Sample collection method for HPV (human papillomavirus) detection and HPV detection method and system

Abstract

The invention discloses a sample collection method for HPV detection and an HPV detection method and system, and relates to the technical field of HPV infection detection. The sample collection method comprises the following steps: enabling the sampling unit to be in contact with a to-be-sampled sampling part; then the samples collected by the sampling unit are stored; the sampling part to be sampled is selected from at least one of an oral cavity, a pharyngeal cavity, a laryngeal cavity and a nasal cavity, and the sampling unit is a swab or a cytobrush. The sample collection method provided by the invention has the technical advantages of noninvasiveness, rapidness and relatively low sampling cost, and is convenient for carrying out conventional HPV sampling work in environments such as outpatient service and ward. According to the present invention, the obtained sample is subjected to nucleic acid extraction and nucleic acid detection, such that the HPV infection state can be accurately and efficiently detected, the subtype identification can be accurately and efficiently performed, and the advantages of high detection accuracy and strong specificity are provided.

Description

Technical Field

[0001] The present invention relates to the technical field of HPV infection detection, and in particular to a sample collection method for HPV detection, an HPV detection method and a system. Background Art

[0002] At present, HPV detection for head and neck tumors mainly includes [1], including: 1) p16 immunohistochemistry detection [2-4], which is the most commonly used HPV detection method in clinical practice. It uses overexpression of p16 protein as a surrogate indicator of HPV infection. It is easy to operate and has low cost, but it is difficult to interpret and is greatly affected by human factors. It is prone to false positive results, resulting in low specificity of the test; 2) HPV DNA nucleic acid detection method [5], which uses universal primers to target amplify HPV DNA sequences and identify HPV genotypes through gel electrophoresis, restriction fragment length polymorphism or direct sequencing. The detection specificity and sensitivity are good; 3) HPV DNA in situ hybridization (ISH) detection method [6], which hybridizes specific labeled probes with DNA in tissue sections to directly observe the targeted DNA in cells in tissue sections. However, in head and neck squamous cell carcinoma, the background signal hinders the recognition of DNA labeling signals, which can easily lead to false negative results; 4) HPV E6 / E7 mRNA RT-qPCR / ISH detection method [7,8], this method has high sensitivity and specificity and is considered the gold standard for HPV detection, but due to its complex detection technology and high cost, it is not suitable for routine clinical practice; 5) Liquid biopsy method based on peripheral blood samples [9,10], this method can evaluate HPV infection status by detecting circulating tumor HPV DNA or HPVE6 / L1 antibodies in the patient's peripheral blood. Existing studies have shown good sensitivity and specificity, but due to the lack of sufficient clinical data, it has not yet been applied to routine clinical testing.

[0003] However, the above-mentioned HPV detection methods for head and neck tumors all rely on surgically removed tissue samples or blood samples, which have limitations such as invasive operations, high costs, and long time consumption. It is not convenient to collect samples and conduct routine HPV infection tests in outpatient clinics. In addition, the diagnostic performance is unstable and false positive / false negative results may occur due to technical reasons such as sample cross-contamination and background signal interference, thus affecting clinical diagnosis and treatment.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The object of the present invention is to provide a sample collection method, an HPV detection method and a system for HPV detection to achieve sample collection in a non-invasive and fast manner and maintain the accuracy of the detection results.

[0006] The present invention is achieved in that:

[0007] In a first aspect, the present invention provides a sample collection method for HPV detection, comprising: contacting a sampling unit with a sampling site to be sampled; then preserving the sample collected by the sampling unit; the sampling site to be sampled is selected from at least one of the oral cavity, pharyngeal cavity, laryngeal cavity and nasal cavity, and the sampling unit is a swab or a cell brush.

[0008] In a preferred embodiment of the present invention, HPV detection is HPV nucleic acid detection.

[0009] In a preferred embodiment of the present invention, the sample collected by the sampling unit is placed in a cell preservation solution or a sample preservation solution for preservation.

