Primer probe combination, kit and application for detecting HPV and / or HPV typing

Abstract

The present invention provides a primer-probe combination for detecting HPV and / or HPV typing, belonging to the field of human papillomavirus (HPV) detection technology. The primer-probe combination includes one or more of HPV16E7, HPV18E7, HPV31E7, HPV45E7, HPV33E7, HPV35E7, HPV59E6, HPV68E6, HPV58E7, HPV51E6, HPV52E7, HPV56E6, HPV66E6, and HPV39E6, and can distinguish different types of HPV, with good specificity, accuracy, and low detection limit. The present invention uses a digital PCR method (dPCR) to detect the fluorescent signal of each droplet, and calculates the copy number of the target gene fragment in the entire reaction system based on the Poisson distribution principle, so that absolute quantification can be achieved without relying on a standard curve.

Description

Technical Field

[0001] The present invention belongs to the technical field of human papillomavirus (HPV) detection, and in particular relates to a primer-probe combination, a kit and applications for detecting HPV and / or HPV typing. Background Art

[0002] HPV-related malignancies include: cervical cancer, vaginal cancer, vulvar cancer, oropharyngeal cancer, penile cancer or anal cancer, among others, with cervical cancer being the main representative of this series of malignant tumors. Tumor recurrence and metastasis are the main factors affecting the five-year survival rate, so monitoring recurrence is key to reducing the risk of death in patients. Imaging examinations and exfoliative cytology are commonly used post-treatment follow-up methods, and histopathological diagnosis is the gold standard. However, imaging is difficult to detect the recurrence of tiny tumors. Secondly, frequent radiation exposure and high costs will reduce compliance with regular recurrence monitoring. Histopathology relies on methods such as surgery or biopsy to obtain tumor tissue, which is an invasive procedure and is not convenient for recurrence identification. High-risk human papillomavirus (HR-HPV), including HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, and 68, can identify HPV-associated malignancies. Furthermore, circulating tumor DNA (ctDNA) carries the same genetic characteristics as the primary tumor and is present in the blood of patients with HPV-associated tumors. Therefore, HPV ctDNA is an ideal marker for HPV-associated tumor recurrence. However, HPV ctDNA in patients with HPV-associated malignancies is diluted by non-tumor DNA and accounts for a low proportion of plasma free DNA. Real-time fluorescence quantitative PCR (QPCR) is not sensitive enough for the detection of HPV ctDNA in plasma. Digital PCR (dPCR) has significantly higher sensitivity and specificity than QPCR and can effectively detect HPV DNA and its concentration in tissues, exfoliated cells, and blood. Summary of the Invention

[0003] In view of this, the object of the present invention is to provide a primer-probe combination, a kit and an application for detecting HPV and / or HPV typing.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] In the present invention, by screening the primer probes HPV16E6, HPV16E7, HPV18E6, HPV18E7, HPV31E7, HPV33E7, HPV35E7, HPV39E6, HPV45E6, HPV45E7, HPV51E6, HPV52E7, HPV56E6, HPV58E7, HPV59E6, HPV66E6, and HPV68E6, it was found that the three groups of primer probes HPV16E6, HPV18E6, and HPV45E6 performed poorly in the test. It is not suitable for detecting HPV and / or HPV typing, but the primer probes HPV16E7, HPV18E7, HPV31E7, HPV45E7, HPV33E7, HPV35E7, HPV59E6, HPV68E6, HPV58E7, HPV51E6, HPV52E7, HPV56E6, HPV66E6 and HPV39E6 have good discrimination for detecting HPV and / or HPV typing using the dPCR method, and have strong specificity, high sensitivity and high accuracy when used to detect HPV and / or HPV typing.

