Early screening and monitoring kit for HPV (human papillomavirus) related tumors

Abstract

The invention relates to the field of gene detection, in particular to an early screening and monitoring kit for HPV (human papillomavirus)-related tumors and application of the early screening and monitoring kit. On the basis of a multiple ddPCR system, twelve free HPV subtypes in plasma can be detected at a time. Compared with previous detection, the application aims at more various HPV subtypes, the detection result is more objective and accurate, and the detection sensitivity and specificity are high.

Description

Technical Field

[0001] The present application relates to the field of gene detection, and specifically to a plasma-based HPV-related tumor early screening and monitoring kit, method and application thereof. Background Art

[0002] There are many methods for detecting human papillomavirus (HPV), 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 results of p16 detection are somewhat non-specific. 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.

[0003] Liquid biopsy is a non-invasive test method. Common analytes include circulating tumor DNA (ctDNA), circulating tumor cells (CTC), mRNA, protein, miRNA, exosomes and metabolites. It is not yet considered 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 the free DNA fragments in the blood, and ctDNA is the part of cfDNA that comes from the tumor. 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. In recent years, the use of ddPCR technology for HPV DNA detection in cervical and head and neck tumors has increased significantly. However, the HPV detection methods commonly used in clinical practice still have problems such as the need for professional operation, high technical requirements, and low sensitivity. The applicant's previous HPV detection technology (CN202410561603.2) has especially solved the sensitivity problem, but the detection range covers relatively few HPV subtypes, and there are certain application limitations in clinical practice. In view of this, this application is proposed. Summary of the invention

[0004] In order to solve the above-mentioned problems of the prior art, the present application proposes a method and system for detecting free HPV in human plasma based on digital PCR. Through mixed probes, free HPV16, HPV18, HPV31, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59 and HPV68 in human plasma can be detected simultaneously in three reaction systems, and they 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.

[0005] Therefore, the first purpose of the present application is to provide a product, especially a kit product, that can be used for early screening and monitoring of HPV-related tumors based on cfDNA samples;

[0006] The second object of the present application is to provide a method for high-sensitivity 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 probes are targeted at the following twelve HPV subtypes: HPV16, HPV18, HPV31, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59 and HPV68.

[0009] Furthermore, the primer probe is also directed to an internal reference gene, and the internal reference gene is RNaseP.

[0010] Furthermore, the primer sequences are specifically designed and screened by the following method:

[0011] 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, 31, 35, 39, 45, 51, 52, 56, 58, 59 and 68, 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 in the genome The conservative sequence segments within the subtype and the specific sequence segments between subtypes were screened one by one, and more than 99% of the sequences within the range greater than 5kb in the human genome sequence could not be matched; no less than 30 target regions were found for each subtype of HPV16, 18, 31, 35, 39, 45, 51, 52, 56, 58, 59 and 68, and the comparison rates were ranked from high to low; in addition, the GC content of the target region was about 50%, and the single / multi-base repeats were less than 5. The target region could not involve the plasmid region, mitochondrial region and 16S conservative region;

[0012] 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

[0013] Furthermore, the sequence of the primer is specifically as shown in SEQ ID NO.1-26, or has at least 90% homology with SEQ ID NO.1-26.

[0014] Furthermore, the sequence of the probe is specifically as shown in SEQ ID NO.27-39, or has at least 90% homology with SEQ ID NO.27-39.

[0015] Furthermore, to ensure the balance and accuracy of the detection results, the primer-probe combination described in the present application is divided into three groups: Group 1 targets four HPV subtypes, namely HPV16, HPV18, HPV52 and HPV56; Group 2 targets four HPV subtypes, namely HPV35, HPV51, HPV59 and HPV68; Group 3 targets four HPV subtypes, namely HPV31, HPV39, HPV45 and HPV58.

