Non-invasive monitoring and prognosis evaluation method for HPV infection and PIK3CA gene mutation of head and neck cancer patient based on multiple digital PCR technology

Abstract

The invention relates to a non-invasive monitoring and prognosis evaluation method for HPV infection and PIK3CA gene mutation of a head and neck cancer patient based on a multiple digital PCR (Polymerase Chain Reaction) technology. By designing a specific primer probe sequence, high-sensitivity detection of E545K / E542K / E545D mutation of the HPV16 / 33 virus and the PIK3CA gene is realized. According to the detection system, the signal-to-noise ratio is increased by adopting a double-quenching-group probe, the detection of five targets can be synchronously completed in two reaction holes, and the sample consumption is only 20 microliters. The method has the following advantages: (1) the specificity is excellent: wild DNA has no false positive; (2) the limit sensitivity reaches 3 copies; (3) high efficiency and economy: single-sample multi-index detection reduces the detection cost; and (4) the operation is convenient, the matched software automatically calculates the sudden change frequency, and complex signal analysis is not needed. Through comparison and verification with an IPC / FISH gold standard, the HPV detection consistency reaches 90.6%. The method provides reliable technical support for revealing the relevance between the HPV / PIK3CA co-mutation mode and head and neck cancer prognosis.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of molecular biology gene detection, and particularly relates to a non-invasive monitoring and prognosis evaluation method for HPV infection and PIK3CA gene mutation of a head and neck cancer patient based on a multiplex digital PCR technology. BACKGROUND

[0002] Head and neck cancer is one of the most common malignancies worldwide, with significant differences in incidence rates across different regions, genders, and age groups. According to the global cancer statistics in 2022, head and neck cancer is the sixth most common malignancy globally. There are approximately 645,000 new cases of head and neck cancer worldwide each year, with Southeast Asia being a high-risk region. It includes many different common and rare entities, the vast majority (90%) of which are squamous cell carcinomas (SCC). There are significant differences in disease progression and overall patient survival across different anatomical sub-sites, which can be explained by its multifactorial etiology, with the main known risk factors being environmental exposure to tobacco products, alcohol, and infection with high-risk human papillomavirus (HPV). Human papillomavirus (HPV) infection, primarily high-risk (HR) HPV16, plays an important role in oropharyngeal cancer. HPV-positive and HPV-negative tumors are clinically recognized as two distinct carcinogenic pathways. It has also been reported that the prognosis of HPV-positive head and neck cancer (especially HPV-related oropharyngeal cancer) is generally better than that of HPV-negative head and neck cancer.

[0003] The American Cancer Genome Atlas (TCGA) data shows that 40% of HPV-positive head and neck cancer carries APOBEC3-related mutation signatures, with a PIK3CA mutation rate of about 15%, while the PIK3CA mutation rate in HPV-negative tumors is about 25%. A study in Portugal, which included 95 cases of head and neck squamous cell carcinoma, found that the PIK3CA mutation rate in HPV-positive patients was 28%, lower than the 42% in HPV-negative patients. Even so, the PIK3CA gene is still one of the most frequently mutated genes in HPV-associated HNSCC. Among the PIK3CA mutations observed in HNSCC, 63% occur at three specific positions that encode the p110a subunit, namely E542, E545, and H1047, known as canonical mutations. In addition, a study in Portugal showed that HPV-positive + PIK3CA wild-type: best prognosis. A study in India showed that HPV-negative + PIK3CA mutant: worst prognosis. Therefore, it is crucial to perfect the PIK3CA mutation characteristics of HPV-positive and HPV-negative HNSCC patients and their relationship with prognosis.

[0004] At present, the methods for HPV detection mainly include cytology, dot blotting, fluorescence in situ hybridization, Southern hybridization, polymerase chain reaction (PCR) and hybrid capture II (HC-II) method, etc. Among them, although the cytology has wide universality, the sensitivity and specificity are low; although the HC-II method has high sensitivity and specificity, it cannot analyze the pathogenic factors of HPV; and the ordinary PCR method can amplify the target gene, but the low sensitivity and high DNA dosage limit its wide application in clinical detection. In order to solve the problems in the above methods, the droplet digital PCR (ddPCR) gradually enters the field of view of researchers. The ddPCR has the advantages of low sample amount and high sensitivity, and can be applied in the detection of tumor cell DNA, so as to effectively screen early tumors, which is particularly meaningful for clinical diagnosis.

