

probe

Probe ID Probe sequence (5'-3') 5' modification Serial number MTB-DR-Rpob-CTRL GCTGGTGCCGAAGAA Amine 182 MTB-DR-KatG-CTRL CGGGGTGTTCGTCCATAC Amine 183 MTB-DR-inhA-CTRL GGTATGTCCACGAGCGTAAC Amine 184 MTB-DR-Rpob-WTc GGTTGTTCTGGTCCATG Amine 185 MTB-DR-Rpob-Wte TGACCCACAAGCGCCGA Amine 186 MTB-DR-Rpob-Wtf GACTGTCGGCGCTGG Amine 187 MTB-DR-KatG-WT GATGCCGCTGGTGATCG Amine 188 MTB-DR-inhA-WT AACCTATCGTCTCGCCG Amine 189 MTB-DR-Rpob-MUTc1 GGTTGTTCTGGACCATGA Amine 190 MTB-DR-Rpob-MUTc2 GGTTGTTCTGGCCCATG Amine 191 MTB-DR-Rpob-MUTe1 GACCGACAAGCGCCGA Amine 192 MTB-DR-Rpob-MUTe2 GCGCTTGTAGGTCAACC Amine 193 MTB-DR-Rpob-MUTe3 GCGCTTGAGGGTCAAC Amine 194 MTB-DR-Rpob-MUTf1 CCCCAGCGCCAACAGT Amine 195 MTB-DR-Rpob-MUTf2 GACTGTGGGCGCTGG Amine 196 MTB-DR-KatG-MUT1 GATGCCGGTGGTGATC Amine 197 MTB-DR-KatG-MUT2 GATGCCTGTGGTGATCG Amine 198 MTB-DR-inhA-MUT1 CAACCTATCATCTCGCCG Amine 199 RS-IAC-P CTCGAATGCCTCGTATCAT Amine 200

Here are some examples for PCR and hybridization:

An example of a hybridization protocol:

Example 3

β-Med genotyping

The present invention provides a system for detecting β-globin gene mutations, wherein the gene mutations include detection of TATA-28 (A>G), TATA-29 (A>G), start codon (G>A), codon 5 (-CT), codon 8/9 (+G), codon 15 (G>A), codon 16 (-C), codon 17 (A>T), codon 19 (A>G), codon 26 (G>A) (hemoglobin E), codon 27/28 (+Three), codon 30G>C, IVS1.1 (G>T), IVS1.1 (G>A), IVS1.5 (G>C), codon 41/42 (-TCTT), codon 43 (G>T), codon 71/72 (+A), IVS2.1 (G>A), IVS2.654 (C>T) and 619bp deletion.

The present invention also provides a method for detecting a β-globin mutation in a sample from a subject, the method comprising the following steps: (a) obtaining a sample comprising a template nucleic acid molecule from the subject; (b) amplifying the template nucleic acid molecule with a primer selected from the group consisting of sequence numbers 201-205, thereby producing an amplified product; and (c) hybridizing the amplified product with a oligonucleotide probe selected from the group consisting of sequence numbers 206-240, the resulting hybridization spectrum indicating the β-thalassemia genotype. In one embodiment, the primer includes a signal-generating tag. In one embodiment, the amplified product is hybridized with a plurality of oligonucleotide probes by a flow-through method, a lateral flow-through method, or a reverse flow-through method. In one embodiment, the above method can detect any one of 21 β-globin mutations.

Figure 5 shows an embodiment of a β-thalassemia cassette and its signal locations (the grid lines in the figure are for illustrative purposes only). Figure 6a shows an example of different β-thalassemia genotypes. Figure 6b shows the test results for various clinical samples using the β-thalassemia cassette, demonstrating that the present invention can identify β-thalassemia genotypes in human samples.

Primer sequences (accession number AF007546.1):

Primer ID direction 5' modification Primer sequence (5' to 3') Serial number HBB primer 1 Forward primer Biotin GTACGGCTGTCATCACTTAGACCTCA 201 HBB primer 2 Reverse primer Biotin TTATCCCCTTCCTATGACATGAAC 202 HBB primer 3 Forward primer Biotin GTGTACACATATTGACCAAA 203 HBB primer 4 Reverse primer Biotin GCAAGAAAGCGAGCTTAG 204 HBB Primer 5 Reverse primer Biotin GTATCTCTAAGCAAGAGAAC 205

Probe sequence (accession number AF007546.1):