[0016] Furthermore, the probes in each group are labeled with a fluorescent reporter group at the 5' end and a quencher group at the 3' end;

[0017] Further preferably, the fluorescent reporter group labeled at the 5' end of the probe for HPV16 subtype is VIC, and the quencher group labeled at the 3' end is MGB; the fluorescent reporter group labeled at the 5' end of the probe for HPV18 subtype is ROX, and the quencher group labeled at the 3' end is MGB; the fluorescent reporter group labeled at the 5' end of the probe for HPV52 subtype is CY5, and the quencher group labeled at the 3' end is MGB; the fluorescent reporter group labeled at the 5' end of the probe for HPV56 subtype is Atto425, and the quencher group labeled at the 3' end is MGB, and the fluorescent reporter group labeled at the 5' end of the probe for the internal reference gene RnaseP is FAM, and the quencher group labeled at the 3' end is MGB;

[0018] For HPV35 subtype, the fluorescent reporter group labeled at the 5' end is ROX, and the quencher group labeled at the 3' end is MGB; for HPV51 subtype, the fluorescent reporter group labeled at the 5' end is FAM, and the quencher group labeled at the 3' end is MGB; for HPV59 subtype, the fluorescent reporter group labeled at the 5' end is CY5, and the quencher group labeled at the 3' end is MGB; for HPV68 subtype, the fluorescent reporter group labeled at the 5' end is Atto425, and the quencher group labeled at the 3' end is MGB;

[0019] The fluorescent reporter group labeled with FAM at the 5' end of the probe for HPV31 subtype is, 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 HPV39 subtype is, 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 HPV45 subtype is, and the quencher group labeled with MGB at the 3' end; the fluorescent reporter group labeled with VIC at the 5' end of the probe for HPV58 subtype is, and the quencher group labeled with MGB at the 3' end.

[0020] Furthermore, the primer concentration ratio of each HPV subtype is equal; the probe concentration ratio of each HPV subtype is: HPV16: HPV18: HPV52: HPV56: HPV35: HPV51: HPV59: HPV68: HPV31: HPV39: HPV45: HPV58 = 1-2:4-6:0.5-1:1-2:1-2:1-2:1-2:1-2:1-2:1-2:1-2:1-2:1-2:1-2.

[0021] Furthermore, the primer-probe combination can be used for high-sensitivity non-invasive early screening and monitoring of HPV-related tumors.

[0022] 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.

[0023] Furthermore, the product is a test kit.

[0024] Furthermore, the finished product also includes dNTPs, Taq enzyme and PCR buffer.

[0025] 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.

[0026] 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.

[0027] A method for amplifying HPV16, HPV18, HPV31, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59 and HPV68 subtypes in cfDNA samples, comprising the following steps: amplifying the sample using any of the above-mentioned primer-probe combinations.

[0028] Preferably, the amplification conditions are 58°C and 45 cycles;

[0029] More preferably, the specific amplification conditions are as follows:

[0030]

[0031] 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; amplification using the above-mentioned amplification method; data reading; and data analysis.

[0032] The present application has at least the following advantages: The present application realizes the multiple simultaneous detection of multiple free HPV subtypes in patient plasma through the design and screening of the primer probe system and the grouping design of different subtypes. The ddPCR system of the present application can detect twelve types of HPV in the blood at one time in a single run, thereby realizing accurate typing of the disease. In terms of detection effect, the kit of the present application has higher sensitivity and stronger specificity, and the minimum detection limit for each subtype can reach 250 copies / ml; thanks to the richer subtype types, the present application also has the clinical advantage of more accurate and objective detection results.

[0033] It is precisely based on the advantages of the kit of this application in terms of sample type, multiple systems and sensitivity that it can not only be used for early screening and diagnosis of HPV-related tumors, but also for effective monitoring of tumor treatment response and prognosis. It has great clinical significance and has been promoted. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] 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.