[0005] Therefore, the application designs a primer probe combination and a detection method for detecting two HPV subtypes HPV16 and HPV33 infection and three PIK3CA gene E545K, E542K and E545D site mutation in two reaction wells by using multiplex digital PCR, so as to lay a foundation for studying the PIK3CA mutation characteristics of HPV positive and HPV negative head and neck cancer patients and the relationship between the PIK3CA mutation characteristics and prognosis. SUMMARY

[0006] The application first provides a primer probe combination for HPV infection and PIK3CA gene mutation detection of head and neck cancer patients, which comprises specific primer probe sequences for HPV16, HPV33, E545K, E542K and E545D targets and primer probes of an internal reference gene GAPDH.

[0007] In some embodiments, the specific primer probe sequences for HPV16, HPV33, E545K, E542K and E545D targets are shown as SEQ ID NO: 1-11, and the primer probes of the internal reference gene GAPDH are shown as SEQ ID NO: 12-15.

[0008] The application further provides a multiplex digital PCR detection system, which comprises: Reaction well 1: HPV16 / 33+E545K three-target detection system, 2x ddPCR Supermix 10 μL+primer probe mixture 1 μL+template DNA; Reaction well 2: E542K / E545D double-target detection system, 2x ddPCR Supermix 10 μL+primer probe mixture 1 μL+template DNA; In some embodiments, the final concentration of the reaction well 1 primer is 250 nM, and the final concentration of the probe is 150 nM. Reaction well 2: the final concentration of the E542K probe is 150 nM, and the final concentration of the E545D probe is 250 nM.

[0009] The present application also provides a method for evaluating the prognosis of a head and neck cancer patient, which comprises detecting the HPV16 / 33 viral load and the PIK3CA gene E545K / E542K / E545D mutation frequency by using the system.

[0010] In some embodiments, a prognosis evaluation model is established: HPV+ / PIK3CA wild type: best prognosis group; HPV- / PIK3CA mutant type: worst prognosis group.

[0011] The present application also provides a detection kit, which comprises the primer probe combination 2xddPCR Supermix, the GAPDH internal control, and the positive control.

[0012] In some embodiments, the positive control contains HPV16; and / or The positive control contains the E545K mutation.

[0013] The present application finally provides a digital PCR detection method, which comprises the following steps: (1) Microdroplet generation: Bio-Rad QX200 system; (2) Amplification procedure: 95℃ 10min→(94℃ 30s→58℃ 1min)×40 cycles→98℃ 10min; (3) Data analysis: automatic identification of FAM / HEX dual-channel signals by QuantaSoft software.

[0014] In some embodiments, the judgment criteria are set as follows: HPV positive: FAM channel≥2 positive microdroplets; PIK3CA mutation: HEX channel≥2 positive microdroplets.

[0015] Compared with the prior art, the present application has at least the following beneficial effects: The application simultaneously detects and analyzes HPV infection status and PIK3CA mutation status by digital PCR, and 2 reaction wells can detect HPV16, HPV33, and E545K, E542K, and E545D PIK3CA gene site mutation types, thereby saving samples. The detection system has good specificity, and no mutation amplification or less than 2 mutation microdroplets are detected in wild-type genomic DNA reactions with high, medium, and low concentrations; no mutation amplification or less than 2 mutation microdroplets are detected at the E545K site when reaction well 1 detects HPV16 standard (provided by Shanghai Measurement and Test Technology Research Institute); no HPV positive amplification or less than 2 positive microdroplets are detected when reaction well 1 detects E545K standard (purchased from Nanjing Kebai Biological Technology Co., Ltd.), and the detection system has no cross-reaction. No mutation amplification or less than 2 mutation microdroplets are detected at the E545D site when reaction well 2 detects E542K standard (purchased from Nanjing Kebai Biological Technology Co., Ltd.); no positive amplification or less than 2 positive microdroplets are detected at the E542K site when reaction well 2 detects E545D plasmid (synthesized by Shanghai Langjing Biological Technology Co., Ltd.), which indicates that the detection system has good specificity and no cross-reaction. Finally, the detection system has high sensitivity, and the detection sensitivity of HPV16 and E545K in reaction well 1 is 3 copies; the detection sensitivity of E542K and E545D in reaction well 2 is also 3 copies. BRIEF DESCRIPTION OF DRAWINGS DETAILED DESCRIPTION OF THE INVENTION BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 FIG. 1 is a result graph of reaction well 1 detecting HPV standard and E545K standard; Figure 2 FIG. 2 is a result graph of reaction well 2 detecting E542K standard and E545D plasmid; Figure 3 FIG. 3 is a sensitivity schematic diagram (3 copies) of reaction well 1; Figure 4 FIG. 4 is a sensitivity schematic diagram (3 copies) of reaction well 2; Figure 5 FIG. 5 is a result graph of reaction well 1 detecting plasma DNA samples; Figure 6 FIG. 6 is a result graph of reaction well 2 detecting plasma DNA samples.