Probe ID Sense/antisense strand 5' modification Probe sequence (5' to 3') Serial number HBB2-A1 sense strand Amine AGACACCATGGTGCATCTG 206 HBB2-A1a sense strand Amine AGACACCATGGTGCACCT 207 HBB2-A2 sense strand Amine CAAACAGACACCAGGGTG 208 HBB2-A3 sense strand Amine GCTGGGCATAAAAGTCAGG 209 HBB2-A4 Antisense strand Amine CCTGACTTCTATGCCCAGC 210 HBB2-A5 Antisense strand Amine CCCTGACTTTCATGCCC 211 HBB2-B1 sense strand Amine GCATCTGACTCCTGAGGA 212 HBB2-B1a sense strand Amine GCACCTGACTCCTGAGG 213 HBB2-B2 Antisense strand Amine ACTTCTCCTCGAGTCAGAT 214 HBB2-B3 Antisense strand Amine CAGGGCCTCACCACCA 215 HBB2-B4 sense strand Amine GTTGGTGGTAAGGCCCT 216 HBB2-B5 Antisense strand Amine CCAGGGGCCTCACCA 217 HBB2-C1 sense strand Amine AGGAGAAGTCTGCCGTTACT 218 HBB2-C2 sense strand Amine AGGAGAAGGTCTGCCGTTA 219 HBB2-C3 sense strand Amine GAGGTTCTTTGAGTCCTTTGG 220 HBB2-C4 Antisense strand Amine CAAAGGACTCAACCTCTGG 221 HBB2-C5 sense strand Amine CCCAGAGGTTCTTTTAGTC 222 HBB2-D1 sense strand Amine TGTGGGGCAAGGTGAAC 223 HBB2-D2 sense strand Amine CCGTTACTGCCCTGTAGG 224 HBB2-D3 sense strand Amine CTGTGGGGAAGGTGAAC 225 HBB2-D4 Antisense strand Amine TGTGGGGCTAGGTGAACG 226 HBB2-D5 Antisense strand Amine CTTCATCCACGCTCACCT 227 HBB2-E1 Antisense strand Amine TGATACCAACCTGCCCAG 228 HBB2-E2 sense strand Amine CTGGGCAGATTGGTATCA 229 HBB2-E3 sense strand Amine CCCTGGGCAGTTTGGTATC 230 HBB2-E4 sense strand Amine GCAGGTTGCTATCAAGGTTAC 231 HBB2-E5 Antisense strand Amine ATACCAACGTGCCCAGG 232 HBB2-F1 sense strand Amine GGTGCCTTTAGTGATGGC 233 HBB2-F2 sense strand Amine TCGGTGCCTTTAAGTGATG 234 HBB2-F3 sense strand Amine TGGGTTAAGGCAATAGCAATAT 235 HBB2-F4 Antisense strand Amine ATATTGCTATTACCTTAACCC 236 HBB2-G1 sense strand Amine CATACCTCTTATCTTCCTCC 237 HBB2-G2 sense strand Amine TGTAACAAGTAGAGATTCAAGTA 238 HBB2-G3 sense strand Amine GAACTTCAGGGTGAGTCTAT 239 HBB2-G4 Antisense strand Amine GAACTTCAGGATGAGTCTATG 240

Below are some example protocols for PCR and hybridization:

Element Capacity (μL) PCR premix 19.6 Taq polymerase 0.4 Template nucleic acid molecule 2 to 5.0* Nuclease-free water variable total 25.00

An example of a hybridization protocol:

Data interpretation as beta-globulin mutations:

Example 4

Hepatitis B virus genotyping

The present invention also provides a method for detecting hepatitis B virus, the method comprising the steps of: (a) obtaining a sample comprising a template nucleic acid molecule; (b) amplifying the template nucleic acid molecule using a group consisting of primers selected from sequence numbers: 241-244, thereby producing an amplified product; and (c) hybridizing the amplified product with a group consisting of oligonucleotide probes selected from sequence numbers: 245-260, and the resulting hybridization spectrum will show the presence of hepatitis B virus. In one embodiment, the primer includes a signal-generating tag. In one embodiment, the amplified product is hybridized with multiple oligonucleotide probes by a flow-through method, a lateral flow-through method, or a reverse flow-through method. In one embodiment, universal probe sequence number 257 is used to capture nucleic acids of various genotypes of hepatitis B virus.

Figure 7 shows an example of eight hepatitis B virus genotypes (hepatitis B virus A, B, C, D, E, F, G and H) detected by a hepatitis B virus kit. Figure 8 shows an example of detecting different hepatitis B virus genotypes.

