A kit for early screening and monitoring of HPV-related tumors

Abstract

The present application relates to the field of DNA detection technology, and specifically to a kit, method and application for early screening and monitoring of HPV-related tumors. The present application is based on multiplex ddPCR and uses mixed probes to detect four HPV subtypes in plasma at one time. Compared with conventional detection technology, the kit is simple and safe to operate, non-invasive, low-cost, and has a short cycle; at the same time, the kit has higher detection sensitivity and strong specificity, with a minimum detection limit of only 250 copies / ml.

Description

Technical Field

[0001] The present application relates to the technical field of DNA detection, and specifically to a kit, method and application thereof for detecting free HPV in plasma, thereby achieving early screening and prognosis monitoring of HPV-related tumors. Background Art

[0002] Human papillomavirus (HPV) is a small molecule circular double-stranded DNA virus. The virus is tissue-specific and is closely related to cervical, head and neck tumors. The types of HPV detection in my country: HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, a total of 13 genotypes are listed as high-risk types. They have a very high risk of causing cancer, especially HPV 16 and 18.

[0003] There are many methods for HPV detection, including HPV E6 / E7 mRNA detection, HPV DNA detection, immunohistochemical p16 detection, etc. HPV E6 / E7 mRNA in situ hybridization technology refers to the process of using a specific labeled known sequence nucleic acid as a probe to hybridize with nucleic acids in cells or tissue sections to accurately and quantitatively locate a specific nucleic acid sequence. E6 / E7 mRNA is observed and located under a light microscope or electron microscope. HPV RNA in situ hybridization can reflect the transcriptional activity level of HPV, which is not available in other detection methods. Therefore, it has gradually become one of the most effective methods for determining HPV status. HPV DNA detection is generally performed by PCR or in situ hybridization. This method generally has a high sensitivity, but it cannot distinguish whether HPV has transcriptional activity. Immunohistochemical p16 detection has become one of the routine detection indicators for cervical cancer and oropharyngeal cancer. Overexpression of p16 protein is not always related to abnormal epithelial cell proliferation. Certain inflammation, injury or other non-pathological factors may also lead to positive expression of p16. Therefore, the detection results of p16 have a certain degree of non-specificity. Although tissue biopsies are the gold standard for tumor diagnosis and screening, they are invasive, painful, and increase the risk of complications such as local infection and bleeding. In addition, tissue biopsies have limited advantages and cannot accurately reflect the dynamics of the tumor or its response to treatment.

[0004] Liquid biopsy is a non-invasive detection method. Common analytes include circulating tumor DNA (ctDNA), circulating tumor cells (CTC), mRNA, protein, miRNA, exosomes and metabolites. It is not yet regarded as a standard test in clinical practice, but its potential applications are rapidly expanding to diagnosis, treatment response, prognosis monitoring and other fields. Circulating free DNA (cell-free DNA, cfDNA) is a type of ctDNA. In 2001, researchers used cervical cancer plasma to start one of the earliest reports of HPV cfDNA in cervical cancer. The detection technology used at that time was quantitative polymerase chain reaction (qPCR), which had defects in the sensitivity of detecting low-copy number viral DNA. With the development of technology, droplet digital PCR (ddPCR) has become a cutting-edge technology for HPV DNA detection. Compared with qPCR, ddPCR has higher accuracy and sensitivity.

[0005] In recent years, the research on HPV DNA detection of cervical and head and neck tumors using ddPCR technology has increased significantly. It is reported that the cfDNA levels of patients with HPV-related tumors are higher than those of healthy individuals, and the levels in plasma are >1000ng / mL. cfDNA in plasma can be used as a biomarker for HPV-related tumors, and its detection is helpful for early cancer screening, treatment response and prognosis monitoring. The HPV detection methods currently used in clinical practice have problems such as the need for professional operation, high technical requirements, and low sensitivity. In particular, there are few existing technologies that can realize HPV multiple typing detection based on a single tube of ddPCR. In practice, there is a need for a detection method that is simple to operate, highly sensitive, safe and non-invasive. In view of this, the present application is proposed. Summary of the invention

[0006] In order to solve the above-mentioned problems of the prior art, the present application proposes a digital PCR-based detection method for free HPV in human plasma. Through mixed probes, free HPV16 / 18 / 52 / 56 in human plasma can be simultaneously detected in one reaction system, and can be accurately typed, thereby achieving a simple, safe, non-invasive and low-cost effect; at the same time, because the detection has the advantages of high sensitivity, strong specificity and low detection limit, the kit of the present application can be effectively used for the early screening and monitoring of HPV-related tumors.