[0017] Figure 7 FIG. 7 is an ROC curve analysis graph of HPV. DETAILED DESCRIPTION

[0018] To make the technical problems, technical solutions, and advantages of the application clearer, specific embodiments will be described in detail below with reference to the drawings.

[0019] HPV16-E6-F1: 5 ，-3 ， , SEQ ID No: 1 ; HPV16-E6-R1: 5 ， -3 ， , SEQ ID No: 2 ; HPV16-E6-P1: 5 ， -3 ， , SEQ ID No: 3 ; HPV33-E6-F1: 5 ， -3 ， , SEQ ID No: 4 ; HPV33-E6-R1: 5 ， -3 ， , SEQ ID No: 5 ; HPV33-E6-P1: 5 ， -3 ， , SEQ ID No: 6 ; PIK-E545K-F1: 5 ， -3 ， , SEQ ID No: 7 ; PIK-E545K-R1: 5 ， -3 ， , SEQ ID No: 8 ; PIK-E545K-MUTP(H): 5 ， -3 ， , SEQ ID No: 9 ; PIK-E542K-MUT-P1(FAM): 5 ， -3 ， , SEQ ID No: 10 ; PIK-E545D-MUTP(H): 5 ， -3 ， , SEQ ID No: 11 ; GAPDH-hDNA-F1: 5 ， -CCCCACACACATGCACT -3 ， , SEQ ID No: 12; GAPDH-hDNA-R1: 5 ， -CCTAGTCCCAGGGCTTTG-3 ， , SEQ ID No: 13; GAPDH-hDNA-P1: 5 ， -TAGGAAGGACAGGCAAC-HEX-3 ， , SEQ ID No: 14; GAPDH-hDNA-P2: 5 ， -TAGGAAGGACAGGCAAC -FAM-3 ， , SEQ ID No: 15; Example 1, Plasma separation 1. Centrifuge blood sample 1600g for 10 min, remove cells.

[0020] 2. Transfer supernatant to a new 1.5ml centrifuge tube, be careful not to suck up the precipitate.

[0021] 3. Centrifuge 16000g for 10 min, remove cell debris.

[0022] 4. Suck up the supernatant and transfer it to a new 1.5ml centrifuge tube, be careful not to suck up the precipitate.

[0023] 5. If used on the same day, store briefly at 4°C, otherwise store the collected plasma at -80°C.

[0024] Example 2, Plasma DNA extraction (take 3ml as an example) 1. Transfer 300μl Proteinase K to a 50ml centrifuge tube.

[0025] 2. Transfer 3ml serum, plasma or other liquid samples to the centrifuge tube containing Proteinase K, mix for 5 seconds.

[0026] 3. Add 2.4ml Buffer ACL / Carrier RNA (1μg) to the sample, vortex for 15 seconds. 60°C water bath for 30 minutes, occasionally mix for several times.

[0027] 4. Add 5.4ml Buffer ACB to the sample. Vortex for 15 seconds. Place on ice for 5 minutes.

[0028] 5. Attach the HiPure CF DNA Mini Column and Vac-Connectors to the vacuum filtration box.

[0029] 6. Insert the Extender Tubes into the column.

[0030] 7. Transfer the mixture from Step 4 to the column and turn on the vacuum pump to begin filtration. Continue to transfer the mixture to the column for filtration until all of the mixture has been transferred to the column and filtered. Turn off the vacuum pump and allow the pressure to drop to zero.

[0031] 8. Remove the column from the filter. Place the column back into the collection tube. Add 600 µl Buffer DCW1 to the column. Centrifuge at 13,000 x g for 60 seconds.

[0032] 9. Discard the flow-through and place the column back into the collection tube. Add 600 µl Buffer DCW2 to the column. Centrifuge at 13,000 x g for 60 seconds.