Primer sequences:

Probe sequence:

Hepatitis B virus positive control sequence [560bp] (pUC57 vector):

TGGGATTCTATATAAGAGGGAAACTACACGTAGCGCCTCATTTTGCGGGTCACCATATTCTTGGGAACAAGAGCTACATCATGGGAGGTTGGTCATCAAAACCTCGCAAAGGCATGGGGACGAACCTTTCTGTTCCCAACCC TCTGGGATTCTTTCCCGATCATCAGTTGGACCCTGCATTCGGAGCCAATTCAAACAATCCAGATTGGGACTTCAACCCCATCAAGGACCACTGGCCACAAGCCAACCAGGTAGGAGTGGGAGCATTCGGGCCAGGGTTCACT CCCCCACACGGAGGTGTTTTGGGGTGGAGCCCTCAGGCTCAGGGCATATTGGCCACAGTGCCAGCAGTGCCTCCTTCCTGCCTCCACCAATCGGCAGTCAGGAAGGCAGCCTACTCCCATCTCTCCACCTCTAAGAGACAGTC ATCCTCAGGCCATGCAGTGGAATTCCACAGCTTTCCACCAAGCTCTGCAAGATCCCAGAGTCAGGGGCCTGTATTTTCCTGCTGGTGGCTCCAGTTCAGGAACACTCAACCCTGTTCCAACTATTGCCTCTCAC (serial number 261)

Internal control sequence [500 bp] (pUC57 vector):

GAGACAGGTTCGTCCAATCCCGTGCCGGCCTTGGCAGGGGGTTCGCAGGCCCCACCCGAAGCGTTGCTGAAGGCTCAGGCCTCTGAGCGACAAAAGCTTTAAACGCGAGTTCCCGCCCATAACCT GGACCGAATGCGGGACCATGCATCGTTCCACTGTGTTTGTCCCATGTAGGACGGGCGCAAGGCGTGCTTAGCTCAGCCTCGAATGCCTCGTATCATTGTGCACCCGCCGGTCACCAGCCAACGATGT GCGGACGGCGTTGCAACTTCCGGGGCCCAACCTGACCGTCCTGGGTACCGCACTCTGGGCAGTGCGAGGTAATGCCAGTCGCCCAGTGCCGAACAACACCTGACCTAACGGTAAGAGGCTCACATAA TGGCTCCGCCGGCGCGCCCAGGGTACATTAGGTCAGCATCGGATGGACTGACATGAACCTTCAACCGAAGCGGAAACGGGTGCGTGGACCAGCGAGGAGCAAACGAAAATTCCTGGCC (serial number 262)

The following are some examples of PCR and hybridization protocols:

Element Capacity (μL) Hepatitis B virus primer premix 19.6 IAC 0.1 Taq polymerase 0.3 Template nucleic acid molecule 5.0* Nuclease-free water variable total 25.00

*It is recommended that the total HBV DNA level in serum be greater than 4000 IU/mL.

This temperature profile is suitable for Applied Biosystems PerkinElmer (ABI-PE) 9600, Gene Amplification PCR System 9700, Veriti (Applied Biosystems), and PTC-200 (MJ Research).

An example of a hybridization protocol:

Example 5

Genotyping and simultaneous detection of multiple STD pathogens

In one embodiment, the present invention provides methods for detecting STD pathogens, including but not limited to protozoa, bacteria, and viruses. In one embodiment, STD pathogens are present in urine, urogenital swabs (urethra, vagina, cervix, and lesions), and liquid-based cytology specimens (PreservCyt ^™ and SurePath ^™ ). In one embodiment, the amplification control (AC) comprises material for detecting human DNA. Target DNA extracted from urogenital swabs (urethra, vagina, cervix, and lesions) and liquid-based cytology specimens (PreservCyt ^™ and SurePath ^™ ) is amplified by PCR and then hybridized to achieve faster, more sensitive, and more specific detection of STDs. In one embodiment, the method can detect 12 common STD pathogens, including one protozoan (Trichomonas vaginalis), seven bacteria (Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealyticum, Sporozoites, and Treponema pallidum), and four viruses (Herpes simplex virus types 1 & 2, and human papillomavirus types 6 and 11). These pathogens are associated with cervicitis, urethritis, trichomoniasis, and pelvic inflammatory disease. In another embodiment, universal primers (S) and/or amplification controls are included in the test.