[0007] Specifically, the technical solutions adopted in this application are as follows:

[0008] The present application first provides a ddPCR primer-probe composition that can be used for high-sensitivity detection and typing of HPV in cfDNA, wherein the primer-probe is targeted at four HPV subtypes in a single-tube system;

[0009] Preferably, the four HPV subtypes are HPV16, HPV18, HPV52 and HPV56.

[0010] Furthermore, the primer probe is also directed to an internal reference gene, and the internal reference gene is RNaseP.

[0011] Furthermore, the primers are specifically designed and screened by the following method:

[0012] Target region selection: Download the genome sequences of 38 HPV subtypes from the public gene database, and download more than 100 sequences for each subtype; evaluate multiple sequences of each subtype of HPV16, 18, 52 and 56, and select the sequences that are conservative within each subtype and specific between the other 37 subtypes of each subtype from each group of subtypes, with a sequence length of 200-500bp; at the same time, use 20bp as a node to screen the conservative and subtype-specific sequence segments in the genome one by one, and more than 99% of the sequences within the 5kb interval of the human genome sequence cannot be matched; find no less than 30 target regions for each subtype of HPV16, 18, 52 and 56, and sort them from high to low according to the matching rate; in addition, the GC content of the target region is about 50%, and the single / multi-base repeats are less than 5, and the target region cannot involve the plasmid region, mitochondrial region and 16S conservative region;

[0013] Primer design, screening and optimization: Preliminary primer design based on the target region, select sequences with small dimer and similar Tm values, and determine the specificity between primer subtypes and conservative compatibility within subtypes through blast comparison; after comparison, evaluate the primer performance through melting curve detection, and select the sequence with the best specificity / compatibility for multiple evaluations

[0014] Furthermore, the sequence of the primer is specifically shown as SEQ ID NO.1-10, or has at least 90% homology with SEQ ID NO.1-10; further, the sequence of the probe is specifically shown as SEQ ID NO.11-15, or has at least 90% homology with SEQ ID NO.11-15.

[0015] Furthermore, the 5' end of the probes are all labeled with a fluorescent reporter group, and the 3' end are all labeled with a quencher group;

[0016] Preferably, the fluorescent reporter group labeled with VIC at the 5' end of the probe for HPV16 type, and the quencher group labeled with MGB at the 3' end; the fluorescent reporter group labeled with ROX at the 5' end of the probe for HPV18 type, and the quencher group labeled with MGB at the 3' end; the fluorescent reporter group labeled with CY5 at the 5' end of the probe for HPV52 type, and the quencher group labeled with MGB at the 3' end; the fluorescent reporter group labeled with Atto425 at the 5' end of the probe for HPV56 type, and the quencher group labeled with MGB at the 3' end; the fluorescent reporter group labeled with FAM at the 5' end of the probe for the internal reference gene RnaseP, and the quencher group labeled with MGB at the 3' end.

[0017] Furthermore, the primer concentration ratio of HPV16, HPV18, HPV52, HPV56 and the internal reference gene is 1:1:1:1:1; the probe concentration ratio is 1-2:4-6:0.5-1:1-2:0.5-1; preferably, the probe concentration ratio is 1:4:0.6:1:0.5.

[0018] Furthermore, the primer-probe combination can be used for high-sensitivity non-invasive early screening and monitoring of HPV-related tumors.