[0010] In a second aspect, the present invention provides an HPV detection method, which is not intended for the diagnosis of a disease. The detection method includes: extracting DNA from a sample obtained by the above-mentioned sample collection method for HPV detection, and then performing PCR amplification and detection of PCR products.

[0011] In a preferred embodiment of the present invention, the PCR amplification program includes: 42°C for 5 minutes; 94°C for 8 minutes; 94°C for 30 seconds, 60°C for 30 seconds, 70°C for 1 minute, a total of 35 cycles; 70-72°C for 1-10 minutes.

[0012] In a preferred embodiment of the present invention, the detection of PCR products includes: analyzing the PCR products using a capillary electrophoresis instrument.

[0013] In a third aspect, the present invention provides an HPV infection detection system, which includes: a sampling unit, a nucleic acid extraction unit, a nucleic acid amplification unit, and a product detection unit;

[0014] The sampling unit includes: a swab or a cell brush, and a cell preservation solution; the nucleic acid extraction unit includes a nucleic acid extraction instrument, a nucleic acid extraction reagent, and a nucleic acid purification reagent.

[0015] The nucleic acid amplification unit includes a nucleic acid detection kit and a PCR instrument, and the product detection unit includes an electrophoresis instrument.

[0016] In a fourth aspect, the present invention provides an HPV typing system, which includes: a sampling unit, a nucleic acid extraction unit, a nucleic acid amplification unit and a typing identification unit;

[0017] The sampling unit includes: a swab or a cell brush, and a sample storage container; the nucleic acid extraction unit includes a nucleic acid extractor, a nucleic acid extraction reagent and a nucleic acid purification reagent; and the sample storage container can contain a cell preservation solution.

[0018] The nucleic acid amplification unit includes a nucleic acid detection kit and a PCR instrument, and the product detection unit includes an electrophoresis instrument for HPV typing and identification.

[0019] In a preferred embodiment of the present invention, the electrophoresis apparatus used for HPV typing and identification is a capillary electrophoresis apparatus.

[0020] In a preferred embodiment of the present invention, the typing standard of the capillary electrophoresis instrument is as follows: if the peak height of the HPV type is measured ≥ 300RFU, it is determined to be positive for the HPV subtype.

[0021] The present invention has the following beneficial effects:

[0022] Unlike the existing invasive operation methods for HPV testing in patients with head and neck tumors, the sample collection method for HPV testing provided by the present invention adopts a swab or a cell brush for sampling, and uses a swab to scrape cells or a cell brush to collect cells to realize the HPV sample collection of the head and neck of the population. It is non-invasive, fast, and has low sampling cost, and is convenient for carrying out routine HPV sampling work in outpatient clinics and wards.

[0023] The present invention finds that the method of scraping cells with a swab or a cell brush to obtain cells uses the obtained samples, and after nucleic acid extraction and nucleic acid detection, can accurately and efficiently detect the HPV infection status and subtype identification, and has the advantages of high detection accuracy and strong specificity. The sample collection method and the detection method also have significant clinical diagnostic application value, which is of great significance for carrying out screening and detection work for key populations with high risk of clinical HPV infection, and is also of great significance for carrying out HPV virus infection detection and accurate typing in basic disease research.

[0024] In addition, the present invention also provides an HPV infection detection system and an HPV typing system, which can quickly and accurately realize HPV infection detection and HPV typing. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0026] Figure 1 This is the ROC curve of the swab HPV test results. DETAILED DESCRIPTION