The present invention also provides a method for detecting the presence of sexually transmitted disease pathogens in a sample from a subject, the method comprising the following steps: (a) obtaining a sample comprising a template nucleic acid molecule from the subject; (b) amplifying the template nucleic acid molecule using a primer selected from the group consisting of SEQ ID NOs: 263-286, thereby producing an amplification product; and (c) hybridizing the amplification product with an oligonucleotide probe selected from the group consisting of SEQ ID NOs: 287-298, the resulting hybridization spectrum indicating the presence of nucleic acid fragments derived from the sexually transmitted disease pathogen. In one embodiment, the primer includes a signal-generating tag. In one embodiment, hybridization of the amplification product with multiple oligonucleotide probes is performed in a flow-through method, a lateral flow-through method, or a reverse flow-through method and/or related equipment.

Primers SEQ ID NOs. 263-284 consist of two parts: a 5' portion containing a synthetic nucleic acid sequence that is not homologous to the target pathogen and does not involve sequences from any organism found in humans; and a 3' portion containing a pathogen-specific sequence that enables the primers to effectively bind to the nucleic acid of the corresponding pathogen, thereby producing amplification products. The 5' portion of all primer sequences is a universal sequence. The universal primer sequences were carefully designed and experimentally designed to exclude the amplification of other organisms in the sample, such as other pathogens and any DNA from the human genome (Figure 1). Specific universal sequences, such as those disclosed in this invention, were selected and validated and then combined with various pathogen-specific sequences to produce pathogen-specific primers capable of producing amplification products containing the universal sequence. These amplification products serve as template nucleic acid molecules for a second PCR reaction, which then uses universal primers SEQ ID NOs. 285 and 286 to amplify all template nucleic acid molecules with the same efficiency. Therefore, the final concentration of amplification products generated linearly is proportional to the initial target DNA concentration. Even if the target copy number in the sample is low, it can still be amplified to reach a considerable concentration before detecting a positive result. The use of universal primers can correct errors and greatly improve sensitivity. In one embodiment, a two-step PCR reaction is performed in a single test tube; the pathogen-specific primers therein bind to the target molecule through their gene-specific 3' region to produce an initial amplification product containing a 5' universal sequence. These amplification products serve as template nucleic acid molecules for the second set of primers (universal primers) to produce amplification products containing signals and are detected by hybridization. Figure 2 shows an example of an amplification scheme.

Because the present invention uses universal primers, it is more accurate and sensitive than other STD pathogen detection kits on the market. As shown in Figure 9a, the present invention can detect STD pathogen copies as low as 50-300, while other manufacturers' kits require at least 500 STD pathogen copies. Therefore, the present invention is capable of detecting samples with lower pathogen concentrations.

Figure 9b shows a performance comparison of the present invention and other STD detection kits on the market. The results were confirmed by quantitative PCR (qPCR) for the four tests and the actual genotype. After confirming the genotype of the pathogen, the present invention (as shown in Tests 3 and 4 in the figure) had fewer false results than other kits (as shown in Tests 1 and 2 in the figure). In addition, the present invention can detect multiple pathogens (as shown in Figure 9b), so compared with other kits on the market, the present invention can detect 12 pathogens at the same time, which is the highest number. The incidence rate after infection with HPV6 and HPV11 is very high, so the coverage of HPV6 and 11 of the present invention is very useful. Genital warts are a highly contagious STD, of which 90% of cases are caused by infection with HPV6 and HPV11.

FIG10 a shows an embodiment of an array for detecting STDs. The array can detect the presence of 12 common STD pathogens: 1 protozoan (Trichomonas vaginalis), 7 bacteria (Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealyticum, Sporozoites, Treponema pallidum), and 4 viruses (Herpes simplex virus type 1 and type 2, Human papillomavirus type 6 and type 11). Amplification controls (AC) indicate whether the results are valid. FIG10 b shows an example of an array for explaining STDs. In one embodiment, protozoa, bacteria, and viruses are detected separately. In another embodiment, protozoa, bacteria, and viruses (multiple infections) are detected simultaneously.

In one embodiment, the template nucleic acid molecule of Treponema pallidum (TP) is provided as a positive control (PC) for monitoring the performance of PCR reagents. If the positive control (PC) signal of TP does not appear in a test, this may be because the DNA has degraded due to long-term storage or nuclease contamination. However, if the positive signal generated by the test sample is any pathogen, the test result is still valid. In the test of the positive control (PC), since the positive control sample only contains Treponema pallidum (TP), but no endogenous human genomic DNA, it will result in a lack of AC signal. The presence of an AC signal in the positive control (PC) test indicates that the control test is invalid, which may be due to contamination and the test should be repeated.