[0006] Based on this, the present invention provides a primer-probe combination for detecting HPV and / or HPV typing, comprising one or more of the primer probes HPV16E7, HPV18E7, HPV31E7, HPV45E7, HPV33E7, HPV35E7, HPV59E6, HPV68E6, HPV58E7, HPV51E6, HPV52E7, HPV56E6, HPV66E6, and HPV39E6;

[0007] The nucleotide sequences of the HPV16E7 probe, upstream primer, and downstream primer are shown in SEQ ID NOs. 4 to 6;

[0008] The nucleotide sequences of the HPV18E7 probe, upstream primer, and downstream primer are shown in SEQ ID NOs. 10 to 12;

[0009] The nucleotide sequences of the HPV31E7 probe, upstream primer, and downstream primer are shown in SEQ ID NOs. 13 to 15;

[0010] The nucleotide sequences of the HPV45E7 probe, upstream primer, and downstream primer are shown in SEQ ID NOs. 25 to 27;

[0011] The nucleotide sequences of the HPV33E7 probe, upstream primer, and downstream primer are shown in SEQ ID NOs. 16 to 18;

[0012] The nucleotide sequences of the HPV35E7 probe, upstream primer, and downstream primer are shown in SEQ ID NOs. 19 to 21;

[0013] The nucleotide sequences of the HPV59E6 probe, upstream primer, and downstream primer are shown in SEQ ID NOs. 43 to 45;

[0014] The nucleotide sequences of the HPV68E6 probe, upstream primer, and downstream primer are shown in SEQ ID NOs. 49 to 51;

[0015] The nucleotide sequences of the HPV58E7 probe, upstream primer, and downstream primer are shown in SEQ ID NOs. 40 to 42;

[0016] The nucleotide sequences of the HPV51E6 probe, upstream primer, and downstream primer are shown in SEQ ID NOs. 31 to 33;

[0017] The nucleotide sequences of the HPV52E7 probe, upstream primer, and downstream primer are shown in SEQ ID NOs. 34 to 36;

[0018] The nucleotide sequences of the HPV56E6 probe, upstream primer, and downstream primer are shown in SEQ ID NOs. 37 to 39;

[0019] The nucleotide sequences of the HPV66E6 probe, upstream primer, and downstream primer are shown in SEQ ID NOs. 46 to 48;

[0020] The nucleotide sequences of the HPV39E6 probe, upstream primer and downstream primer are shown in SEQ ID NOs. 22 to 24.

[0021] In the present invention, the 5' end of the probe is modified with a fluorescent group. The fluorescent groups modified at the 5' end of the probe nucleotide sequences in HPV16E7, HPV18E7, HPV31E7, HPV45E7, HPV33E7, HPV35E7, HPV59E6, HPV68E6, HPV58E7, HPV51E6, HPV52E7, HPV56E6, HPV66E6, and HPV39E6 are VIC, ROX, CY5, ATTO425, VIC, ROX, CY5, CY5, ATTO425, VIC, ROX, CY5, CY5, and ATTO425, respectively.

[0022] The primer-probe combination of the present invention can accurately detect a variety of different high-risk HPV types, and the HPV type is one or more of HPV16, HPV18, HPV31, HPV45, HPV33, HPV35, HPV58, HPV59, HPV68, HPV51, HPV52, HPV56, HPV66 and HPV39.

[0023] In the present invention, as a preferred embodiment, the primer probe combination is divided into primer probe combination 1, primer probe combination 2 or primer probe combination 3; the probe combination 1 is HPV16E7, HPV18E7, HPV31E7 and HPV45E7; the probe combination 2 is HPV33E7, HPV35E7, HPV59E6, HPV68E6 and HPV58E7; the probe combination 3 is HPV51E6, HPV52E7, HPV56E6, HPV66E6 and HPV39E6.

[0024] The present invention also provides an application of the above primer-probe combination in the preparation of a product for detecting HPV and / or HPV typing.

[0025] The product of the present invention includes a reagent or a kit. The primer-probe combination of the present invention is used to detect HPV and / or HPV typing with high specificity, high sensitivity and high accuracy.

[0026] The present invention also provides a kit for detecting HPV and / or HPV typing, comprising the above primer-probe combination.

[0027] In the present invention, the kit also includes an internal reference gene and a PCR reaction mixture. The internal reference gene preferably includes RPP30, and the nucleotide sequences of the RPP30 probe, upstream primer, and downstream primer are shown in SEQ ID NOs. 52 to 54. The PCR reaction mixture preferably includes the following components at final concentrations: 1× PCR buffer, 50 to 400 μM dNTPs, 1 to 2.5 mM MgCl2, 0.1 U to 5 U of Taq DNA polymerase, 400 to 750 nM upstream primers and downstream primers, and 150 to 500 nM probe. The present invention uses a PCR reaction mixture for PCR amplification, and in particular, the dosage of the upstream primer, downstream primer, and probe provided in the present invention improves the specificity, sensitivity, and accuracy of HPV detection and / or HPV typing.