[0027] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0028] At present, clinical detection of HPV infection in patients with head and neck tumors mainly relies on surgical tissue samples, which are all invasive and invasive operations. The sample collection operation is complicated and the cost is high. It is not convenient to take samples in the outpatient clinic and carry out routine HPV infection screening; and the diagnostic performance is unstable, and false positive / false negative results are easily produced due to technical reasons such as sample cross contamination and background signal interference, thereby affecting clinical diagnosis and treatment. The present invention is aimed at the problems and shortcomings of the above-mentioned existing detection methods, and studies a method that can conveniently, quickly, accurately and efficiently detect the HPV infection status and its subtype identification (typing) and sample collection. The method combines swab scraping with HPV nucleic acid detection, and can complete sampling in the outpatient clinic and ward without surgical sampling, and carry out HPV infection and subtype identification. The results show that the detection sensitivity and specificity are high, the cost is low, and the requirements for convenience, sensitivity, specificity, etc. in the HPV infection detection and screening work can be met. It is of great significance for carrying out the detection of HPV infection status and subtype identification in patients with head and neck tumors.

[0029] In the first aspect, the present invention provides a sample collection method for HPV detection, which comprises: contacting a sampling unit with a sampling site to be sampled; and then preserving the sample collected by the sampling unit; the sampling site to be sampled is selected from at least one of the oral cavity, pharyngeal cavity, laryngeal cavity and nasal cavity, and the sampling unit is a swab and a cell brush.

[0030] The sample collection method provided by the present invention has the technical advantages of being non-invasive, simple, low-cost, easy to promote, and able to meet the sampling requirements of nucleic acid testing.

[0031] The above-mentioned contact methods include but are not limited to: rotating a swab in the oral cavity to obtain oral epithelial cells; scraping a swab in the pharyngeal cavity or laryngeal cavity to obtain cells; scraping a swab in the nasal cavity to obtain cells; or using a cell brush to brush cells in the oral cavity, pharyngeal cavity, laryngeal cavity or nasal cavity.

[0032] In a preferred embodiment of the present invention, HPV detection is HPV DNA nucleic acid detection. HPV nucleic acid detection includes but is not limited to DNA detection, mRNA detection, etc. DNA detection includes but is not limited to: gel electrophoresis, restriction fragment length polymorphism or direct sequencing to identify HPV genotype, DNA in situ hybridization, etc. mRNA detection includes but is not limited to RT-qPCR detection, RT-qPCR / ISH detection, etc.

[0033] In a preferred embodiment of the present invention, the sample collected by the sampling unit is placed in a sample storage container containing a cell preservation solution (Zheyong Medical Equipment No. 20150197, Haiershi Gene) for storage. In other embodiments, other commercially available buffers or stabilizing solutions that can achieve sample storage may also be used.

[0034] In a second aspect, the present invention provides an HPV detection method, which is not intended for the diagnosis of a disease. The detection method includes: extracting DNA from a sample obtained by the above-mentioned sample collection method for HPV detection, and then performing PCR amplification and detection of PCR products.

[0035] Methods that are not intended for disease diagnosis include but are not limited to: HPV prevalence survey methods in the population, HPV infection detection and precise typing in basic disease research.

[0036] The reagent for DNA extraction or purification is, for example, a reagent selected from (PN.1060056, Hirsch Gene).

[0037] In a preferred embodiment of the present invention, the PCR amplification procedure includes: 42°C for 5 minutes; 94°C for 8 minutes; 94°C for 30 seconds, 60°C for 30 seconds, 70°C for 1 minute, a total of 35 cycles; 70-72°C for 1-10 minutes. Under this amplification procedure, it has good detection accuracy and typing effect.

[0038] In a preferred embodiment of the present invention, the detection of PCR products includes: analyzing the PCR products using a capillary electrophoresis instrument.

[0039] In a third aspect, the present invention provides an HPV infection detection system, which includes: a sampling unit, a nucleic acid extraction unit, a nucleic acid amplification unit, and a product detection unit;

[0040] The sampling unit includes: a swab or a cell brush, and a sample storage container; the nucleic acid extraction unit includes a nucleic acid extraction instrument, a nucleic acid extraction reagent, and a nucleic acid purification reagent.

[0041] The nucleic acid amplification unit includes a nucleic acid detection kit and a PCR instrument, and the product detection unit includes an electrophoresis instrument.