If the AC signal is absent, this indicates insufficient or absent DNA in the test sample. However, if the test sample generates a positive signal for any pathogen, the test is still valid. Due to competition from other pathogen DNA, the AC signal may not be present in some samples. In this case, as long as there is one or more positive signals on the array, the test result is still valid.

In another embodiment, the array cartridges and/or devices used in the diversion process have different formats and sizes. In addition to using a 10-spot array, a 35-spot array can also be used, and additional array spots can be added to detect, for example, additional pathogen subtypes and/or genetic mutations that indicate drug-resistant strains. The detection signal can also be digitized using an imaging system that accompanies the FT-Pro hybridization device. In another embodiment, the hybridization device used is an automated device that combines analysis and digitization capabilities.

This invention is more convenient than existing detection methods and kits on the market because it can simultaneously amplify and detect multiple targets, has high sensitivity, and covers 12 STD pathogens, including HPV subgenotypes excluded by most existing kits. By combining it with flow-through hybridization, this invention allows for rapid, sensitive, and high-throughput STD pathogen detection.

Primer sequences:

Probe sequence:

Below are some example PCR and hybridization protocols:

Element Capacity (μL) PCR premix 18.6 STD Primer Premix 1.0 DNA Taq polymerase 0.4 STD DNA is under control Up to 5.0* Nuclease-free water variable total 25.0

*Recommended range of total DNA: 5ng–100ng.

The above thermal cycling program is suitable for use with the Thermal Cycler (Life technologies) and the S1000 ^™ Thermal Cycler (Bio-rad). The above thermal cycling program can be modified for use with other thermal cyclers (ramp rates up to 3°C/second).

An example of a hybridization protocol:

Example 6

Genetic typing related to thrombotic tendency

The present invention also provides a method for detecting a gene mutation associated with a thrombotic tendency, the method comprising the following steps: (a) obtaining a sample comprising a template nucleic acid molecule; (b) amplifying the template nucleic acid molecule using a primer selected from sequence numbers 299-306 to obtain an amplified product; and (c) hybridizing the obtained amplified product with an oligonucleotide probe selected from sequence numbers 307-314, the hybridization result indicating the presence or absence of a gene mutation associated with a thrombotic tendency. In one embodiment, the primer comprises a tag that generates a signal. In one embodiment, the amplified product is hybridized with multiple oligonucleotide probes using a flow-through method, a lateral flow-through method, or a reverse flow-through method. In one embodiment, the above method can detect a gene mutation selected from Factor V Leiden (FVL), Factor II (FII or Factor II), and Methylenetetrahydrofolate Reductase (MTHFR).

Figure 11 shows an example of an array of a thrombophilia test kit. Figure 12 shows an example of interpretation of the test results of a thrombophilia test kit.

Primer sequences:

Probe sequence:

probe Probe sequence (5'-3') 5' modification Serial number FVL_WT GGACAGGCGAGGAAT Amine 307 FVL_MUT TGGACAGGCAAGGAAT Amine 308 FII_WT TCTCAGCGAGCCTC Amine 309 FII_MUT ACTCTCAGCAAGCC Amine 310 MTH677_WT CGGGAGCCGATTTCA Amine 311 MTH677_MUT ATGATGAAATCGACTCC Amine 312 MTH1298_WT CAGTGAAGAAAGTGTCT Amine 313 MTH1298_MUT GACCAGTGAAGCAAGTG Amine 314

Reaction mixture:

Element Capacity (μL) Thrombophilia PCR Master Mix 21.6 Thrombophilia Primer Premix 1.0 Enzyme mixture 0.4 Template nucleic acid molecule Up to 2.0 Nuclease-free water variable total 25.00

*The recommended range of total DNA amount is 10ng–100ng.

An example of a hybridization procedure:

Sequence Listing

<110> DiagCor Biotechnology Co., Ltd.

