A primer probe set, a kit and an application for rapidly detecting human papilloma virus

Abstract

The application discloses a primer probe group, a kit and application for rapidly detecting human papilloma virus. The kit discloses that by optimizing a lysis system and RPA reaction conditions, the rapid release of nucleic acid in a sample is realized, a traditional extraction step is not needed, and amplification can be directly carried out after the rapid nucleic acid release of a cervical swab; three target primers and probes are designed for type 16, type 18 and a human reference gene, and the three target primers and probes are colored in T zones, respectively; the test strip can also be more accurate and reliable through T zones (detection of enzyme-cut nucleic acid) and C zones (detection of non-enzyme-cut nucleic acid) results, and there is no cross reaction with 27 non-target HPV types; gold-labeled antibody-probe complexes and nitrocellulose membrane flow rate control technology are adopted, the result visualization is realized through composite labeled test strips, the sensitivity of detection of HPV16 and HPV18 can reach 5 copies / reaction, and the whole detection only needs 25 minutes, and the kit has the advantages of high sensitivity, simple operation and rapid result output.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection technology, specifically relating to a primer and probe set, reagent kit and application for rapid detection of human papillomavirus, and particularly to visual detection technology for human papillomavirus nucleic acid. Background Technology

[0002] Cervical cancer is a malignant tumor that seriously threatens the health of women worldwide, and its occurrence is closely related to persistent infection with high-risk human papillomavirus (HPV). Among the many high-risk types, HPV types 16 and 18 are particularly critical. Epidemiological data shows that approximately 70% of cervical cancer cases are directly caused by these two types. They not only have high morbidity rates but also have distinct clinical characteristics: HPV type 16 is known for its extremely aggressiveness and viral persistence, and is the most important pathogen inducing precancerous lesions such as cervical intraepithelial neoplasia (CIN); while HPV type 18 is significantly associated with the more aggressive cervical adenocarcinoma. Because its early symptoms are more subtle and not sensitive to routine cytological examinations, it has a higher risk of missed diagnosis in clinical testing and is more difficult to diagnose.

[0003] Accurate genotyping of the two key HPV types mentioned above is crucial for early screening, risk stratification, and prognostic assessment of cervical cancer. Currently, the mainstream high-risk HPV16 / 18 genotyping tests rely on nucleic acid detection kits based on polymerase chain reaction (PCR) technology. This technology, thanks to its molecular biological characteristics, does achieve high detection sensitivity. However, its clinical application has significant limitations: First, the entire testing process heavily relies on expensive specialized thermal cyclers and laboratory environments, resulting in high equipment investment and maintenance costs; second, the operation steps are cumbersome, involving multiple stages such as nucleic acid extraction, amplification, and product analysis, requiring a high level of expertise from operators and being susceptible to significant human error; finally, the complete testing cycle typically takes more than 3 hours, making it difficult to meet the urgent needs of outpatient rapid screening or primary healthcare institutions for timely testing.

[0004] Therefore, there is an urgent need in this field to develop a new HPV16 / 18 genotyping solution that can overcome the bottlenecks of existing technologies and has high accuracy, speed, ease of operation and low dependence on equipment. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention discloses a method and application for detecting human papillomavirus (HPV) nucleic acid based on the combination of recombinase polymerase amplification (RPA) and lateral flow chromatography test strips. This invention employs an isothermal amplification-test strip method, utilizing a recombinase-primer complex to achieve isothermal amplification, nucleic acid chain exchange, and extension at 39℃ to 42℃. It eliminates the need for thermal cycling equipment and uses immunochromatography for color development, presenting results directly through chromogenic bands. This method is rapid, convenient, and low-cost, reducing detection time to within 25 minutes, eliminating the need for expensive equipment, and lowering detection costs by over 60%, while maintaining a detection sensitivity of 99.5%. The advantages of this invention include specific primers designed for HPV16 / 18 types to avoid cross-reactions; optimized stability using lyophilized bulbs to extend reagent shelf life to over 12 months; and the requirement for complex training, making it suitable for large-scale screening scenarios. It can be widely applied to cervical cancer initial screening, postoperative follow-up, and vaccination efficacy evaluation.