[0042] In a fourth aspect, the present invention provides an HPV typing system, which includes: a sampling unit, a nucleic acid extraction unit, a nucleic acid amplification unit and a typing identification unit;

[0043] The sampling unit includes: a swab or a cell brush, and a sample storage container, and the nucleic acid extraction unit includes a nucleic acid extraction instrument, a nucleic acid extraction reagent, and a nucleic acid purification reagent;

[0044] The nucleic acid amplification unit includes a nucleic acid detection kit and a PCR instrument, and the product detection unit includes an electrophoresis instrument for HPV typing and identification.

[0045] In a preferred embodiment of the present invention, the electrophoresis instrument used for HPV typing is a capillary electrophoresis instrument. The kit used for typing includes but is not limited to: Human papillomavirus (HPV) nucleic acid detection and genotyping kit (PCR capillary electrophoresis fragment analysis method) (PN.1060037, Hirsch Gene).

[0046] In a preferred embodiment of the present invention, the typing standard of the capillary electrophoresis instrument is as follows: if the peak height of the HPV type is ≥300RFU, it is determined to be HPV positive. The presence of pcDNA and β-globin specific peaks in the normal sample test results and the pcDNA peak height ≥500RFU are used as effective result quality control indicators (if the β-globin specific peak does not appear due to too few cells, but the specific HPV type peak appears, it is still considered a valid sample); the positive control peak height of 750-15600RFU is used as a positive control quality control indicator.

[0047] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0048] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0049] Example 1

[0050] This embodiment provides a sampling method for HPV infection detection, which includes the following steps:

[0051] 1. Materials and Reagents

[0052] Equipment and materials: disposable sterile sampler (Su Xie No. 20182660365, Haiershi Gene), cell preservation fluid (Zhejiang Yong Xie Bei No. 20150197, Haiershi Gene).

[0053] 2. Sampling methods and steps

[0054] Sample collection: Medical staff rotate the swab clockwise for 5 times at the tumor lesion through the mouth to scrape enough epithelial cell samples, and store the swab brush head in cell preservation solution.

[0055] Example 2

[0056] This embodiment provides a sampling method for HPV infection detection, which includes the following steps:

[0057] 1. Materials and Reagents

[0058] Equipment and materials: disposable sterile sampler (Su Xie No. 20182660365, Haiershi Gene), cell preservation fluid (Zhejiang Yong Xie Bei No. 20150197, Haiershi Gene).

[0059] 2. Sampling methods and steps

[0060] Sample collection: Medical staff rotate the swab clockwise for 5 times at the tumor lesion through the nasal cavity to scrape enough epithelial cell samples, and store the swab brush head in cell preservation solution.

[0061] Example 3

[0062] This embodiment provides a method for detecting HPV.

[0063] 1. Materials and Reagents

[0064] Equipment and materials: fully automatic nucleic acid extraction instrument Smart LabAssist-16 / Smart LabAssist-32 (Hangzhou Precision Biotechnology Co., Ltd.), 3500Dx / 3500xL Dx genetic analyzer (Applied Biosystems), microcentrifuge, vortex mixer, PCR tube, disposable sterile sampler (Su Xie No. 20182660365, Haiershi Gene), cell preservation solution (Zheyong Xie Bei No. 20150197, Haiershi Gene).

[0065] Reagents: nucleic acid extraction or purification reagent (PN.1060056, Hirsch Gene), human papillomavirus (HPV) nucleic acid detection and genotyping kit (PCR capillary electrophoresis fragment analysis method) (PN.1060037, Hirsch Gene), 1× PBS Buffer.