<120> Rapid and sensitive genotyping and nucleic acid detection

<130> 897-F-PCT-CN

<140>

<141>

<150> PCT/CN2014/000275

<151> 2014-03-17

<150> 61/791,933

<151> 2013-03-15

<160> 141

<210> 174

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 174

tcaacatccg gccggtggtc 20

<210> 175

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 175

ccggcacgct cacgtgacag a 21

<210> 176

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 176

tggcaccgga accggtaagg a 21

<210> 177

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 177

cgccagcagg gctcttcgt 19

<210> 178

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 178

aagttcccgc cggaaatcgc ag 22

<210> 179

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 179

ccggtaacca ggactgaacg ggat 24

<210> 180

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 180

cgagttcccg cccataacc 19

<210> 181

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 181

ggaagttgca acgccgtcc 19

<210> 182

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 182

gctggtgccg aagaa 15

<210> 183

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 183

cggggtgttc gtccatac 18

<210> 184

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 184

ggtatgtcca cgagcgtaac 20

<210> 185

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 185

ggttgttctg gtccatg 17

<210> 186

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 186

tgacccacaa gcgccga 17

<210> 187

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 187

gactgtcggc gctgg 15

<210> 188

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 188

gatgccgctg gtgatcg 17

<210> 189

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 189

aacctatcgt ctcgccg 17

<210> 190

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 190

ggttgttctg gaccatga 18

<210> 191

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 191

ggttgttctg gcccatg 17

<210> 192

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 192

gaccgacaag cgccga 16

<210> 193

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 193

gcgcttgtag gtcaacc 17

<210> 194

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 194

gcgcttgagg gtcaac 16

<210> 195

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 195

ccccagcgcc aacagt 16

<210> 196

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 196

gactgtgggc gctgg 15

<210> 197

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 197

gatgccggtg gtgatc 16

<210> 198

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 198

gatgcctgtg gtgatcg 17

<210> 199

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 199

caacctatca tctcgccg 18

<210> 200

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 200

ctcgaatgcc tcgtatcat 19

<210> 201

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 201

gtacggctgt catcacttag acctca 26

<210> 202

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 202

ttatcccctt cctatgacat gaac 24

<210> 203

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 203

gtgtacacat attgaccaaa 20

<210> 204

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 204

gcaagaaagc gagcttag 18

<210> 205

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 205

gtatctctaa gcaagagaac 20

<210> 206

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 206

agacaccatg gtgcatctg 19

<210> 207

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 207

agacaccatg gtgcacct 18

<210> 208

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 208

caaacagaca ccagggtg 18

<210> 209

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 209

gctgggcata aaagtcagg 19

<210> 210

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 210

cctgacttct atgcccagc 19

<210> 211

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 211

ccctgacttt catgccc 17

<210> 212

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 212

gcatctgact cctgagga 18

<210> 213

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 213

gcacctgact cctgagg 17

<210> 214

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 214

acttctcctc gagtcagat 19

<210> 215

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 215

cagggcctca ccacca 16

<210> 216

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 216

gttggtggta aggccct 17

<210> 217

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 217

ccaggggcct cacca 15

<210> 218

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 218

aggagaagtc tgccgttact 20

<210> 219

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 219

aggagaaggt ctgccgtta 19

<210> 220

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 220

gaggttcttt gagtcctttg g 21

<210> 221

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 221

caaaggactc aacctctgg 19

<210> 222

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 222

cccagaggtt cttttagtc 19

<210> 223

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 223

tgtggggcaa ggtgaac 17

<210> 224

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 224

ccgttactgc cctgtagg 18

<210> 225

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 225

ctgtggggaa ggtgaac 17

<210> 226

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 226

tgtggggcta ggtgaacg 18

<210> 227

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 227

cttcatccac gctcacct 18

<210> 228

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 228

tgataccaac ctgcccag 18

<210> 229

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 229

ctgggcagat tggtatca 18

<210> 230

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 230

ccctgggcag tttggtatc 19

<210> 231

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 231