[0028] The present invention also provides a method for detecting HPV and / or HPV typing using a dPCR method, which is used for non-diagnostic purposes and comprises performing PCR amplification using the above-mentioned primer-probe combination or kit using the dPCR method. As a preferred embodiment, the method comprises the following steps:

[0029] (1) extracting DNA from the sample to be tested;

[0030] (2) using the DNA from step (1) as a template, performing dPCR amplification using primer and probe combinations;

[0031] (3) Determine whether the sample is HPV positive based on the fluorescence amplification signals of different channels and perform HPV typing.

[0032] In the present invention, the sample type of the sample to be tested includes any one of a tissue sample, an exfoliated cell sample and a plasma sample from a patient with HPV-related lesions.

[0033] The present invention provides an application of a dPCR method in preparing a detection product for HPV and / or HPV typing in a sample.

[0034] In the present invention, the exfoliated cells include cervical exfoliated cells, penis exfoliated cells, vulvar exfoliated cells, oropharyngeal exfoliated cells or anal exfoliated cells. In the present invention, the above-mentioned sample includes any one of a tissue sample, an exfoliated cell sample and a plasma sample of a patient with HPV-related lesions.

[0035] dPCR is a technique for absolute nucleic acid quantification that divides the PCR reaction system into tens of thousands of independent droplets, i.e., tens of thousands of independent reactions. The dPCR instrument used in this invention uses vibration injection technology to divide the reaction system into tens of thousands of microdroplets, each of which can independently complete the PCR amplification reaction.

[0036] Compared with the prior art, the present invention has the following advantages:

[0037] The present invention provides a primer-probe combination for detecting HPV and / or HPV typing. This combination can distinguish different high-risk HPV subtypes with good specificity, accuracy, and a low limit of detection. After amplification, the present invention's dPCR method uses microdroplets containing target nucleic acid molecules to produce fluorescence, while microdroplets without target nucleic acid molecules do not produce fluorescence. By detecting the fluorescence signal of each droplet, the copy number of the target gene fragment in the entire reaction system is calculated based on the Poisson distribution principle, achieving absolute quantification without relying on a standard curve. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1The results of the corresponding HPV typing were detected by dPCR using different primer probes, where 1A in A is the result of the primer probe HPV16E6 detecting HPV16-positive samples, and 1E is the result of the primer probe HPV16E7 detecting HPV16-positive samples; 2A in B is the result of the primer probe HPV18E6 detecting HPV18-positive samples, and 2E is the result of the primer probe HPV18E7 detecting HPV18-positive samples; 1A in C is the result of the primer probe HPV31E7 detecting negative samples, and 1B is the result of the primer probe HPV 1D in D is the result of primer probe HPV45E6 detecting a negative sample of HPV45, and 1F is the result of primer probe HPV45E7 detecting a positive sample of HPV45; 1G in E is the result of primer probe HPV52E7 detecting a negative sample, and 1H is the result of primer probe HPV52E7 detecting a positive sample of HPV52; 2B in F is the result of primer probe HPV58E7 detecting a negative sample, and 2D is the result of primer probe HPV58E7 detecting a positive sample of HPV58;

[0039] Figure 2 In Figure 1, 2E in A is the result of primer probe HPV51E6 detecting negative samples, and 2F is the result of primer probe HPV51E6 detecting HPV51-positive samples; 1A and 1B in B are the results of primer probe HPV33E7 detecting HPV33-positive samples; 1C and 1D in C are the results of primer probe HPV35E7 detecting HPV35-positive samples; 1E and 1F in D are the results of primer probe HPV39E6 detecting HPV39-positive samples; 1G and 1H in E are the results of primer probe HPV59E6 detecting HPV59-positive samples; 2A and 2C in F are the results of primer probe HPV56E6 detecting HPV56-positive samples; 1E and 1F in G are the results of primer probe HPV66E6 detecting HPV66-positive samples; 1G and 1H in H are the results of primer probe HPV68E6 detecting HPV68-positive samples;