[0019] The present application also provides a product that can be used for high-sensitivity detection and typing of HPV in cfDNA, the product comprising any of the primer-probe combinations described above; the product can be used for high-sensitivity, non-invasive early screening and monitoring of HPV-related tumors.

[0020] Furthermore, the product is a kit; further, the finished product also includes dNTPs, Taq enzyme and PCR buffer.

[0021] Furthermore, in the product, the primer concentration ratio of HPV16, HPV18, HPV52, HPV56 and the internal reference gene is 1:1:1:1:1; the probe concentration ratio is 1-2:4-6:0.5-1:1-2:0.5-1; preferably, the probe concentration ratio is 1:4:0.6:1:0.5.

[0022] The present application also provides the use of any of the above-mentioned primer-probe combinations in the preparation of high-sensitivity non-invasive early screening and monitoring products for HPV-related tumors.

[0023] The present application also provides the use of any of the above-mentioned primer-probe combinations in high-sensitivity non-invasive early screening and monitoring of HPV-related tumors.

[0024] A method for amplifying HPV16, HPV18, HPV52, and HPV56 subtypes in a cfDNA sample, comprising the following steps: amplifying the sample using any of the primer-probe combinations described above.

[0025] Preferably, the amplification conditions are 58° C. and 45 cycles; more preferably, the specific amplification conditions are as follows:

[0026]

[0027] The present application also provides a method for high-sensitivity non-invasive early screening and monitoring of HPV-related tumors, which specifically includes the following steps: plasma separation and extraction of cfDNA; preparation of a digital PCR reaction system; chip loading; the above-mentioned amplification method; data reading; and data analysis.

[0028] Compared with the prior art, this application has at least the following advantages: Traditional ddPCR HPV detection can usually only be performed in a single tube and single multiplex detection, and the sample types are mostly for tissue samples. This application realizes the direct detection of cfDNA samples, that is, free HPV in plasma, through the design and optimization of the system, and realizes ddPCR single-tube multiplex detection. The use of multiple mixed primer probes can not only detect four types of HPV in the blood at one time, but also accurately type them. This method is not only simple, safe, non-invasive, but also has the advantages of lower cost; and in terms of detection effect, the sensitivity and specificity of the kit of this application are high, and the minimum detection limit can reach 250 copies / ml, which is significantly better than the detection limit of current market products; the detection cycle of this application is shorter, and it can be completed within ≤1 natural day after the sample arrives. It is precisely based on the advantages of the kit of this application in terms of sample type, multiple system and sensitivity, so that it can not only be used for early screening and diagnosis of HPV-related tumors, but also can effectively monitor the response and prognosis of tumor treatment, which has great clinical significance and has been promoted. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1 , select primer melting curve specific result data;

[0031] Figure 2 , gDNA single subtype + internal reference mixed detection results;

[0032] Figure 3 , gDNA single tube multiplex detection results at different probe concentrations;

[0033] Figure 4 , cfDNA single tube single multiplex test results;

[0034] Figure 5 , cfDNA single tube multiplex test results;

[0035] Figure 6 , Detection results under different PCR conditions

[0036] Figure 7 , gDNA detection limit test results;

[0037] Figure 8 , cfDNA detection limit test results. DETAILED DESCRIPTION

[0038] The technical solution of the present application will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

[0039] Experimental Example 1: Design, screening and optimization of primer probes

[0040] 1) Selection of HPV subtype;

[0041] HPV16 and 18 genotypes are high-risk types with a very high risk of cancer; HPV 52 is the dominant type in Asian women; in addition, combined with in-hospital clinical practice and existing data statistics, HPV56 is a more common type in northern my country. Taking the above into consideration and considering the technical feasibility of the amplification process, this application finally determines to select HPV16, 18, 52 and 56 subtypes as the basis for detection, and designs the primer probe system of ddPCR accordingly.