[0035] Figure 1 , candidate primer melting curve specificity result data;

[0036] Figure 2 , gDNA single subtype + internal reference mixed detection results;

[0037] Figure 3 , gDNA single tube double detection results;

[0038] Figure 4 , gDNA single tube multiplex detection results at different probe concentrations;

[0039] Figure 5 , gDNA single tube multiple detection results;

[0040] Figure 6 , cfDNA single tube single-plex test results for HPV16, HPV18, HPV52 and HPV56;

[0041] Figure 7 , cfDNA single tube single-plex test results for HPV35, HPV51, HPV59, HPV68, HPV31, HPV39, HPV45 and HPV58;

[0042] Figure 8, single-tube multiplex test results of cfDNA for HPV16, HPV18, HPV52, and HPV56;

[0043] Fig. 9 , single-tube multiplex test results of cfDNA for HPV35, HPV51, HPV59, HPV68, HPV31, HPV39, HPV45, and HPV58;

[0044] Fig.10 , Detection results under different PCR conditions

[0045] Fig.11 , gDNA detection limit test results;

[0046] Fig.12 , cfDNA detection limit test results for HPV16, HPV18, HPV52, and HPV56;

[0047] Fig.13 , cfDNA detection limit test results for HPV35, HPV51, HPV59, HPV68, HPV31, HPV39, HPV45 and HPV58. DETAILED DESCRIPTION

[0048] 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.

[0049] Experimental Example 1: Design, screening and optimization of primer probes

[0050] 1) Selection of HPV subtype;

[0051] In order to improve the accuracy and comprehensiveness of early screening and monitoring of HPV-related tumors, and in combination with common high-risk subtypes in China and internationally, this application enriches the HPV subtypes and selects a total of twelve clinically significant HPV subtypes, including HPV16, HPV18, HPV31, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59 and HPV68.

[0052] 2) Design, screening and optimization of corresponding primers

[0053] Target region selection and preliminary primer design

[0054] 1. Targeting region selection

[0055] The genome sequences of 38 HPV subtypes were downloaded from the public gene database, and more than 100 sequences of each subtype were downloaded. Multiple sequences of each subtype of HPV16, 18, 31, 35, 39, 45, 51, 52, 56, 58, 59 and 68 were evaluated. From each group of subtypes, the sequences of each strain within the subtype were selected, and the sequences of each subtype that were conserved and specific between the other 37 subtypes of each subtype were selected. The sequence length was 200-500bp. At the same time, 20bp was used as a node, and the sequences were selected one by one in the genome. The sequence segments that are conservative within the subtype and specific between subtypes were screened, and more than 99% of the sequences within the range greater than 5kb that could not be matched with the entire human genome sequence were found; no less than 30 target regions were found for each subtype of HPV16, 18, 31, 35, 39, 45, 51, 52, 56, 58, 59 and 68, and they were sorted 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 could not involve the plasmid region, mitochondrial region and 16S conserved region.

[0056] 2. Primer design, screening and optimization:

[0057] 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.

[0058] Based on the above steps, the candidate primer systems preliminarily designed by this application for the above 12 subtypes are as follows in Table 1:

[0059]

[0060]

[0061]

[0062] 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:

[0063]

[0064]

[0065] Example 2: Primer-probe feasibility evaluation and system optimization

[0066] Considering 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 control samples gDNA and cfDNA. The preparation of the quality control 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 the cfDNA quality control product with the expected fragment length.

[0067] The specific evaluation and operation steps are as follows:

[0068] 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:

[0069]

[0070]

[0071] The results are as follows:

[0072]

[0073] It can be seen that: in the first plate, only the samples with the template HPV16 have a starting line; in the second plate, only the samples with the template HPV18 have a starting line; in the third plate, only the samples with the template HPV52 have a starting line; the primers for each subtype have good specificity, all samples in the fourth plate have a starting line, and the internal reference control gene is well selected.

[0074] 2) Using positive quality control gDNA of four subtypes of HPV16 / 18 / 52 / 56, primers for each subtype and internal reference gene primers were mixed in different proportions for testing to determine the feasibility of primer probes.

[0075] Sample Information

[0076]

[0077]

[0078] The results are as follows:

[0079]

[0080] Digital PCR results Figure 2 As shown, it can be seen that in the QPCR methodology, plate 1: the influence of the change in the probe ratio on the Ct value is greater than the change in the primer ratio; plate 2: the hpv52 probe ratio increases, the Ct value decreases, and the gap with the Ct values ​​of other subtypes 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.