[0040] Figure 3These are the results of different concentration groups of primer probe HPV45E7 detecting HPV45-positive samples, among which wells 1A and 1B are the results of group 1 primer probe HPV45E7 detecting HPV45-positive samples; wells 1C and 1D are the results of group 2 primer probe HPV45E7 detecting HPV45-positive samples; wells 1E and 1F are the results of group 3 primer probe HPV45E7 detecting HPV45-positive samples; wells 1G and 1H are the results of group 4 primer probe HPV45E7 detecting HPV45-positive samples; wells 2A and 2C are the results of group 5 primer probe HPV45E7 detecting HPV45-positive samples;

[0041] Figure 4 Results of testing mixed samples (prepared from gDNA, HPV16, HPV18, HPV31, and HPV45) at different concentrations using primer-probe internal reference, HPV16E7, HPV18E7, HPV31E7, and HPV45E7 in tube 1. The target gene concentration for 1E and 1F tests was 1.25E+05 copies / mL, and the target gene concentration for 1G and 1H tests was 2.5E+03 copies / mL.

[0042] Figure 5 The results of detecting different concentrations of mixed samples (prepared from gDNA, HPV33, HPV35, HPV59, HPV68, and HPV58) in tube 2 using primer-probe internal control, HPV33E7, HPV35E7, HPV59E6, HPV68E6, and HPV58E7. The target gene concentrations for (2E, 2F) and (1A, 1B) were 1.25E+05 copies / mL, and those for (2G, 2H) and (1C, 1D) were 2.5E+03 copies / mL and 1.25E+04 copies / mL, respectively.

[0043] Figure 6 Results of tube 3 using primers, probes, internal reference, HPV51E6, HPV52E7, HPV56E6, HPV66E6, and HPV39E6 to detect mixed samples (prepared from gDNA, HPV51, HPV52, HPV56, HPV66, and HPV39) at different concentrations. The target gene concentrations for (2A, 2B) and (2E, 2F) were 1.25E+05 copies / mL, and those for (2C, 2D) and (2G, 2H) were 2.5E+03 copies / mL.

[0044] Figure 7Results from tube 4 using primer-probe internal control, HPV51E6, HPV35E7, HPV59E6, and HPV58E7 to detect mixed samples (prepared from gDNA, HPV51, HPV35, HPV59, and HPV58) at different concentrations. The target gene concentrations for (1C, 1D) and (1E, 1F) were 1.25E+05 copies / mL and 2.5E+03 copies / mL, respectively.

[0045] Figure 8 Results from tube 5 using primer-probe internal reference, HPV33E7, HPV52E7, HPV56E6, and HPV39E6 to detect mixed samples (gDNA, HPV33, HPV52, HPV56, and HPV39 mixed preparation) at different concentrations. The target gene concentrations for (2A, 2C) and (2E, 2G) were 1.25E+05 copies / mL and 2.5E+03 copies / mL, respectively.

[0046] Figure 9 The following are the results of three replicates of primer-probe combination 1 testing HPV16-positive simulated samples. (1A, 1B, 1C), (1D, 1E, 1F), and (1G, 1H, 2A) are the test results of low-, medium-, and high-concentration simulated samples, respectively; 2C and 2E are positive and negative controls.

[0047] Figure 10 The following are the results of three replicates of primer-probe combination 1 testing HPV18-positive simulated samples. (1A, 1B, 1C), (1D, 1E, 1F), and (1G, 1H, 2A) are the test results of low-, medium-, and high-concentration simulated samples, respectively; 2C and 2E are positive and negative controls.

[0048] Figure 11 The following are the results of three replicates of primer-probe combination 1 testing HPV31-positive simulated samples. (1A, 1B, 1C), (1D, 1E, 1F), and (1G, 1H, 2A) are the test results of low-, medium-, and high-concentration simulated samples, respectively; 2C and 2E are positive and negative controls.

[0049] Figure 12 The following are the results of three replicates of primer-probe combination 1 testing HPV45-positive simulated samples. (1A, 1B, 1C), (1D, 1E, 1F), and (1G, 1H, 2A) are the test results of low-, medium-, and high-concentration simulated samples, respectively; 2C and 2E are positive and negative controls.

[0050] Figure 13The following are the results of three repeated tests of HPV16-positive simulated samples using primer-probe combination 1-1. (1A, 1B, 1C), (1D, 1E, 1F), and (1G, 1H, 2A) are the test results of low-, medium-, and high-concentration simulated samples, respectively; 2C and 2E are positive and negative controls.