[0006] This invention designs and screens specific primer-probe combinations for human papillomavirus (HPV) types 16 and 18, introduces an internal reference gene to ensure the reliability and accuracy of results, and finally, the amplified products are analyzed by colorimetric assay using a test strip. This invention triggers test strip colorimetry by isothermal amplification of the nfo enzyme to the probe breakage state. The test strip's triple-target detection mechanism simultaneously detects both the T region (detecting enzyme-digested nucleic acids) and the C region (detecting undigested nucleic acids), resulting in more accurate and reliable results, whereas conventional nucleic acid test strips rely on only a single target. Colorimetry in the C region indicates sample validity, colorimetry in the T region confirms the presence of the target analyte, and the disappearance of the C region quickly determines complete enzyme digestion, avoiding false negatives / positives. The latex particles of the labeled antibodies enhance signal sensitivity and are easier to visually interpret than traditional gold or fluorescent labels. When using the primer and probe set of this invention to detect human papillomavirus (HPV) types, it has high sensitivity for types 16 and 18, and no cross-reactivity with HPV types 6, 11, 26, 40, 42, 43, 44, 53, 54, 61, 67, 69, 70, 71, 72, 73, 81, 82, and 83, herpes simplex virus type II, Treponema pallidum, Ureaplasma urealyticum, Mycoplasma hominis, Neisseria gonorrhoeae, Chlamydia trachomatis, Candida albicans, and Trichomonas vaginalis. It has strong specificity and is suitable for rapid screening in primary healthcare institutions.

[0007] To achieve the above objectives, this invention discloses a primer and probe set for rapid detection of human papillomavirus, the primer and probe set comprising forward and reverse primers and probes for human papillomavirus types 16 and 18 and a human internal reference gene. The nucleotide sequence of the human papillomavirus type 16 forward primer is shown in SEQ ID No. 2; The nucleotide sequence of the reverse primer for human papillomavirus type 16 is shown in SEQ ID No. 5; The nucleotide sequence of the human papillomavirus type 16 probe is shown in SEQ ID No. 7; The nucleotide sequence of the human papillomavirus type 18 forward primer is shown in SEQ ID No. 9; The nucleotide sequence of the reverse primer for human papillomavirus type 18 is shown in SEQ ID No. 12; The nucleotide sequence of the human papillomavirus type 18 probe is shown in SEQ ID No. 14; The nucleotide sequence of the forward primer for the human internal reference gene is shown in SEQ ID No. 17; The nucleotide sequence of the reverse primer for the human internal reference gene is shown in SEQ ID No. 20; The nucleotide sequence of the human internal reference gene probe is shown in SEQ ID No. 21.

[0008] Preferably, the 5' end of the reverse primers for human papillomavirus type 16, human papillomavirus type 18, and human internal reference is labeled with biotin; Preferably, the probes of human papillomavirus type 16, human papillomavirus type 18 and human internal reference are connected to antigenic molecular markers at their 5' ends, and have polymerase extension blocking groups as termination structures at their 3' ends. Furthermore, the antigenic molecular markers attached to the probes of human papillomavirus type 16, human papillomavirus type 18, and human internal reference are different; The antigenic molecular markers are selected from fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (FAM), 6-carboxytetramethylrhodamine (TAMRA), or digoxin (Dig). The blocking group is a phosphate, a C3-spacer, an amino group, or a dideoxynucleotide, preferably a phosphate or a C3-spacer.

[0009] In some preferred embodiments, the 5' end of the human papillomavirus type 16 probe is modified with an antigenic molecular marker, preferably any one of fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (FAM), 6-carboxytetramethylrhodamine (TAMRA), or digoxigenin (Dig), or other antigenic molecular markers that match antibodies or specific binding molecules on multiple target nucleic acid test strips; the 3' end of the human papillomavirus type 16 probe is modified with a blocking group to prevent amplification extension, which is any one of phosphate, C3-spacer, amino, or dideoxynucleotide.

[0010] Further preferably, the 5' end of the human papillomavirus type 16 probe is modified with FAM; and the 3' end of the human papillomavirus type 16 probe is modified with C3-spacer.

[0011] In some preferred embodiments, the 5' end of the human papillomavirus type 18 probe is modified with an antigenic molecular marker, preferably any one of fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (FAM), 6-carboxytetramethylrhodamine (TAMRA), or digoxigenin (Dig), or other antigenic molecular markers that match antibodies or specific binding molecules on multiple target nucleic acid test strips; the 3' end of the human papillomavirus type 18 probe is modified with a blocking group to prevent amplification extension, which is any one of phosphate, C3-spacer, amino, or dideoxynucleotide.

[0012] Further preferably, the 5' end of the human papillomavirus type 18 probe is modified with TAMRA; and the 3' end of the human papillomavirus type 18 probe is modified with C3-spacer.

[0013] In some preferred embodiments, the 5' end of the human internal control probe is modified with an antigenic molecular marker, preferably any one of fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (FAM), 6-carboxytetramethylrhodamine (TAMRA), or digoxigenin (Dig), or other antigenic molecular markers that match antibodies or specific binding molecules on multiple target nucleic acid test strips; the 3' end of the human internal control probe is modified with a blocking group to prevent amplification extension, which is any one of phosphate, C3-spacer, amino, or dideoxynucleotide.