gcaggttgct atcaaggtta c 21

<210> 232

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 232

ataccaacgt gcccagg 17

<210> 233

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 233

ggtgccttta gtgatggc 18

<210> 234

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 234

tcggtgcctt taagtgatg 19

<210> 235

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 235

tgggttaagg caatagcaat at 22

<210> 236

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 236

atattgctat taccttaacc c 21

<210> 237

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 237

catacctctt atcttcctcc 20

<210> 238

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 238

tgtaacaagt agagattcaa gta 23

<210> 239

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 239

gaacttcagg gtgagtctat 20

<210> 240

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 240

gaacttcagg atgagtctat g 21

<210> 241

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 241

acgyagcgcc tcattttgtg 20

<210> 242

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 242

cactgcatgg cctgaggat 19

<210> 243

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 243

cgagttcccg cccataacc 19

<210> 244

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 244

ggaagttgca acgccgtcc 19

<210> 245

<211> 14

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 245

ccacaagcca acca 14

<210> 246

<211> 14

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 246

ccagcagcca acca 14

<210> 247

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 247

gccaactcag aaaatc 16

<210> 248

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 248

gccaactccg acaatc 16

<210> 249

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 249

agaggcaaat caggta 16

<210> 250

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 250

agcggcacac caggta 16

<210> 251

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 251

ccaacaagga cacctg 16

<210> 252

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 252

ccaacaagga cccctg 16

<210> 253

<211> 14

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 253

ccactggaca gaag 14

<210> 254

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 254

cagaccatca gctgg 15

<210> 255

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 255

gcaaatacca acaatc 16

<210> 256

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 256

caacctcgcc accaga 16

<210> 257

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 257

gtcaccatat tcttgg 16

<210> 258

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 258

ctcgaatgcc tcgtatcat 19

<210> 259

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 259

gttccaacta ggaacatca 19

<210> 260

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 260

gttccaacta ggaacatca 19

<210> 261

<211> 560

<212> DNA

<213> Artificial sequence

<220>

<223> HBV positive control sequence

<400> 261

tgggattcta tataagaggg aaactacacg tagcgcctca ttttgcgggt caccatattc 60

ttgggaacaa gagctacatc atgggaggtt ggtcatcaaa acctcgcaaa ggcatgggga 120

cgaacctttc tgttcccaac cctctgggat tctttcccga tcatcagttg gaccctgcat 180

tcggagccaa ttcaaacaat ccagattggg acttcaaccc catcaaggac cactggccac 240

aagccaacca ggtaggagtg ggagcattcg ggccagggtt cactccccca cacggaggtg 300

ttttggggtg gagccctcag gctcagggca tattggccac agtgccagca gtgcctcctc 360

ctgcctccac caatcggcag tcaggaaggc agcctactcc catctctcca cctctaagag 420

acagtcatcc tcaggccatg cagtggaatt ccacagcttt ccaccaagct ctgcaagatc 480

ccagagtcag gggcctgtat tttcctgctg gtggctccag ttcaggaaca ctcaaccctg 540

ttccaactat tgcctctcac 560

<210> 262

<211> 500

<212> DNA

<213> Artificial sequence

<220>

<223> Internal amplification control sequence

<400> 262

gagacaggtt cgtccaatcc cgtgccgcgg ccttggcagg gggttcgcag gccccacccg 60

aagcgttgct gaaggctcag gcctctgagc gacaaaagct ttaaacgcga gttcccgccc 120

ataacctgga ccgaatgcgg gaccatgcat cgttccactg tgtttgtccc atgtaggacg 180

ggcgcaaggc gtgcttagct cagcctcgaa tgcctcgtat cattgtgcac ccgccggtca 240

ccagccaacg atgtgcggac ggcgttgcaa cttccggggc ccaacctgac cgtcctgggt 300

accgcactct gggcagtgcg aggtaatgcc agtcgcccag tgccgaacaa cacctgacct 360

aacggtaaga ggctcacata atggctccgc cggcgcgccc agggtacatt aggtcagcat 420

cggatggact gacatgaacc ttcacaccga agcggaaacg ggtgcgtgga ccagcgagga 480

gcaaacgaaa attcctggcc 500

<210> 263

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 263

gaccacaacg ctacgacgct aatcaatgcc cgggattggt 40

<210> 264

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 264

ttctgcttgt ccggctacga tccggagcga gttacgaaga 40

<210> 265

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 265

gaccacaacg ctacgacgct aacgaggcat tgaagcaaag 40

<210> 266

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 266

ttctgcttgt ccggctacga ctgctgtttc aagtcgtcca 40

<210> 267

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 267

gaccacaacg ctacgacgct actgtggtag ataccacacg c 41

<210> 268

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 268

ttctgcttgt ccggctacga acaggtaatg gcctgtgact g 41

<210> 269

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 269

gaccacaacg ctacgacgct gttgatgaaa ccttaacccc ttg 43

<210> 270

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 270

ttctgcttgt ccggctacga gttgaggggt tttccatttt tgc 43

<210> 271

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 271

gaccacaacg ctacgacgct gtcaggatca tcaaatcaat tcac 44

<210> 272

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 272

ttctgcttgt ccggctacga caacttggat aggacggtca 40

<210> 273

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 273

gaccacaacg ctacgacgct cagagccatc agcccttttc a 41