[0042] 2) Design, screening and optimization of corresponding primers

[0043] Target region selection and preliminary primer design

[0044] 1. Targeting region selection

[0045] The genome sequences of 38 HPV subtypes were downloaded from the public gene database, with more than 100 sequences downloaded for each subtype; multiple sequences of each subtype of HPV16, 18, 52 and 56 were evaluated, and sequences that were conservative within each subtype and specific between the other 37 subtypes of each subtype were selected from each group of subtypes, with a sequence length of 200-500bp; at the same time, using 20bp as a node, the sequence segments that were conservative within the subtype and specific between subtypes were screened one by one in the genome, and more than 99% of the sequences within the 5kb interval with the full human genome sequence could not be matched; no less than 30 target regions were found for each subtype of HPV16, 18, 52 and 56, and they were ranked from high to low according to the matching rate; in addition, the GC content of the target region was about 50%, and the single / multi-base repeats were less than 5, and the target region did not involve plasmid, mitochondrial and 16S conservative regions.

[0046] 2. Primer design, screening and optimization:

[0047] The primers were initially designed based on the above-mentioned target regions. The design required the selection of sequences with small dimer and similar Tm values. The primers were then blasted to determine the specificity between primer subtypes and the conservative compatibility within subtypes. The aligned sequences were used for primer synthesis, and the primer performance was determined by melting curve detection. The sequences with the best specificity / compatibility were selected for subsequent evaluation.

[0048] Based on the above steps, the candidate primer systems preliminarily designed by this application for the above 4 subtypes are as follows in Table 1:

[0049]

[0050]

[0051] Some results of the primer specificity screening by candidate primer melting curves are shown in Figure 1 In addition, the present application also screened RnaseP as an internal reference control gene. Based on the above screening and optimization, the primer probe system finally determined by the present application is shown in the following table:

[0052]

[0053] Example 2: Feasibility evaluation and system optimization of primer probes

[0054] Taking into account the sensitivity of single-tube multiplex ddPCR to the primer-probe system and the complexity of detecting multiple HPV subtypes, this application further verifies the feasibility of the screened primer-probe system for positive accused samples gDNA and cfDNA.

[0055] Among them, the positive test sample gDNA information used in this application is as shown in the following table; in addition, the preparation of the quality control product cfDNA is to use an ultrasonic interrupter to interrupt the gDNA positive HPV quality control product, and pass the Qsep fragment quality inspection to prepare a cfDNA quality control product with a fragment length that meets the expected requirements.

[0056] Positive accused gDNA and other information;

[0057]

[0058] The specific evaluation and operation steps are as follows:

[0059] 1) Use HPV16 / 18 / 52 / 56 four subtypes of positive quality control gDNA for separate testing (each subtype and internal reference gene data were repeated 3 times), the sample information is as follows:

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066] The results are as follows:

[0067]

[0068] It can be seen that only samples with HPV16 template have starting lines in the first plate; only samples with HPV18 template have starting lines in the second plate; only samples with HPV52 template have starting lines in the third plate; the primers for each subtype have good specificity, all samples in the fourth plate have starting lines, and the internal reference control gene is well selected.

[0069] 2) Using positive quality control gDNA of four subtypes of HPV16 / 18 / 52 / 56, primers of each subtype and internal reference gene primers were mixed in different proportions for testing to determine the feasibility of primer probes. Sample information is as follows:

[0070]

[0071]

[0072]

[0073]

[0074] The results are as follows:

[0075]

[0076] Digital PCR results Figure 2 As shown, it can be seen that in the methodology, plate 1: the effect of the change in probe ratio on the Ct value is greater than the change in primer ratio; plate 2: the hpv52 probe ratio increases, the Ct value decreases, and the gap with other subtype Ct values ​​decreases. The digital PCR results show that the internal reference gene is mixed with each subtype gene separately, and the results of different mixing ratios are displayed. Each subgroup can be clearly distinguished, and the ratio is relatively appropriate.

[0077] 3) Use the positive quality control gDNA of four subtypes of HPV16 / 18 / 52 / 56 to mix, and use single-tube multiple primers to test three ratios (3 repeated data) to determine the feasibility of the primer probe (ratio 1 = 0.5μM). The sample information is as follows:

[0078]

[0079] The results are as follows Figure 3 As shown, the probe ratio was adjusted by setting the concentration of HPV16:HPV18:HPV52:HPV56:internal reference = 1:4:0.6:1:0.5 for the best effect.