[0014] Further preferably, the 5' end of the human internal reference probe is modified with Dig; and the 3' end of the human internal reference probe is modified with C3-spacer.

[0015] In embodiments of the present invention, the probes for human papillomavirus type 16, human papillomavirus type 18, and the human internal reference gene are nfo probe structures, with a debased nucleotide analog (tetrahydrofuran [THF] residue) modified in the middle segment of the sequence as a nucleotide substitute; the probe is 46-52 nucleotides in length; at least 29 nucleotides are located at the 5' end of the THF site; at least 15 nucleotides are located at the 3' end of the THF site; the THF residue replaces a nucleotide that can normally pair with the complementary sequence, serving as a recognition site for exonuclease.

[0016] Another aspect of the present invention discloses a kit for rapid detection of human papillomavirus, comprising primers or probes having 80% or more homology with any of the nucleotide sequences or their complementary sequences described in SEQ ID No. 1 to 9; It may contain a nucleotide sequence that is functionally similar to any of the nucleotide sequences shown in SEQ ID No. 1 to 9, or a nucleotide sequence obtained by modifying, substituting, deleting, or adding one or more bases to any of the nucleotide sequences shown in SEQ ID No. 1 to 9.

[0017] Preferably, the kit also includes a nucleic acid release agent, RPA amplification reaction lyophilized buffer beads, RPA lyophilized enzyme MIX beads, and flow chromatography test strips; The flow chromatography test strip has T1, T2, and T3 detection lines drawn sequentially on the detection area; the T1, T2, and T3 detection lines are used to detect human papillomavirus type 16, human papillomavirus type 18, and human internal reference, respectively.

[0018] The flow chromatography test strip has the following structure: it includes a sample application pad area, a colloidal gold-labeled antibody area, a nitrocellulose membrane layer, and a reaction chromatography membrane absorption area; the nitrocellulose membrane layer contains an anti-nucleic acid marker anti-FAM antibody pre-immobilized in detection area 1 (T1), an anti-nucleic acid marker anti-TAMRA antibody pre-immobilized in detection area 2 (T2), an anti-nucleic acid marker anti-Dig antibody pre-immobilized in detection area 3 (T3) on the NC membrane, and a control line sprayed with anti-biotin antibody.

[0019] Preferably, the RPA amplification reaction lyophilized buffer spheres contain 1.5-5 wt% polyethylene glycol, 10-50 mM Tris, 200-240 mM sodium acetate, 1-10 mM dithiothreitol, 1.5-3.5 mM ATP, 20-75 mM disodium creatine phosphate, 10-200 ng / μL phosphokinase, 450-500 μM deoxyribonucleoside triphosphate, 2.5-7.5 wt% trehalose, 40-50 ng / μL mannitol, and magnesium acetate as the activator; the RPA lyophilized enzyme MIX spheres contain recombinant enzymes, single-stranded DNA-binding proteins, DNA polymerases, and endonucleases.

[0020] Preferably, the nucleic acid releasing agent comprises the following components: 5 mM tris(hydroxymethyl)aminomethane hydrochloride at pH 8.0, 0.2 mM ethylenediaminetetraacetic acid, 10 mM sodium hydroxide, 0.05% Triton X-100, and 1 mg / mL bovine serum albumin.

[0021] In another aspect, the present invention provides a method for using the above-mentioned kit, comprising the following steps: immersing the swab head in the nucleic acid release agent, thoroughly shaking and mixing, allowing it to stand at room temperature for 5 minutes, aspirating the sample supernatant and adding it to a reaction tube containing RPA amplification reaction lyophilized buffer beads and RPA lyophilized enzyme MIX beads, and amplifying the amplification product in the RPA amplification reaction system of the primer and probe set described in claim 1, with the amplification temperature at 42°C and the time at 20 min; diluting the amplification product and adding it dropwise to a flow chromatography test strip, and reading the detection result.

[0022] Given the role of the RPA primer and probe combination provided by this invention, its application in the preparation of products for detecting human papillomavirus also falls within the scope of protection of this invention.