[0081] Using positive quality control gDNA of eight subtypes of HPV31 / 35 / 39 / 45 / 51 / 58 / 59 / 68, primers and probes of each subtype were mixed in equal ratios for detection to determine the feasibility of primers and probes (ratio 1 = 0.5 μM).

[0082] Sample information:

[0083]

[0084] Digital PCR results Figure 3 As shown, it can be seen from the results that the HPV31+HPV58 combination test can clearly distinguish each subgroup, and the HPV51+HPV58 combination test, HPV58 will inhibit the amplification of HPV51, resulting in HPV51 can not be detected, so these two subtype combinations are excluded; HPV39+HPV45 combination test and HPV59+HPV45 combination test, each subgroup can be separated, the effect is consistent; HPV39+HPV68 combination test 2D map can not distinguish the HPV39 (CY5) single positive area, so the combination is excluded, HPV59+HPV68 combination test each subgroup can be clearly distinguished. Based on the above results, HPV31+HPV58; HPV39+HPV45; HPV59+HPV68 are selected for the two-by-two combination.

[0085] 3) Positive quality control gDNA of four subtypes of HPV16 / 18 / 52 / 56 was mixed, and three ratios were tested with a single tube of multiple primers (3 repeated data) to determine the feasibility of the primer probe (ratio 1 = 0.5 μM).

[0086] Sample information:

[0087]

[0088] The results are as follows Figure 4 As shown, the probe ratio was adjusted, and HPV16 was set to 0.5μM; HPV18 was 2μM; HPV52 was 0.3μM and 0.35μM; HPV56 was 0.5μM and 0.6μM; the internal reference was 0.25μM and 0.1μM, and according to the threshold division, the final probe mixing ratio was determined to be HPV16+HPV18+HPV52+HPV56+internal reference (1:4:0.6:1:0.5), that is, HPV16 was 0.5μM; HPV18 was 2μM; HPV52 was 0.3μM; HPV56 was 0.5μM; and the internal reference was 0.25μM.

[0089] According to the pairwise combination results, positive quality control gDNA of eight subtypes of HPV31 / 35 / 39 / 45 / 51 / 58 / 59 / 68 were mixed in 3, 4 and 5 combinations respectively to determine the feasibility of primers and probes (ratio 1 = 0.5 μM).

[0090] Sample information:

[0091]

[0092] Digital PCR results Figure 5 As shown, the template uses a mixture of three subtypes, HPV51+HPV39+HPV45, among which HPV51 and HPV45 will cause the fluorescence shift of HPV51, which is not preferred; the four subtypes, HPV51+HPV59+HPV68+HPV35, are mixed, and each subgroup can be clearly distinguished, which is preferred; the remaining HPV31+HPV58+HPV39+HPV45, each subgroup can also be clearly distinguished, which is preferred. Based on the above results, the present application divides the eight subtype primer probes into two tubes, one tube is HPV51 (FAM) + HPV59 (CY5) + HPV68 (ATTO425) + HPV35 (ROX), and the other tube is HPV31 (FAM) + HPV58 (VIC) + HPV39 (CY5) + HPV45 (ATTO425). The probe ratio in the above experiment is appropriate, so it is determined that the two tubes of probes are added in equal proportions, both of which are 0.5 μM.

[0093] 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.

[0094] Sample information:

[0095]

[0096] The results are as follows Figure 6 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.

[0097] Tube 1: Primers and probes of four subtypes, HPV51 (FAM) + HPV59 (CY5) + HPV68 (ATTO425) + HPV35 (ROX), were mixed; Tube 2: Primers and probes of four subtypes, HPV31 (FAM) + HPV58 (VIC) + HPV39 (CY5) + HPV45 (ATTO425), were mixed. Eight cfDNA positive quality control products, HPV31 / 35 / 39 / 45 / 51 / 58 / 59 / 68, were tested separately to determine the feasibility of the primers and probes.

[0098] Sample information:

[0099]

[0100] Digital PCR results Figure 7 As shown, the cfDNA positive quality control samples of each subtype were detected correctly and the primer probes were usable.

[0101] 5) Mix the positive 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.

[0102] Sample information:

[0103]

[0104] The results are shown as Figure 8 As shown, the four subtypes of positive quality control cfDNA were mixed for detection, and each subgroup could be clearly distinguished with an appropriate ratio.