[0033] 10. Discard the flow-through and place the column back into the collection tube. Add 600 µl absolute ethanol to the column. Centrifuge at 13,000 x g for 60 seconds.

[0034] 11. Discard the flow-through and place the column back into the collection tube. Centrifuge at 13,000 x g for 3 minutes.

[0035] 12. Remove the column and place it into a new 1.5 ml collection tube. Place the column in a 56oC oven to dry for 10 minutes.

[0036] 13. Add 50-60 µl Nuclease Free Water or Buffer TE to the center of the membrane in the column. Let stand for 3 minutes. Centrifuge at >13,000 x g for 1 minute.

[0037] 14. Transfer the eluate to the center of the membrane in the column. Let stand for 1 minute. Centrifuge at >13,000 x g for 1 minute.

[0038] 15. Discard the DNA binding column. Measure the DNA concentration by Qubit and store the DNA at -20oC or -80oC.

[0039] Example 3, PCR Mix Preparation 1. Use both Reaction Well 1 and Reaction Well 2 for each sample.

[0040] 2. Prepare each reaction as shown in Table 1: Table 1, reaction system The primer probe mixture in the reaction well 1 is the primer probe dry powder diluted to a concentration of 100 μM, and each of the target genes takes 4 μL of upstream and downstream primers and 2.4 μL of probes, with a total of 31.2 μL (the final concentration of primers is 250 nM, and the final concentration of probes is 150 nM); the GAPDH internal reference gene takes 4 μL of upstream and downstream primers and 2.4 μL of probes, with a total of 12.8 μL (the final concentration of primers is 250 nM, and the final concentration of probes is 150 nM); 36 μL of water is added to a total of 80 μL of the mixed liquid system, and 1 μL is added to each reaction.

[0041] The primer probe mixture in the reaction well 2 is the primer probe dry powder diluted to a concentration of 100 μM, and each of the target genes takes 4 μL of upstream and downstream primers and 2.4 μL of E542K probes and 4 μL of E545D probes, with a total of 14.4 μL (the final concentration of primers is 250 nM, the final concentration of E542K probes is 150 nM, and the final concentration of E545D probes is 250 nM); the GAPDH internal reference gene takes 4 μL of upstream and downstream primers and 2.4 μL of probes, with a total of 12.8 μL (the final concentration of primers is 250 nM, and the final concentration of probes is 150 nM); 52.8 μL of water is added to a total of 80 μL of the mixed liquid system, and 1 μL is added to each reaction.

[0042] Example 4, microdroplet generation 1. Add 20 μL of the above-mentioned PCR reaction liquid and 70 μL of Bio-Rad microdroplet generation special oil to the corresponding holes on the DG8TM microdroplet generation card, cover the microdroplet generation card sealing pad, and put it into the QX200 microdroplet generator for reaction. The reaction time is about 1 min, and the QX200 microdroplet generator can process up to 8 samples at the same time; 2. After the sample is prepared by microdroplet, it is sequentially sucked by a manual 8-channel adjustable pipette and slowly transferred to the corresponding 96-well plate; 3. After the sample transfer is completed, place the 96-well plate on the heat sealer, and when the red indicator light on the heat sealer is on, the film can be sealed. Cover the heat sealing film, press the handle, and count 5-10 seconds. Before the timing is over, the pressure must be maintained. When the timing is over, release the handle and take out the 96-well plate. Example 5, PCR amplification 1. Place the 96-well plate confirmed to be sealed in the T100 PCR instrument for amplification; 2. The reaction program is shown in Table 2: Table 2, reaction program Example 6, microdroplet detection 1. Turn on the QX200 Droplet Analyzer and allow it to warm up for 30 minutes. Then turn on the computer and start the QuantaSoft™ software. 2. Set up the sample name in the "Sample" tab, enter the sample name in "Name", and select the type of detection in "Experiment" by using the drop-down menu. After completion, check the "Apply" option.

[0043] 3. When the sample name and detection type selection is complete, click "Apply" in the lower right corner to complete the setup.

[0044] Example 7, Data Analysis 1. Analyze the data, click "Load" in the settings panel, select the file to be analyzed, and click the "Analyze" key in the left navigation bar.

[0045] 2. Click "2D Amplitude" to view the CH1 and CH2 cluster map. This map allows manual or automatic adjustment of the threshold value to designate positive and negative droplets for each detection channel.

[0046] 3. In the results table, check the droplet count of each well, usually the droplet count is 10000-20000.