[0023] The RPA amplification reaction system includes RPA amplification reaction lyophilized buffer spheres, RPA lyophilized enzyme MIX spheres, and forward and reverse primers and probes for human papillomavirus (HPV) types 16 and 18, and a human internal reference gene. Each 6 μL RPA amplification reaction system, in addition to the RPA amplification reaction lyophilized buffer spheres and RPA lyophilized enzyme MIX spheres, also includes: 0.6 μL of HPV type 16 forward primer, 0.6 μL of HPV type 16 reverse primer, 0.2 μL of HPV type 16 probe, 1.5 μL of HPV type 18 forward primer, 1.5 μL of HPV type 18 reverse primer, 0.45 μL of HPV type 18 probe, 0.5 μL of human internal reference gene forward primer, 0.5 μL of human internal reference gene reverse primer, and 0.15 μL of human internal reference gene probe. All primers and probes are at a concentration of 10 μM.

[0024] As a preferred embodiment, a method for using a rapid detection kit for human papillomavirus specifically includes the following steps: First, a cervical swab is used to collect a sample; After sampling, immerse the cervical swab head in the release agent, break off the swab tail, discard the tail, and tighten the cap of the tube containing the sample. Vortex to mix thoroughly, let stand at room temperature for 5 minutes, briefly incubate for 3-5 seconds, and take 44 μL of the sample supernatant for direct nucleic acid detection. Prepare a 6 μL RPA amplification reaction system and add it to the supernatant to obtain the amplification product. The amplification temperature is 37–42℃, and the time is 20 min.

[0025] Take 10 μL of the RPA amplification product and dilute it in 190 μL of water. Add 65 μL of the diluted product to the sample pad and observe the results after 5 minutes. C is the control line; T1 is the HPV16 detection line; T2 is the HPV18 detection line; T3 is the human internal control detection line. The control line (C) and the corresponding detection lines (T1 / T2) develop color simultaneously. This indicates that the amplification product contains the HPV16 / 18 nucleic acid fragment to be detected, and its content is greater than or equal to the minimum detection limit of the test strip. The control line (C) and the human internal control (T3) develop color, and T1 / T2 show no bands. This indicates that the amplification product does not contain the HPV16 / 18 nucleic acid fragment to be detected, or its content is lower than the minimum detection limit of the test strip. If no color band appears at the control line (C), the result is invalid regardless of whether the detection lines (T1, T2, T3) show red bands, and the test strip needs to be retested. This result indicates that the test strip used was invalid, damaged, or the operation was incorrect.

[0026] This invention is based on RPA isothermal rapid amplification technology. It uses specific primers and probes to target the conserved regions of the human papillomavirus (HPV) type 16 E6 gene, HPV type 18 L1 gene, and the human RPP30 internal reference gene, rapidly amplifying the target gene fragments under isothermal conditions without the need for complex temperature control equipment. The amplified products are detected using lateral flow chromatography test strips. The binding pad contains colloidal gold-labeled streptavidin, and the gold nanoparticle binding pad integrates anti-FAM / anti-TAMRA / anti-Dig antibodies and biotinylated antibody control lines, enabling visualization of multiple signals. This technology features high sensitivity (5 copies / reaction for HPV type 16, 5 copies / reaction for HPV type 18) and speed (results within 25 minutes), significantly superior to traditional PCR or hybridization capture techniques (which require 1-3 days and rely on laboratory equipment). Its lack of specialized equipment makes it particularly suitable for grassroots screening scenarios, reducing testing costs and increasing coverage, thus contributing to the early prevention and control of cervical cancer. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the color development of the test strip in Example 1. Detailed Implementation

[0028] The present invention will be further illustrated by the following embodiments. It should be noted that the following embodiments are only for further illustration of the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above-described invention, and these improvements and adjustments will still fall within the scope of protection of the present invention.

[0029] Example 1: A rapid human papillomavirus kit This protocol provides a rapid human papillomavirus (HPV) kit, comprising cervical swab nucleic acid release agent, RPA amplification reaction lyophilized buffer spheres, RPA lyophilized enzyme MIX spheres, HPV type 16 and 18 primer probes, human internal reference gene primer probes, and flow chromatography test strips.

[0030] The cervical swab nucleic acid release agent comprises the following components: 5mM tris(hydroxymethyl)aminomethane hydrochloride at pH 8.0, 0.2mM ethylenediaminetetraacetic acid, 10mM sodium hydroxide, 0.05% Triton X-100, and 1 mg / mL bovine serum albumin.

[0031] The RPA amplification reaction lyophilized buffer spheres and RPA lyophilized enzyme MIX spheres were purchased from Anpu Future Biotechnology Co., Ltd. The RPA amplification reaction lyophilized buffer spheres comprise polyethylene glycol, Tris, sodium acetate, dithiothreitol, ATP, creatine phosphate disodium salt, phosphokinase, deoxyribonucleoside triphosphate, trehalose, mannitol, and magnesium acetate activator. The RPA lyophilized enzyme MIX spheres comprise recombinase, single-stranded DNA-binding protein, DNA polymerase, and exonuclease. The exonuclease is used to recognize and cleave the THF site in the human papillomavirus probe.