[0080] 4) Use the positive quality control cfDNA of four subtypes of HPV16 / 18 / 52 / 56 for separate detection, according to the primer mixing ratio HPV16+HPV18+HPV52+HPV56 internal reference (1:1:1:1:1), that is, all are 0.2μM. The probe mixing ratio is HPV16+HPV18+HPV52+HPV56+internal reference (1:4:0.6:1:0.5), that is, HPV16 is 0.5μM; HPV18 is 2μM; HPV52 is 0.3μM; HPV56 is 0.5μM; internal reference is 0.25μM, and the detection (3 repeated data) is carried out to determine the feasibility of the primer probe.

[0081] The sample information is as follows:

[0082]

[0083] The results are as follows Figure 4 As shown, the four subtypes of positive quality control cfDNA were detected according to the above primer-probe ratio, and each subgroup could be clearly distinguished with an appropriate ratio.

[0084] 5) Mix the positive quality control cfDNA of four subtypes of HPV16 / 18 / 52 / 56, and test the single-tube multiple primers according to the above ratio to determine the feasibility of the primer probe. The sample information is as follows:

[0085]

[0086] The results are shown as Figure 5 As shown, the four subtypes of positive quality control cfDNA were mixed and tested, and each subgroup could be clearly distinguished.

[0087] 6) Use positive control cfDNA and gDNA of 21 subtypes of HPV genes 6, 11, 26, 31, 33, 35, 39, 42, 43, 44, 45, 51, 53, 58, 59, 66, 68, 73, 81, 82 and 83 for testing respectively, and mix the primers and probes in the proportions according to 3) to determine the feasibility of the primers and probes.

[0088] Sample information: Positive quality control cfDNA and gDNA of 21 subtypes of HPV genes: 6, 11, 26, 31, 33, 35, 39, 42, 43, 44, 45, 51, 53, 58, 59, 66, 68, 73, 81, 82 and 83.

[0089] The specific results are as follows:

[0090]

[0091]

[0092]

[0093] It can be seen that the primer probe of the present application has no cross-reactivity to other subtypes and can show good inter-subtype specificity.

[0094] To sum up the test results, the four sets of primer probes determined in this application can not only effectively perform single-multiplex specific amplification of gDNA / cfDNA samples, but also do not cross with other subtypes; in addition, when the primers of HPV16: HPV18: HPV52: HPV56: internal reference in the amplification system are mixed in equal proportions (1:1:1:1:1), the probe ratio is 1-2:4-6:0.5-1:1-2:0.5-1 (preferably 1:4:0.6:1:0.5), the effect is optimal.

[0095] Example 3: Optimization of PCR amplification reaction program

[0096] This embodiment sets different PCR reaction procedures to determine the most suitable reaction procedure for this application. The detailed steps are as follows:

[0097] 1) Using cfDNA as a template, add primer probe premix and digital PCR premix to prepare the reaction system. The configuration system is as follows:

[0098] Reagents Volume(uL) 4X dPCR Probe Master Mix(cy5.5) 6 Primer probe premix X DNA sample to be tested Y Nuclease-Free Water Replenish to 22uL

[0099] 2) Chip loading: Use Sniper DQ24 / DQ24 Plus / DQ24 Pro ddPCR instrument.

[0100] 3) PCR amplification: Place the chip symmetrically into the PCR amplification instrument, set the reaction program, and start amplification. In this embodiment, the following 6 reaction programs are set to determine the appropriate process.

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108] The sample information is as follows:

[0109]

[0110]

[0111] The results are as follows Figure 6 As shown, when the amplification conditions are set at 58 degrees and 45 cycles, the effect is optimal. Therefore, the optimal amplification conditions of this application are determined as follows:

[0112]

[0113] Example 4. Test kit performance verification

[0114] A corresponding detection kit was constructed by combining the primer probe obtained in the present application with a digital PCR premix (containing dNTPs, Taq enzyme and PCR buffer), and the performance of the kit was further verified.