[0047] 4. Check the NTC, allow <2 positive droplets. Otherwise, contamination may occur.

[0048] 5. Check the negative control, allow <2 positive droplets.

[0049] 6. Check the positive control and check if it is within the reference range.

[0050] 7. If all of these are valid, manually set the threshold value and apply it to all samples.

[0051] Example 8, Specificity, Blank Detection Limit, Sensitivity and HPV Consistency Evaluation of Detection System Specificity: Reaction well 1: Use high, medium and low gradient HPV-16 standard, E545K mutation reference (Nanjing Kebai, product number CBP10517, containing internal reference) and genomic gDNA as template respectively, to detect the cross reaction specificity of the reaction system.

[0052] The results are shown in Table 3 below: Table 3, detection results Results show: HPV-16 reference high, low, three concentrations, the detection results only HPV-16 positive, E545K and GAPDH are negative; E545K reference high, low, three concentrations, the detection results E545K sample, GAPDH positive, HPV-16 negative; gDNA reference high, low, three concentrations, the detection results GAPDH positive, E545K and HPV-16 are negative; NTC blank control results are negative. The detection system has no cross reaction, good specificity.

[0053] Reaction well 2: use high, medium and low gradient E542K positive reference (Nanjing Kebai, item number CBP10324, containing internal reference), E545D mutant plasmid and genomic gDNA as template respectively, detect the cross reaction specificity of the reaction system.

[0054] The results are shown in Table 4 as follows: Table 4, detection results Results show: E542K positive reference high, low, three concentrations, the detection results E542K sample, GAPDH positive, E545D negative; E545D mutant plasmid high, low, three concentrations, the detection results E545D sample positive, E542K, GAPDH are negative; gDNA reference high, low, three concentrations, the detection results GAPDH positive, E542K and E545D are negative; NTC blank control results are negative. The detection system has no cross reaction, good specificity.

[0055] Blank detection limit evaluation: Reaction well 1: use 28 clinical negative samples (verified by other means) as template, use the detection system for detection. Use Poisson distribution probability function to calculate the cumulative frequency of false positive droplets (i.e. false positive rate). Calculate the clinical positive interpretation threshold. The results are shown in Table 5 as follows: Table 5, detection results Note: The numbers in parentheses represent the number of positive droplets.

[0056] Results show that among the 28 negative samples, one sample has 2 HPV false positive droplets, one sample has 1 E545K false positive droplet, and the cumulative frequency of false positive droplets is calculated by Poisson distribution probability function. The blank detection limit of the detection system for HPV and E545K mutation is: when FAM channel (HPV) ≥ 2 positive droplets and when HEX channel (E545K) ≥ 2 positive droplets are judged as positive. At this time, the detection system detects the authenticity of positive droplets > 99%.

[0057] Reaction well 2: using 26 clinical negative samples (verified by other means) as templates, the optimized detection system was used for detection. The cumulative frequency of false positive microdroplets detected was calculated using the Poisson distribution probability function (i.e. false positive rate). The clinical positive interpretation threshold was calculated.

[0058] The results are shown in Table 6 below: Table 6, detection results Note: The numbers in parentheses represent the number of positive microdroplets.

[0059] The results show that among the 26 negative samples, 3 samples each detected 1 E542K false positive microdroplet, and 2 samples each detected 1 E545D false positive microdroplet. The cumulative frequency of false positives detected was calculated using the Poisson distribution probability function. The detection system E542K and E545D mutation blank detection limit is: when the FAM channel (E542K) ≥ 2 positive microdroplets and when the HEX channel (E545D) ≥ 2 positive microdroplets are interpreted as positive. At this time, the detection system detects positive microdroplets with a true rate of > 99%.

[0060] Sensitivity: Reaction well 1: using HPV-16 and E545K standard, diluted to 1 copy, 3 copies, 10 copies three gradients, each gradient was detected 20 times, the lowest copy group with two target detection rate of more than 95% was defined as the sensitivity of the detection system.

[0061] The results are shown in Table 7 below: Table 7, sensitivity The results show that when the positive reference is 1 copy, the HPV detection rate is 90%, and the E545K detection rate is 70%; when the positive reference is 3 copies, the HPV detection rate is 100%, and the E545K detection rate is 100%; when the positive reference is 10 copies, the HPV detection rate is 100%, and the E545K detection rate is 100%. The sensitivity of the detection system is defined as 3 copies.