[0066] 2. Sample Collection (Same as Example 1)

[0067] 3. HPV DNA extraction:

[0068] 1. Vortex the cell preservation solution sample on a vortex mixer for 10 seconds, take 1 ml of the preservation solution sample into a 1.5 ml centrifuge tube, centrifuge at 12000g for 5 minutes, discard the supernatant, and retain the cell pellet;

[0069] 2. Add 200 μl 1×PBS Buffer to the centrifuge tube and gently pipette to resuspend the cells for use as the sample to be extracted;

[0070] 3. Add the sample to be extracted into nucleic acid extraction or purification reagent, use the fully automatic nucleic acid extractor SmartLabAssist-16 / Smart LabAssist-32 to extract and purify the nucleic acid, and use a pipette to aspirate the purified nucleic acid and transfer it to a 1.5ml centrifuge tube.

[0071] IV. PCR amplification, nucleic acid detection and genotyping:

[0072] The human papillomavirus (HPV) nucleic acid detection and genotyping kit (PN.1060037, Hirsch Genetics) was used for subsequent HPV subtype detection and identification;

[0073] 1. According to the number of samples N to be tested, prepare 1.1×N reaction solution (if 1.1×N is a non-integer, take the nearest integer > 1.1×N) with 9μl HPV PCR premix F488 and 2μl TaqDNA polymerase C for each sample, flick the sample tube to mix thoroughly, centrifuge for 10 seconds in a microcentrifuge to make the reaction solution fall to the bottom of the tube, and dispense into PCR reaction tubes at 11μl / tube;

[0074] 2. Add 9 μl of the DNA sample to be tested, 9 μl of HPV positive control F488 and 9 μl of water (nuclease-free) as positive control and negative control respectively, record the sample number sequence, and cover the tube tightly;

[0075] 3. Tap to mix and centrifuge briefly to allow the liquid to fall to the bottom of the tube, and press the following temperature to perform PCR thermal cycling reaction: a. 42°C for 5 minutes; b. 94°C pre-denaturation for 8 minutes; c. 94°C denaturation for 30 seconds, 60°C annealing for 30 seconds, 70°C extension for 1 minute, a total of 35 cycles; d. 70°C extension for 1 minute; e. PCR products are stored at 4°C.

[0076] 4. Analyze the PCR products using 3500Dx / 3500xL Dx Genetic Analyzer (Applied Biosystems). Add 9 μl of Hi-Di and 1 μl of PCR product to each sample well to obtain the HPV typing detection peak pattern for subsequent analysis.

[0077] Result analysis: The peak height of the specific HPV type ≥ 300RFU is judged as positive for the HPV type. The normal sample test results show the presence of pcDNA and β-globin specific peaks and the pcDNA peak height ≥ 500RFU, which is used as a quality control indicator for effective results (if the β-globin specific peak does not appear due to too few cells, but the specific HPV type peak appears, it is still considered a valid sample); the positive control peak height of 750-15600RFU is used as a quality control indicator for the positive control.

[0078] Comparative Example 1

[0079] This embodiment provides a sampling method for HPV infection detection, which includes the following steps:

[0080] 1. Materials and Reagents

[0081] Equipment and Materials: Tissue cryopreservation tubes.

[0082] 2. Sampling methods and steps

[0083] Sample collection: Medical staff will cut a piece of tumor tissue the size of a rice grain to a mung bean during the operation and store it in a tissue cryopreservation tube.

[0084] Experimental Example 1

[0085] The method of Example 3 was used to perform HPV infection and subtype identification detection. A total of 33 head and neck tumor patients who visited the Department of Otorhinolaryngology and Head and Neck Surgery of West China Hospital of Sichuan University were tested, and lesion samples scraped by oral or nasal swabs were collected for HPV infection and subtype identification detection; at the same time, surgical tissue samples of patients were collected according to the method of Comparative Example 1 for HPV detection as the gold standard for diagnostic tests. The test results are shown in Tables 1 and 2.