[0032] As a reference, this invention precisely identified base sequence markers of specific pathogen target genes using publicly available human papillomavirus (HPV) gene sequences (GenBank). Based on this, this invention comprehensively utilized Primer Premier 5, DNAMAN software, and NCBI's Primer-BLAST platform to design forward and reverse primers and probes for HPV types 16 and 18, as well as a human internal reference gene. The primer and probe design principles followed those published on the TwistDx Inc. website. Both primers and probes were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0033] In this embodiment, the 5' end of the reverse primer for human papillomavirus type 16 is connected to the antigen molecule biotin; the 5' end of the probe for human papillomavirus type 16 is connected to carboxyfluorescein (FAM), and the 3' end is connected to the blocking group C3-spacer.

[0034] The 5' end of the reverse primer for human papillomavirus type 18 is linked to the antigen molecule biotin; the 5' end of the probe for human papillomavirus type 18 is linked to TAMRA, and the 3' end is linked to the blocking group C3-spacer.

[0035] The human internal control reverse primer has an antigen molecule biotin attached to its 5' end; the human internal control probe has an antigenic molecular marker, digoxigenin, modified to its 5' end, and a blocking group C3-spacer attached to its 3' end.

[0036] For reference, the flow chromatography test strip was purchased from AMP Future Biotechnology Co., Ltd., and its structure is as follows: it includes a sample application pad area, a colloidal gold-labeled antibody area, a nitrocellulose membrane layer, and a reaction chromatography membrane absorption area; the nitrocellulose membrane layer contains an anti-nucleic acid marker anti-FAM antibody pre-immobilized to detection area 1 (T1), an anti-nucleic acid marker anti-TAMRA antibody pre-immobilized to detection area 2 (T2), an anti-nucleic acid marker anti-Dig antibody pre-immobilized to detection area 3 (T3) on the NC membrane, and a control line sprayed with anti-biotin antibody.

[0037] In this embodiment, T1 is the streak anti-FAM antibody, T2 is the streak anti-TAMRA antibody, and T3 is the streak anti-Dig antibody. The colloidal gold-labeled antibody region is coated with colloidal gold-labeled streptavidin.

[0038] For reference, the sample nucleic acid amplification method of this kit is as follows: Immerse the cervical swab head in the cervical swab nucleic acid release agent after sampling, break off the swab tail, discard the tail, tighten the cap of the tube containing the sample, shake thoroughly to mix, and let stand at room temperature for 5 minutes to obtain the sample supernatant.

[0039] As a reference, 10 μL of sample supernatant can be mixed with the RPA amplification reaction system, and water can be added to a final volume of 50 μL for amplification to obtain the amplification product. The amplification temperature is 42℃ and the time is 20 min.

[0040] The RPA amplification reaction system includes RPA amplification reaction lyophilized buffer spheres, RPA lyophilized enzyme MIX spheres, human papillomavirus types 16 and 18, and human internal reference gene forward and reverse primers and probes. In addition to the RPA amplification reaction lyophilized buffer spheres and RPA lyophilized enzyme MIX spheres, the RPA amplification reaction system also includes primer and probe MIX: 2-0.4 μL of human papillomavirus type 16 forward primer, 2-0.4 μL of human papillomavirus type 16 reverse primer, 0.6-0.2 μL of human papillomavirus type 16 probe, 2-0.4 μL of human papillomavirus type 18 forward primer, 2-0.4 μL of human papillomavirus type 18 reverse primer, 0.6-0.2 μL of human papillomavirus type 18 probe, 2-0.4 μL of human internal reference gene forward primer, 2-0.4 μL of human internal reference gene reverse primer, and 0.5-0.1 μL of human internal reference gene probe. All primer and probe concentrations are 10 μM.

[0041] As a reference, 10 μL of sample supernatant can be mixed with the RPA amplification reaction system, and water can be added to a final volume of 50 μL for amplification to obtain the amplification product. The amplification temperature is 42℃ and the time is 20 min. The RPA amplification reaction system includes RPA amplification reaction lyophilized buffer spheres, RPA lyophilized enzyme MIX spheres, human papillomavirus types 16 and 18, and human internal reference gene forward and reverse primers and probes. Each 6 μL RPA amplification reaction system, in addition to the RPA amplification reaction lyophilized buffer spheres and RPA lyophilized enzyme MIX spheres, also includes: 0.6 μL of human papillomavirus type 16 forward primer, 0.6 μL of human papillomavirus type 16 reverse primer, 0.2 μL of human papillomavirus type 16 probe, 1.5 μL of human papillomavirus type 18 forward primer, 1.5 μL of human papillomavirus type 18 reverse primer, 0.45 μL of human papillomavirus type 18 probe, 0.5 μL of human internal reference gene forward primer, 0.5 μL of human internal reference gene reverse primer, and 0.15 μL of human internal reference gene probe. The concentration of all primers and probes is 10 μM.