[0105] Positive quality control cfDNA of eight subtypes of HPV31 / 35 / 39 / 45 / 51 / 58 / 59 / 68 were mixed, and single-tube multiple primers were tested according to the above-mentioned ratio to determine the feasibility of the primer probe.

[0106] Sample information:

[0107]

[0108] Digital PCR results Fig. 9 As shown, the four subtypes of positive quality control cfDNA were mixed for detection, and each subgroup could be clearly distinguished, with an appropriate ratio, and primers and probes were available.

[0109] 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.

[0110] 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.

[0111] The results are as follows:

[0112]

[0113]

[0114]

[0115] 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.

[0116] In summary, the multiple 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 effect is optimal when 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 primer concentration ratio of HPV51, HPV59, HPV68 and HPV35 is also mixed in equal proportions, and the probe concentration ratio is preferably 1:1:1:1, which has the best effect; the primer concentration ratio of HPV31, HPV58, HPV39 and HPV45 is also mixed in equal proportions, and the probe concentration ratio is preferably 1:1:1:1, which has the best effect.

[0117] Example 3: Optimization of PCR amplification reaction program

[0118] Set up different PCR reaction programs to determine the most suitable reaction program for this application. The detailed steps are as follows:

[0119] 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:

[0120] 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

[0121] 2) Chip loading: Use Sniper DQ24 / DQ24 Plus / DQ24 Pro ddPCR instrument.

[0122] 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.

[0123]

[0124]

[0125] The sample information is as follows:

[0126]

[0127] The results are as follows Fig.10 As shown, when the amplification conditions are set at 58 degrees and 45 cycles, the effect is optimal and obvious, and under this condition, the detection of HPV51, HPV59, HPV68, HPV35, HPV31, HPV58, HPV39 and HPV45 is also very good. Therefore, the optimal amplification conditions of this application are determined as follows:

[0128]

[0129] Example 4: Test kit performance verification

[0130] 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.

[0131] 1. Minimum detection limit test

[0132] 1. Single genotype detection

[0133] 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 Fig.11 .

[0134]

[0135]

[0136] 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 Fig.12 and Fig.13 shown.

[0137]

[0138] stencil Expected value (copies / mL) Actual value (copies / mL) cfDNA-HPV51 1000 629 cfDNA-HPV59 1000 1000 cfDNA-HPV68 1000 1000 cfDNA-HPV35 1000 676 cfDNA-HPV31 1000 1430 cfDNA-HPV58 1000 849 cfDNA-HPV39 1000 817 cfDNA-HPV45 1000 1600

[0139]

[0140]

[0141] 2. Multiple detection of four subtypes:

[0142] 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.

[0143]

[0144]

[0145]

[0146] 2) cfDNA: The four subtypes are mixed according to the concentration in the table below. The weak positive is 300-500 copies / mL, and the strong positive is 1-4E+07 copies / mL. Determine whether the weak positive detection will be affected. The sample information is as follows:

[0147]

[0148]

[0149] The results are as follows:

[0150]

[0151]

[0152]

[0153]

[0154] The results showed that for single genotype detection, the gDNA and cfDNA samples of four subtypes (HPV16 / 18 / 52 / 56) were detected at three concentration gradients of 1000 copies / mL, 500 copies / mL and 250 copies / mL, with a detection rate of 100%; for mixed multiple detection of four subtypes (HPV16 / 18 / 52 / 56), three concentration gradients of 1000 copies / mL, 500 copies / mL and 250 copies / mL were set respectively. At a concentration of 1000 copies / mL, the detection rate was 100%, at a concentration of 500 copies / mL, the detection rate was 100%, and at a concentration of 250 copies / mL, the detection rate was 90%; for four subtypes (HPV16 / 18 / 52 / 56), the detection rate was 100%, 100% and 90% respectively. V51 / 59 / 68 / 35) cfDNA samples were set at two concentrations of 1000 copies / mL and 250 copies / mL, and both could be detected, with a detection rate of 100%; the cfDNA samples of the four subtypes (HPV51 / 59 / 68 / 35) were mixed and multiplexed, and the detection rate of the samples was 100% at a concentration of 250 copies / mL; the cfDNA samples of the four subtypes (HPV31 / 58 / 39 / 45) were set at two concentrations of 1000 copies / mL and 250 copies / mL, and both could be detected, with a detection rate of 100%; the cfDNA samples of the four subtypes (HPV31 / 58 / 39 / 45) were mixed and multiplexed, and the detection rate of the samples was 100% at a concentration of 250 copies / mL.