[0086] Table 1. Summary of HPV test results for 33 patients with head and neck cancer

[0087]

[0088]

[0089] Table 2. Comparison of swab test results with the “gold standard” diagnosis results

[0090]

[0091] The results showed that this study included 11 HPV-positive (gold standard diagnostic results) patients, of which 9 patients tested positive by swab and 2 patients tested negative. The virus subtype identification results of patients with HPV-positive swabs were consistent with the tissue sample test results; 22 HPV-negative patients were included, and their swab test results were all negative, with no false positive results. In summary, the sensitivity of swab HPV detection was 81.82% (the accuracy of HPV subtype detection was 100%), the specificity was 100%, the missed diagnosis rate (false negative rate) was 18.18%, the misdiagnosis rate (false positive rate) was 0%, the negative likelihood ratio was 18.18%, the Youden index γ was 0.818, the positive predictive value was 100%, and the negative predictive value was 91.67%. The ROC curve was drawn and the area under the curve AUC was calculated to be 0.909 (p < 0.001) (ROC curve is shown in Figure 1 shown).

[0092] Therefore, the HPV infection detection method and virus subtype identification method provided by the present invention have high accuracy, high predictive value for the actual disease status, have clinical diagnostic application value, and are conducive to carrying out screening and detection work for high-risk and low-risk populations for HPV infection.

[0093] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0094] References:

[0095] 1. Kim, KY; Lewis, JS; Chen, Z., Current status of clinical testing for human papillomavirus in oropharyngeal squamous cell carcinoma. J Pathol ClinRes 2018, 4(4), 213-226.

[0096] 2.Prigge,E.-S.;Arbyn,M.;von Knebel Doeberitz,M.;Reuschenbach,M.,Diagnostic accuracy of p16INK4a immunohistochemistry in oropharyngealsquamous cell carcinomas:Asystematic review and meta-analysis.Int JCancer2017,140(5),1186-1198.

[0097] 3.Lewis,J.S.;Beadle,B.;Bishop,J.A.;Chernock,R.D.;Colasacco,C.;Lacchetti,C.;Moncur,J.T.;Rocco,J.W.;Schwartz,M.R.;Seethala,R.R.;Thomas,N.E.;Westra,W.H.;Faquin,W.C.,Human Papillomavirus Testing in Head and NeckCarcinomas:Guideline From the College of American Pathologists.Arch PatholLab Med 2018,142(5),559-597.

[0098] 4.Torres-Ibarra,L.;Lorincz,A.T.;Wheeler,C.M.;Cuzick,J.;Hernández-López,R.;Spiegelman,D.;León-Maldonado,L.;Rivera-Paredez,B.;Méndez-Hernández,P.;Lazcano-Ponce,E.;Salmerón,J.,Adjunctive testing by cytology,p16 / Ki-67dual-stained cytology or HPV16 / 18E6 oncoprotein for the management of HPV16 / 18screen-positive women.Int JCancer 2021,148(9),2264-2273.

[0099] 5.Boscolo-Rizzo,P.;Pawlita,M.;Holzinger,D.,From HPV-positivetowardsHPV-driven oropharyngeal squamous cell carcinomas.Cancer TreatRev 2016,42,24-29.

[0100] 6.Zhao,P.-Y.;Jiang,H.-C.;Li,Y.;Wang,J.-B.;Zhang,T.-T.;Liu,C.-H.;Song,L.-W.;Cheng,J.-J.,Comparison of the cervista HPV HR test andluminex XMAPtechnology for the diagnosis of cervical intraepithelial neoplasia.Eur JObstet Gynecol Reprod Biol 2017,214,150-155.

[0101] 7.Schache,A.G.;Liloglou,T.;Risk,J.M.;Filia,A.;Jones,T.M.;Sheard,J.;Woolgar,J.A.;Helliwell,T.R.;Triantafyllou,A.;Robinson,M.;Sloan,P.;Harvey-Woodworth,C.;Sisson,D.;Shaw,R.J.,Evaluationof human papilloma virusdiagnostic testing in oropharyngeal squamous cellcarcinoma:sensitivity,specificity,and prognostic discrimination.Clin CancerRes 2011,17(19),6262-6271.