[0042] This solution also provides a test strip detection method, which includes dropping the amplification product onto a flow chromatography test strip and displaying the detection result.

[0043] As a reference, dilute 10 μL of RPA amplification product with 190 μL of water. After dilution, add 65 μL to the sample pad and observe the results after 5 minutes. Interpret the results. C is the control line; T1 is the HPV16 detection line; T2 is the HPV18 detection line; T3 is the human internal control detection line. The color development of the test strip is as follows: Figure 1 As shown. Simultaneous color development of the control line (C) and the corresponding test lines (T1 / T2) indicates that the amplification product contains the HPV16 / 18 nucleic acid fragment to be detected, and its content is greater than or equal to the minimum detection limit of the test strip. Color development of the control line (C) and the human internal control (T3), with no bands on T1 / T2, indicates that the amplification product does not contain the HPV16 / 18 nucleic acid fragment to be detected, or its content is lower than the minimum detection limit of the test strip. No color band appears on the control line (C). In this case, regardless of whether red bands appear on the test lines (T1, T2, T3), the result is invalid, and the test strip needs to be retested. This result suggests that the test strip used is invalid, damaged, or the operation was incorrect.

[0044] Example 2: Screening of primer and probe sets Using publicly available human papillomavirus (HPV) gene sequences (GenBank), this invention identifies base sequence markers of specific pathogen target genes. Based on this, the invention comprehensively utilizes Primer Premier 5, DNAMAN software, and NCBI's Primer-BLAST platform. Primer and probe design principles are based on those published on the RPA company's website. Three pairs of forward and reverse primers and one probe were designed for each conserved region of the HPV 16 E6 gene, HPV 18 L1 gene, and the human RPP30 internal reference gene (Table 1).

[0045] Table 1

[0046] A mixed sample of HPV16 (1×10^5 copies / mL) and HPV18 (1×10^5 copies / mL) was used as a template (purchased from a human cell line with an integrated HPV genome sequence from Jingliang Biotechnology). 10 μL of template was added, and water was added to a final volume of 50 μL. 40.8 μL of purified water was used as a negative control. RPA amplification was performed in RPA lyophilized enzyme reaction tubes according to the RPA amplification method described in Example 1. 2 μL each of the forward and reverse primers for HPV16 and 18 were used, along with 0.6 μL of probe. Human internal control primers and probes were not added to the amplification system. The obtained RPA amplification products were detected using nucleic acid detection strips. The results of the dual screening for HPV16 and 18 are shown in Table 2.

[0047] Table 2

[0048] The results showed that in groups 1, 2, 3, 7, and 9 (negative controls), abnormal color bands were observed in both the HPV16 test line (T1) and the HPV18 test line (T2). In groups 4 and 6 (negative controls), abnormal color bands were observed in the HPV16 test line (T1), while no abnormal bands were observed in the HPV18 test line (T2). In groups 5 and 8, both the positive HPV16 test line (T1) and the HPV18 test line (T2) showed effective color development, while the negative control samples showed no abnormal color development in either the HPV16 or HPV18 test lines (T2), allowing for further screening and subsequent experiments.

[0049] The above experiments were repeated in optimized systems 5 and 8 by adding human internal control primers and probes for further triple system screening and optimization. All primer amounts were 2 μL and probe amounts were 0.6 μL. The results of the triple screening are shown in Table 3.

[0050] Table 3

[0051] The results showed that abnormal color development bands appeared on the human internal reference gene detection line T3 in groups 13, 14, and 15 (negative controls). In groups 10, 11, and 12, the positive HPV16 detection line T1 and HPV18 detection line T2 showed effective color development. In the negative control samples, neither the HPV16 nor HPV18 detection lines T1 and T2 showed abnormal color development, and no abnormal color development bands were observed on the human internal reference gene detection line T3. Further screening is possible for subsequent experiments.

[0052] Example 3: Sensitivity Test Sensitivity tests were performed using primer and probe sets 10, 11, and 12 from Example 2, which had been optimized for primer and probe dosage. Each primer and probe set was tested using a mixed sample of HPV16 (1×10^6 copies / mL) and HPV18 (500 copies / mL), as well as a reverse-prepared mixed sample of HPV18 and HPV16. The reaction systems for each primer and probe set were prepared as shown in Table 4.