[0062] Reaction well 2: using E542K standard and E545D plasmid, diluted to 1 copy, 3 copies, 10 copies three gradients, each gradient was detected 20 times, the lowest copy group with two target detection rate of more than 95% was defined as the sensitivity of the detection system.

[0063] The results are shown in Table 8 below: Table 8, sensitivity The results show that: when the positive reference is 1 copy, the detection rate of E542K is 80%, and the detection rate of E545D is 90%; when the positive reference is 3 copies, the detection rate of E542K is 100%, and the detection rate of E545D is 100%; when the positive reference is 10 copies, the detection rate of E542K is 100%, and the detection rate of E545D is 100%. The sensitivity of the detection system is 3 copies.

[0064] HPV consistency evaluation: FISH detected 15 positive samples (15 / 32, 46.9%) and 17 negative samples (17 / 32, 53.1%) from 32 tissue section samples, and ddPCR detected 16 positive samples (16 / 32, 50%) and 16 negative samples (16 / 32, 50%). Compared with FISH detection, the consistency rate of ddPCR detection was 90.6%, and the results of the two detection methods were as shown in Table 9: Table 9: Evaluation of ddPCR method for detecting HPV copy number by FISH as gold standard Example 9: Detection of clinical samples New 113 tumor tissue samples and corresponding plasma of head and neck cancer patients were collected, and the ROC analysis of ddPCR detection of HPV results was compared with the diagnosis standard of tumor tissue immunohistochemistry (Immunohistochemistry, IHC) P16 staining (results as shown in Table 10). Figure 7 ddPCR detection of HPV has high consistency with P16 staining diagnosis standard, AUC=0.92, specificity 100%, sensitivity 80.2%, and it is determined that the diagnosis value of ddPCR detection of HPV is 2 positive droplets.

[0065] The above is the preferred embodiment of the present application. It should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, which should also be considered within the scope of protection of the present application.

Claims

1. A primer-probe combination for detecting HPV infection and PIK3CA gene mutation in patients with head and neck cancer, characterized in that, It contains specific primer and probe sequences for targets of HPV16, HPV33, E545K, E542K, and E545D, as well as primers and probes for the internal reference gene GAPDH.

2. The primer-probe combination as described in claim 1, characterized in that: The specific primer and probe sequences for the targets of HPV16, HPV33, E545K, E542K, and E545D are shown in SEQ ID NO:1-11, and the primer and probe sequences for the internal reference gene GAPDH are shown in SEQ ID NO:12-15.

3. A multiplex digital PCR detection system, characterized in that, Include: Reaction well 1: HPV16 / 33+E545K triple target detection system, 10 μL of 2xddPCR Supermix + 1 μL of primer and probe mixture + template DNA; Reaction well 2: E542K / E545D dual-target detection system, 10 μL of 2xddPCR Supermix + 1 μL of primer and probe mixture + template DNA.

4. The detection system as described in claim 3, characterized in that: The final primer concentration in reaction well 1 was 250 nM, and the final probe concentration was 150 nM. The final concentration of the E542K probe in reaction well 2 was 150 nM, and the final concentration of the E545D probe was 250 nM.

5. A method for prognostic assessment of head and neck cancer patients, characterized in that, The HPV16 / 33 viral load and the mutation frequency of the PIK3CA gene E545K / E542K / E545D were detected using the system described in claim 3.

6. The method as described in claim 5, characterized in that, Establish a prognostic assessment model: HPV+ / PIK3CA wild-type: best prognostic group; HPV- / PIK3CA mutant: worst prognostic group.

7. A test kit, characterized in that, It includes: the primer and probe combination 2xddPCRSupermix premix as described in claim 1, GAPDH internal reference gene control, and positive control.

8. The kit as described in claim 7, characterized in that: Positive control samples contain HPV16; and / or The positive control sample contained the E545K mutation.

9. A digital PCR detection method, characterized in that, The method comprises the following steps: (1) Droplet generation: Bio-Rad QX200 system; (2) Amplification program: 95℃ 10min → (94℃ 30s → 58℃ 1min) × 40 cycles → 98℃ 10min; (3) Data analysis: QuantaSoft software automatically identifies FAM / HEX dual-channel signals.

10. The method as described in claim 9, characterized in that, Set interpretation criteria: HPV positive: ≥2 positive droplets in the FAM channel; PIK3CA mutation: ≥2 positive droplets in the HEX channel.

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