[0102] 8.Chernesky,M.;Jang,D.;Schweizer,J.;Arias,M.;Doerwald-Munoz,L.;Gupta,M.;Jackson,B.;Archibald,S.;Young,J.;Lytwyn,A.;Smieja,M.;Severini,A.;Ecobichon-Morris,A.;Hyrcza,M.,HPV E6 oncoproteins and nucleic acids in necklymph node fine needleaspirates and oral samples from patients withoropharyngeal squamous cellcarcinoma.Papillomavirus Res 2018,6,1-5.

[0103] 9.Siravegna,G.;O'Boyle,C.J.;Varmeh,S.;Queenan,N.;Michel,A.;Stein,J.;Thierauf,J.;Sadow,P.M.;Faquin,W.C.;Perry,S.K.;Bard,A.Z.;Wang,W.;Deschler,D.G.;Emerick,K.S.;Varvares,M.A.;Park,J.C.;Clark,J.R.;Chan,A.W.;Andreu Arasa,V.C.;Sakai,O.;Lennerz,J.;Corcoran,R.B.;Wirth,L.J.;Lin,D.T.;Iafrate,A.J.;Richmon,J.D.;Faden,D.L.,Cell-Free HPV DNA Provides an Accurate andRapidDiagnosis of HPV-Associated Head and Neck Cancer.Clin Cancer Res2022,28(4),719-727.

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Claims

1. A sample collection method for HPV detection, characterized in that: It includes: placing the sampling unit in contact with the sampling site to be sampled; Then the sample collected by the sampling unit is stored; the sampling site to be sampled is selected from at least one of the oral cavity, pharyngeal cavity, laryngeal cavity and nasal cavity, and the sampling unit is a swab or a cell brush.

2. The sample collection method for HPV detection according to claim 1, characterized in that: The HPV detection is HPV nucleic acid detection.

3. The sample collection method for HPV detection according to claim 1, characterized in that: The sample collected by the sampling unit is placed in a cell preservation solution or a sample preservation solution for preservation.

4. A method for detecting HPV, characterized in that: The HPV detection method is not intended for the diagnosis of a disease, and the detection method comprises: extracting DNA from a sample obtained by the sample collection method for HPV detection according to any one of claims 1 to 3, and then performing PCR amplification and detection of PCR products.

5. The HPV detection method according to claim 4, characterized in that: The PCR amplification program includes: 42°C for 5 minutes; 94°C for 8 minutes; 94°C for 30 seconds, 60°C for 30 seconds, 70°C for 1 minute, a total of 35 cycles; 70-72°C for 1-10 minutes.

6. The HPV detection method according to claim 4, characterized in that: The detection of the PCR product includes: analyzing the PCR product using a capillary electrophoresis instrument.

7. A HPV infection detection system, characterized in that: It includes: a sampling unit, a nucleic acid extraction unit, a nucleic acid amplification unit and a product detection unit; The sampling unit includes a swab or a cell brush and a sample storage container, and the nucleic acid extraction unit includes a nucleic acid extraction instrument, a nucleic acid extraction reagent and a nucleic acid purification reagent. The nucleic acid amplification unit includes a nucleic acid detection kit and a PCR instrument, and the product detection unit includes an electrophoresis instrument.

8. An HPV typing system, characterized in that: It includes: a sampling unit, a nucleic acid extraction unit, a nucleic acid amplification unit and a typing identification unit; The sampling unit includes a swab or a cell brush and a sample storage container, and the nucleic acid extraction unit includes a nucleic acid extraction instrument, a nucleic acid extraction reagent and a nucleic acid purification reagent. The nucleic acid amplification unit includes a nucleic acid detection kit and a PCR instrument, and the product detection unit includes an electrophoresis instrument for HPV typing and identification.

9. The HPV typing system according to claim 8, characterized in that: The electrophoresis instrument used for HPV typing and identification is a capillary electrophoresis instrument.

10. The HPV typing system according to claim 9, characterized in that: The typing standard of the capillary electrophoresis instrument is as follows: if the peak height of the HPV type measured is ≥300RFU, it is determined to be HPV positive.

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