[0053] Table 4

[0054] The prepared reaction system tube was placed in a constant temperature metal bath and incubated at 42°C for 20 min. 10 µL of the amplification product was added to a centrifuge tube containing 190 µL of water and mixed. The test strip was then removed, and 65 µL of the mixture was dropped onto the end containing the sample pad. The detection results are shown in Table 5.

[0055] Table 5

[0056] Experimental Conclusions: In the sensitivity verification experiment, by comparing the detection results, the recognition sensitivity of this detection system for HPV16 / 18 at different concentration gradients can be comprehensively evaluated, especially verifying its ability to detect low concentrations of viral DNA. The experimental design adopted a concentration cross-configuration, which not only avoided the limitations of a single concentration combination, but also ensured the reliability of the detection results through data cross-verification. The experimental results show that the 12-group primer-probe combination has the highest sensitivity, with a detection sensitivity of up to 5 copies / reaction for both HPV16 and HPV18.

[0057] Example 4: Specificity Test The specificity of the primer-probe set 12 in Example 2 for detecting other pathogens was tested. Specifically, its specificity against HPV types 6, 11, 26, 40, 42, 43, 44, 53, 54, 61, 67, 69, 70, 71, 72, 73, 81, 82, and 83 (derived from human cell lines integrating the complete HPV genome sequence), herpes simplex virus type II, Treponema pallidum, Ureaplasma urealyticum, Mycoplasma hominis, Neisseria gonorrhoeae, Chlamydia trachomatis, Candida albicans, and Trichomonas vaginalis (with cross-pathogen nucleic acid DNA extracted using a commercial extraction kit (Kangwei Century)) was tested. RPA amplification reactions were performed in RPA lyophilized enzyme powder reaction tubes according to the RPA-LPS method in Example 1, and specificity was detected using test strips. The detection results are shown in Table 6 below.

[0058] Table 6

[0059] Experimental conclusions: No bands were observed for any of the other HPV subtypes, and no nonspecific amplification was observed. Human internal reference genes all showed bands. No bands were observed for other pathogens related to the human urinary and reproductive tracts, sexually transmitted pathogens, or other common pathogens, and no nonspecific amplification was observed. The primer-probe set 12 in Example 2 demonstrated satisfactory specificity.

[0060] Experiment Example 5: Reagent kit testing cervical swab samples Following the composition of Example 1, a rapid human papillomavirus kit containing primer and probe set 12 as described in Example 2 was prepared. Human testing was performed using this kit in comparison with the PCR-HPV method.

[0061] Make the following preparations before data collection.

[0062] Timing: Avoid menstruation; it is recommended to collect samples 3-7 days after menstruation ends. Avoid sexual intercourse for 24 hours before sampling and avoid vaginal douching or topical medication for 48 hours before sampling. There should be at least a one-month interval between abortion and childbirth.

[0063] Item preparation: Self-sampling kit (including dedicated cervical swab, sample processing solution, and instruction manual). Ensure the kit is not expired and the sample processing solution is properly sealed.

[0064] Detailed operation demonstration diagrams can be found in the cervical swab sampling kit instruction manual. The sampling kit was purchased from Guangdong Medical Device Registration Certificate No. 20162221108, and the sample processing solution was purchased from Jiangsu Changzhou Medical Device Registration Certificate No. 20220492.

[0065] The detection method is the same as in Example 1.

[0066] The test results are shown in Table 7.

[0067] Table 7

[0068] The experimental results of the above embodiments show that the rapid detection kit for human papillomavirus (isothermal amplification test strip method) provided by the present invention has high specificity, no cross-reactivity with other high-risk types of clinical HPV, high sensitivity, and is easy to operate, enabling rapid visual detection of human papillomavirus.

[0069] The above embodiments are merely preferred embodiments of the present invention and should not be used to limit the scope of protection of the present invention. Any modifications or refinements made to the main design concept and spirit of the present invention that are not of substantial significance, but which still solve the same technical problem as the present invention, should be included within the scope of protection of the present invention. The present invention can be used for the auxiliary diagnosis of human papillomavirus infection. The test results are for reference only and cannot be used as the sole basis for confirming or excluding cases.