[0155] 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 multi-subtype mixed multiple detection.

[0156] 2. Specificity Detection

[0157] In this embodiment, interferences are added to determine whether the detection situation will be affected.

[0158] 1) Using 5 types of composite human papillomavirus (HPV cells), the viral nucleic acid was obtained by extraction and tested:

[0159] GW-IPF055: HPV16, HPV18, HPV52, HPV58, HPV6, HPV11;

[0160] GW-IPF056: HPV66, HPV51, HPV56, HPV16, HPV44;

[0161] GW-IPF057: HPV31, HPV59, HPV35, HPV81, HPV83;

[0162] GW-IPF058:HPV39, HPV73, HPV53, HPV45, HPV43;

[0163] GW-IPF059: HPV68, HPV26, HPV82, HPV33, HPV42.

[0164] The results are shown in the following table:

[0165]

[0166]

[0167] The results showed that the internal reference genes could be detected in all five cell types. HPV16, HPV18, HPV52 and HPV58 detected by GW-IPF055 cells were consistent with the quality control information; HPV16, HPV56 and HPV51 detected by GW-IPF056 cells were consistent with the quality control information; HPV31, HPV59 and HPV35 detected by GW-IPF057 cells were consistent with the quality control information; HPV39 and HPV45 detected by GW-IPF058 cells were consistent with the quality control information; HPV68 detected by GW-IPF059 cells was 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.

[0168] 3. Interference Detection

[0169] Five quality control products were used, namely, a cfDNA positive quality control product mixed with four subtypes of HPV16 / 18 / 52 / 56, a cfDNA weakly positive quality control product mixed with four subtypes of HPV16 / 18 / 52 / 56, a cfDNA positive quality control product mixed with four subtypes of HPV51 / 59 / 68 / 35, a cfDNA positive quality control product mixed with four subtypes of HPV31 / 58 / 39 / 45, and normal human plasma as a negative quality control product. Albumin (final concentration 200 g / L), triglycerides (final concentration 37 mmol / L), cytomegalovirus nucleic acid (2.4E+05IU / mL), hepatitis B virus nucleic acid (5E+05IU / mL), and Epstein-Barr virus (5.4E+05IU / mL) were added to these five quality control products. After adding albumin and triglycerides, 1.5X magnetic beads were purified, and the rest were directly added to the five quality control product nucleic acids.

[0170] The results are shown in the following table:

[0171]

[0172]

[0173]

[0174] 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.

[0175] 4. Precision Testing

[0176] Evaluate the precision of the test process in the following aspects: the impact of the precision between different operators, the intra-day precision of the same operator and the inter-day precision on the test results. The specific arrangements are shown in the following table:

[0177]

[0178] The results are shown in the following table:

[0179] The precision results between different personnel in the same batch are as follows:

[0180]

[0181]

[0182] The intra-day precision results of the same personnel are as follows:

[0183]

[0184]

[0185] The daytime precision results of the same personnel are as follows:

[0186]

[0187]

[0188] The results showed that the precision between different operators, within the same operator or between days reached a CV value of less than 35%.

[0189] Example 5: Real clinical sample testing

[0190] A total of 14 samples with known clinical typing were collected in the hospital and tested using this kit. The specific test results are shown in the following table:

[0191]

[0192]

[0193] This embodiment uses 14 clinical samples with clinical gold standards to verify the ability to detect HPV16 / 18 / 52 / 56 / 51 / 59 / 68 / 35 / 31 / 58 / 39 / 45. The test statistical results are shown in the table above. For clinical samples, the total compliance rate is 100%, the positive compliance rate is 100%, and the negative compliance rate is 100%. The results reflect the reliability advantage of this application for actual clinical sample detection.