<210> 274

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 274

ttctgcttgt ccggctacga gaagtttgtc ccagttgcgg tt 42

<210> 275

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 275

gaccacaacg ctacgacgct actccatgaa gtcggaatcg 40

<210> 276

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 276

ttctgcttgt ccggctacga cccacgttct cgtagggata 40

<210> 277

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 277

gaccacaacg ctacgacgct gttggagctg gaatagttgc t 41

<210> 278

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 278

ttctgcttgt ccggctacga cagctgatgt aagtgcagca t 41

<210> 279

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 279

gaccacaacg ctacgacgct ccaccatgta ctacaaagac g 41

<210> 280

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 280

ttctgcttgt ccggctacga tggtcgtccc ggtgaaac 38

<210> 281

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 281

gaccacaacg ctacgacgct gtcgaacatt ggtcttaccc tc 42

<210> 282

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 282

ttctgcttgt ccggctacga tgtgccgtct tcaagtatgc 40

<210> 283

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 283

gaccacaacg ctacgacgct gaagagccaa ggacaggtac 40

<210> 284

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 284

ttctgcttgt ccggctacga ccttgatacc aacctgccca g 41

<210> 285

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 285

gaccacaacg ctacgacgct 20

<210> 286

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 286

ttctgcttgt ccggctacga 20

<210> 287

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 287

agtgcataaa cttctgagg 19

<210> 288

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 288

tataaacgcc cggcagttac 20

<210> 289

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 289

aaagattact ggagagaacc 20

<210> 290

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 290

tgaacgaagg tagagaagca 20

<210> 291

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 291

tgcggtttac gaaattgaaa 20

<210> 292

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 292

actcatgacg aacgaagaag 20

<210> 293

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 293

gcagaaaaac tatcctcag 19

<210> 294

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 294

cagctatgct gcggtgaata 20

<210> 295

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 295

cttgtcgatc acctcctcg 19

<210> 296

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 296

ttatgtgcat ccgtaacta 19

<210> 297

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 297

gtagcagatt tagacacaga tg 22

<210> 298

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 298

aaggtgaacg tggatgaagt tggtgg 26

<210> 299

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 299

gaaaatgatg cccagtgctt 20

<210> 300

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 300

ttgaaggaaa tgccccatta 20

<210> 301

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 301

gaaccaatcc cgtgaaagaa 20

<210> 302

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 302

agctgcccat gaatagcact 20

<210> 303

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 303

ggttacccca aaggccacc 19

<210> 304

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 304

aagcggaaga atgtgtcagc 20

<210> 305

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 305

tttggggagc tgaaggacta 20

<210> 306

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 306

ctttgtgacc attccggttt 20

<210> 307

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 307

ggacaggcga ggaat 15

<210> 308

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 308

tggacaggca aggaat 16

<210> 309

<211> 14

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 309

tctcagcgag cctc 14

<210> 310

<211> 14

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 310

actctcagca agcc 14

<210> 311

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 311

cgggagccga tttca 15

<210> 312

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 312

atgatgaaat cgactcc 17

<210> 313

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 313

cagtgaagaa agtgtct 17

<210> 314

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 314

gaccagtgaa gcaagtg 17

Claims (4)

1. The use of primers with sequence numbers 263-286 and oligonucleotide probes with sequence numbers 287-298 in the preparation of a kit for detecting the presence of sexually transmitted pathogens in a subject, the detection comprising the following steps: (a) Obtain a sample containing a template nucleic acid molecule from the subject; (b) The template nucleic acid molecule was amplified using primers with sequence numbers 263-286 to obtain the amplification product; and (c) The obtained amplification product was hybridized with oligonucleotide probes with sequence numbers 287-298, and the hybridization results showed whether the subject had sexually transmitted pathogens. 2. The use according to claim 1, wherein the amplification product is hybridized with a plurality of oligonucleotide probes by a flow-through method, a lateral flow-through method, or a reverse flow-through method. 3. The use according to claim 1, wherein the use can detect sexually transmitted pathogens selected from Trichomonas vaginalis, Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealyticum, Lysimachia spirochetes, Treponema pallidum, Herpes simplex virus type 1, Herpes simplex virus type 2, Human papillomavirus type 6 and Human papillomavirus type 11. 4. A kit for detecting sexually transmitted pathogens, comprising primers with sequence numbers 263-286 and oligonucleotide probes with sequence numbers 287-298.