Claims

1. A primer and probe set for rapid detection of human papillomavirus, characterized in that, The primer and probe set includes forward and reverse primers and probes for human papillomavirus types 16 and 18 and a human internal reference gene. The nucleotide sequence of the human papillomavirus type 16 forward primer is shown in SEQ ID No. 2; The nucleotide sequence of the reverse primer for human papillomavirus type 16 is shown in SEQ ID No. 5; The nucleotide sequence of the human papillomavirus type 16 probe is shown in SEQ ID No. 7; The nucleotide sequence of the human papillomavirus type 18 forward primer is shown in SEQ ID No. 9; The nucleotide sequence of the reverse primer for human papillomavirus type 18 is shown in SEQ ID No. 12; The nucleotide sequence of the human papillomavirus type 18 probe is shown in SEQ ID No. 14; The nucleotide sequence of the forward primer for the human internal reference gene is shown in SEQ ID No. 17; The nucleotide sequence of the reverse primer for the human internal reference gene is shown in SEQ ID No. 20; The nucleotide sequence of the human internal reference gene probe is shown in SEQ ID No.

21.

2. The primer-probe set according to claim 1, characterized in that, The reverse primers for human papillomavirus type 16, human papillomavirus type 18, and human internal reference are labeled with biotin at their 5' ends.

3. The primer-probe set according to claim 1, characterized in that, The probes of human papillomavirus type 16, human papillomavirus type 18 and human internal reference are connected to antigenic molecular markers at their 5' ends, and have polymerase extension blocking groups at their 3' ends as termination structures. Furthermore, the antigenic molecular markers attached to the probes of human papillomavirus type 16, human papillomavirus type 18, and human internal reference are different; The antigenic molecular markers are selected from FITC, FAM, TAMRA, or Dig; The blocking group is a phosphate, a C3-spacer, an amino group, or a dideoxynucleotide, preferably a phosphate or a C3-spacer.

4. The primer-probe set according to claim 3, characterized in that, The 5' end of the human papillomavirus type 16 probe is connected to FAM; the 5' end of the human papillomavirus type 18 probe is connected to TAMRA; and the 5' end of the human internal reference probe is modified with Dig.

5. The primer-probe set according to claim 3, characterized in that, The probes of human papillomavirus type 16, human papillomavirus type 18, and human internal reference are connected to a C3-spacer at their 3' ends.

6. A kit for rapid detection of human papillomavirus, characterized in that, Primers or probes containing 80% or more homology to any of the nucleotide sequences or their complementary sequences described in SEQ ID No. 1 to 9; It may contain a nucleotide sequence that is functionally similar to any of the nucleotide sequences shown in SEQ ID No. 1 to 9, or a nucleotide sequence obtained by modifying, substituting, deleting, or adding one or more bases to any of the nucleotide sequences shown in SEQ ID No. 1 to 9.

7. The kit for rapid detection of human papillomavirus according to claim 4, characterized in that, The kit also includes a nucleic acid release agent, RPA amplification reaction lyophilized buffer beads, RPA lyophilized enzyme MIX beads, and flow chromatography test strips; The flow chromatography test strip has T1, T2, and T3 detection lines drawn sequentially on the detection area; the T1, T2, and T3 detection lines are used to detect human papillomavirus type 16, human papillomavirus type 18, and human internal reference, respectively.

8. A kit for rapid detection of human papillomavirus according to claim 7, characterized in that, The RPA amplification reaction lyophilized buffer spheres contain polyethylene glycol 1.5-5 wt%, Tris 10-50 mM, sodium acetate 200-240 mM, dithiothreitol 1-10 mM, ATP 1.5-3.5 mM, creatine phosphate disodium salt 20-75 mM, phosphokinase 10-200 ng / μL, deoxyribonucleoside triphosphate 450-500 μM, trehalose 2.5-7.5 wt%, mannitol 40-50 ng / μL, and magnesium acetate as the activator; the RPA lyophilized enzyme MIX spheres contain recombinant enzyme, single-stranded DNA-binding protein, DNA polymerase, and endonuclease.

9. A kit for rapid detection of human papillomavirus according to claim 7, characterized in that, The nucleic acid release agent comprises the following components: 5 mM tris(hydroxymethyl)aminomethane hydrochloride at pH 8.0, 0.2 mM ethylenediaminetetraacetic acid, 10 mM sodium hydroxide, 0.05% Triton X-100, and 1 mg / mL bovine serum albumin.

10. A method of using the reagent kit according to claim 7, characterized in that, The procedure includes the following steps: immersing the swab head in the nucleic acid release agent, thoroughly shaking and mixing, allowing it to stand at room temperature for 5 minutes, then briefly detaching for 3-5 seconds to obtain the sample supernatant; adding the sample supernatant to an RPA amplification reaction system containing RPA amplification reaction lyophilized buffer beads, RPA lyophilized enzyme MIX beads, and the primer and probe set described in claim 1 to amplify the amplification product at a temperature of 42℃ for 20 minutes; diluting the amplification product and adding it to a flow chromatography test strip to read the detection result.

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