[0194] The foregoing description of the specific exemplary embodiments of the present application is for the purpose of illustration and illustration. 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 for high-sensitivity detection and typing of HPV in cfDNA, characterized in that: The primer probes are directed to twelve HPV subtypes, including HPV16, HPV18, HPV31, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59 and HPV68; Preferably, the primer probe also targets the internal reference gene RnaseP.

2. The primer-probe combination according to claim 1, characterized in that The sequences of the primers are shown in SEQ ID NO.1-26, or have at least 90% homology to SEQ ID NO.1-26; the sequences of the probes are shown in SEQ ID NO.27-39, or have at least 90% homology to SEQ ID NO.27-39.

3. The primer-probe combination according to any one of claims 1-2, characterized in that: The primer-probe combination is divided into three groups: Group 1 is for HPV16, HPV18, HPV52 and HPV56; Group 2 is for HPV35, HPV51, HPV59 and HPV68; Group 3 is for HPV31, HPV39, HPV45 and HPV58.

4. The primer-probe combination according to any one of claims 1 to 3, characterized in that: 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; Preferred: The fluorescent reporter group labeled with VIC at the 5' end of the probe for HPV16 subtype, 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 subtype, 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 subtype, 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 subtype, 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; For HPV35 subtype, the fluorescent reporter group labeled at the 5' end is ROX, and the quencher group labeled at the 3' end is MGB; for HPV51 subtype, the fluorescent reporter group labeled at the 5' end is FAM, and the quencher group labeled at the 3' end is MGB; for HPV59 subtype, the fluorescent reporter group labeled at the 5' end is CY5, and the quencher group labeled at the 3' end is MGB; for HPV68 subtype, the fluorescent reporter group labeled at the 5' end is Atto425, and the quencher group labeled at the 3' end is MGB; The fluorescent reporter group labeled with FAM at the 5' end of the probe for HPV31 subtype is, 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 HPV39 subtype is, 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 HPV45 subtype is, and the quencher group labeled with MGB at the 3' end; the fluorescent reporter group labeled with VIC at the 5' end of the probe for HPV58 subtype is, and the quencher group labeled with MGB at the 3' end.

5. The primer-probe combination according to any one of claims 1 to 4, characterized in that: The primer concentration ratio of each HPV subtype is equal; the probe concentration ratio of each HPV subtype is: HPV16: HPV18: HPV52: HPV56: HPV35: HPV51: HPV59: HPV68: HPV31: HPV39: HPV45: HPV58 = 1-2:4-6:0.5-1:1-2:1-2:1-2:1-2:1-2:1-2:1-2:1-2:1-2:1-2:1-2.

6. The primer-probe combination according to any one of claims 1 to 5, characterized in that: The primer-probe combination can be used for high-sensitivity non-invasive early screening and monitoring of HPV-related tumors.

7. A product that can be used for high-sensitivity detection and typing of HPV in cfDNA, the product comprising the primer-probe combination of any one of claims 1-6; preferably, the product is a kit; more preferably, the finished product also includes dNTPs, Taq enzyme and PCR buffer.

8. Use of the primer-probe combination described in any one of claims 1-6 in the preparation of a high-sensitivity, non-invasive early screening and monitoring product for HPV-related tumors, or use of the primer-probe combination described in any one of claims 1-6 in the high-sensitivity, non-invasive early screening and monitoring of HPV-related tumors.

9. A method for amplifying HPV16, HPV18, HPV52, HPV56, HPV35, HPV51, HPV59, HPV68, HPV31, HPV39, HPV45 and HPV58 subtypes in cfDNA samples, characterized in that: The method comprises the following steps: amplifying a sample using the primer-probe combination according to any one of claims 1 to 6.

10. A highly sensitive non-invasive early screening and monitoring method for HPV-related tumors, characterized in that: The method comprises the following steps: separating plasma and extracting cfDNA; preparing a digital PCR reaction system; loading samples onto a chip; amplifying using the amplification method described in claim 9; reading data; and analyzing data.

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