[0115] 1. Minimum detection limit test

[0116] 1. Single genotype detection

[0117] 1) gDNA: gradient dilution into three gradients of 1000 copies / ml, 500 copies / ml and 250 copies / ml of standard (four subtypes), and determine the detection limit. The results are shown in the following table and Figure 7 .

[0118]

[0119] 2) cfDNA: gradient dilution into three gradients of 1000 copies / ml, 500 copies / ml and 250 copies / ml of standard (four subtypes), and determine the detection limit. The results are shown in the following table and Figure 8 shown.

[0120]

[0121]

[0122] 2. Multiple detection of four subtypes:

[0123] 1) cfDNA: Dilute the standard into three gradients of 1000 copies / mL, 500 copies / mL and 250 copies / mL (four subtypes were mixed) and determine the detection limit. The results are shown in the following table.

[0124]

[0125]

[0126]

[0127]

[0128] 2) cfDNA: The four subtypes are mixed according to the concentration in the table below. The weak positive is 500 copies / mL and the strong positive is 0.7-1E+07 copies / mL to determine whether the weak positive detection will be affected. The sample information is as follows:

[0129]

[0130] The results are as follows:

[0131]

[0132] The results showed that for single genotype detection, the four subtypes of gDNA and cfDNA samples were detected at three concentration gradients of 1000 copies / mL, 500 copies / mL and 250 copies / mL, with a detection rate of 100%; for four-subtype mixed multiplex detection, the three concentration gradients of 1000 copies / mL, 500 copies / mL and 250 copies / mL were set, with a detection rate of 100% at 1000 copies / mL, a detection rate of 100% at 500 copies / mL and a detection rate of 90% at 250 copies / mL.

[0133] The above overall results show that the kit of the present application can achieve a detection limit of 250 copies / mL regardless of single gene detection or four-subtype mixed multiple detection.

[0134] 2. Specificity Detection

[0135] In this embodiment, interferences are added to determine whether the detection situation will be affected.

[0136] 1) Using 5 types of composite human papillomavirus (HPV cells), the viral nucleic acid was obtained by extraction and tested:

[0137] GW-IPF055: HPV16, HPV18, HPV52, HPV58, HPV6, HPV11;

[0138] GW-IPF056: HPV66, HPV51, HPV56, HPV16, HPV44;

[0139] GW-IPF057: HPV31, HPV59, HPV35, HPV81, HPV83;

[0140] GW-IPF058:HPV39, HPV73, HPV53, HPV45, HPV43;

[0141] GW-IPF059: HPV68, HPV26, HPV82, HPV33, HPV42.

[0142] The results are shown in the following table:

[0143]

[0144] The results showed that the internal reference genes could be detected in all five cell types. HPV16, HPV18 and HPV52 were detected in GW-IPF055 cells, which were consistent with the quality control information; HPV16 and HPV56 were detected in GW-IPF056 cells, which were consistent with the quality control information; no four subtypes were detected in GW-IPF057, GW-IPF058 and GW-IPF059 cells, which were consistent with the quality control information. The primers and probes did not have cross-reactivity with other subtypes and could show good inter-subtype specificity.

[0145] 2) Interference detection

[0146] Three quality control products were used, namely, a cfDNA positive quality control product mixed with four subtypes of HPV16 / 18 / 52 / 56, a weak positive quality control product mixed with four subtypes of HPV16 / 18 / 52 / 56, and a normal person as a negative quality control product. Albumin (final concentration 200g / L), triglycerides (final concentration 37mmol / L), cytomegalovirus nucleic acid ((2.4E+05IU / mL), hepatitis B virus nucleic acid (5E+05IU / mL), Epstein-Barr virus (5.4E+05IU / mL), human genome CB male and human genome CB female were added to these three quality control products, and albumin and triglycerides were added before 1.5X magnetic bead purification, and the rest were directly added to the three quality control nucleic acids.

[0147] The results are shown in the following table:

[0148]

[0149]

[0150] The results showed that the added interfering substances had no effect on the detection of positive quality control products, weak positive quality control products and negative quality control products.

[0151] 3. Precision Testing

[0152] Evaluate the precision of the test process in the following aspects: the impact of precision between different operators, intra-day precision of the same operator and inter-day precision on the test results. The specific arrangement is shown in the following table:

[0153]

[0154] The results are shown in the following table:

[0155] The precision results between different personnel in the same batch are as follows:

[0156]

[0157] The intra-day precision results of the same personnel are as follows:

[0158]

[0159] The daytime precision results of the same personnel are as follows:

[0160]

[0161]

[0162] The above results show that the precision between different operators or within or between days of the same operator reaches a CV value of less than 30%.

[0163] The foregoing description of the specific exemplary embodiments of the present application is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present application to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present application and its practical application, so that those skilled in the art can realize and utilize the various exemplary embodiments of the present application and various selections and changes. The scope of the present application is intended to be limited by the claims and their equivalents.

Claims

1. A ddPCR primer-probe composition that can be used for HPV detection and typing in cfDNA, characterized in that: The primer probes simultaneously target four HPV subtypes in a single tube system; The four HPV subtypes are HPV16, HPV18, HPV52 and HPV56; The primer probe also targets an internal reference gene, which is RnaseP; The sequences of the primers are shown in SEQ ID NO.1-10; The sequences of the probes are shown in SEQ ID NOs. 11-15; The 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a quencher group: the fluorescent reporter group labeled with VIC at the 5' end of the probe for HPV16 type, and the quencher group labeled with MGB at the 3' end; the fluorescent reporter group labeled with ROX at the 5' end of the probe for HPV18 type, and the quencher group labeled with MGB at the 3' end; the fluorescent reporter group labeled with CY5 at the 5' end of the probe for HPV52 type, and the quencher group labeled with MGB at the 3' end; the fluorescent reporter group labeled with Atto425 at the 5' end of the probe for HPV56 type, and the quencher group labeled with MGB at the 3' end; the fluorescent reporter group labeled with FAM at the 5' end of the probe for the internal reference gene RnaseP, and the quencher group labeled with MGB at the 3' end; The primer concentration ratios of HPV16, HPV18, HPV52, HPV56 and the internal reference gene are equal; The probe concentration ratio of HPV16, HPV18, HPV52, HPV56 and the internal reference gene is 1-2:4-6:0.5-1:1-2:0.5-1; The primer-probe combination can be used for non-invasive early screening and prognosis monitoring of HPV-related tumors.

2. The primer-probe combination according to claim 1, characterized in that The primers are specifically designed and screened by the following method: Target region selection: Download the genome sequences of 38 HPV subtypes from the public gene database, and download more than 100 sequences for each subtype; evaluate multiple sequences of each subtype of HPV16, 18, 52 and 56, and select sequences that are conservative within each subtype and specific to the other 37 subtypes from each group of subtypes, with a sequence length of 200-500bp; at the same time, use 20bp as a node to screen the conservative and subtype-specific sequence segments in the genome one by one, and more than 99% of the sequences within the 5kb interval of the human genome sequence cannot be matched; find no less than 30 target regions for each subtype of HPV16, 18, 52 and 56, and sort them from high to low according to the matching rate; in addition, the GC content of the target region is about 50%, and the single / multi-base repeats are less than 5, and the target region cannot involve the plasmid region, mitochondrial region and 16S conservative region; Primer design, screening and optimization: preliminary primer design was performed based on the target region, and sequences with small dimer and similar Tm values ​​were selected. The specificity between primer subtypes and conservative compatibility within subtypes were determined by blast comparison; After alignment, the primer sequences were tested by melting curves to evaluate primer performance, and the sequences with the best specificity / compatibility were selected for multiple evaluations.

3. A product that can be used for HPV detection and typing in cfDNA, the product comprising the primer-probe combination described in any one of claims 1-2; the product is a kit.

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