[0051] Figure 14 The following are the results of three repeated tests of HPV18-positive simulated samples using primer-probe combination 1-1. (1A, 1B, 1C), (1D, 1E, 1F), and (1G, 1H, 2A) are the test results of low-, medium-, and high-concentration simulated samples, respectively; 2C and 2E are positive and negative controls.

[0052] Figure 15 The following are the results of three repeated tests of HPV31-positive simulated samples using primer-probe combination 1-1. (1A, 1B, 1C), (1D, 1E, 1F), and (1G, 1H, 2A) are the test results of low-, medium-, and high-concentration simulated samples, respectively; 2C and 2E are positive and negative controls.

[0053] Figure 16 The following are the results of three repeated tests of HPV45-positive simulated samples using primer-probe combination 1-1. (1A, 1B, 1C), (1D, 1E, 1F), and (1G, 1H, 2A) are the test results of low-, medium-, and high-concentration simulated samples, respectively; 2C and 2E are positive and negative controls;

[0054] Figure 17 The dPCR test results of the present invention for high and low concentration HPV16 samples;

[0055] Figure 18 The dPCR test results of the present invention for high and low concentration HPV18 samples;

[0056] Figure 19 The dPCR test results of the present invention for high and low concentration HPV31 samples are shown;

[0057] Figure 20 The dPCR test results of the present invention for high and low concentration HPV45 samples are shown;

[0058] Figure 21 The dPCR test results of the present invention for high and low concentration HPV33 samples;

[0059] Figure 22 The dPCR test results of the present invention for high and low concentration HPV35 samples are shown;

[0060] Figure 23 The dPCR test results of the present invention for high and low concentration HPV59 samples;

[0061] Figure 24 The dPCR test results of the present invention for high and low concentration HPV68 samples;

[0062] Figure 25 The dPCR test results of the present invention for high and low concentration HPV58 samples are shown;

[0063] Figure 26 The dPCR test results of the present invention for high and low concentration HPV51 samples are shown;

[0064] Figure 27 The dPCR test results of the present invention for high and low concentration HPV52 samples are shown;

[0065] Figure 28 The dPCR test results of the present invention for high and low concentration HPV56 samples are shown;

[0066] Figure 29 The dPCR test results of the present invention for high and low concentration HPV66 samples are shown;

[0067] Figure 30 The dPCR test results of the present invention for high and low concentration HPV39 samples;

[0068] Figure 31 These are the QPCR test results for high and low concentrations of HPV16 samples;

[0069] Figure 32 These are the QPCR test results for high and low concentrations of HPV18 samples;

[0070] Figure 33 These are the QPCR test results for high and low concentrations of HPV31 samples;

[0071] Figure 34 These are the QPCR test results for high and low concentrations of HPV45 samples;

[0072] Figure 35 These are the QPCR test results for high and low concentrations of HPV33 samples;

[0073] Figure 36 These are the QPCR test results for high and low concentrations of HPV35 samples;

[0074] Figure 37 These are the QPCR test results for high and low concentrations of HPV59 samples;

[0075] Figure 38 These are the QPCR test results for high and low concentrations of HPV68 samples;

[0076] Figure 39These are the QPCR test results for high and low concentrations of HPV58 samples;

[0077] Figure 40 These are the QPCR test results for high and low concentrations of HPV51 samples;

[0078] Figure 41 These are the QPCR test results for high and low concentrations of HPV52 samples;

[0079] Figure 42 These are the QPCR test results for high and low concentrations of HPV56 samples;

[0080] Figure 43 These are the QPCR test results for high and low concentrations of HPV66 samples;

[0081] Figure 44 These are the QPCR test results for high and low concentrations of HPV39 samples. DETAILED DESCRIPTION

[0082] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0083] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0084] Example 1

[0085] (1) Design and screening of primers and probes for HPV typing detection using dPCR

[0086] The E6 and E7 gene sequences for all variants of HPV16, 18, 31, 45, 33, 35, 58, 59, 68, 51, 52, 56, 66, and 39 were downloaded from NCBI. Conserved regions were screened using MAGE7, and primers and probes were designed within these conserved regions to ensure coverage of all variants of HPV16, 18, 31, 45, 33, 35, 58, 59, 68, 51, 52, 56, 66, and 39. The system includes one upstream primer, one downstream primer, and one probe for each of HPV16, 18, 31, 45, 33, 35, 58, 59, 68, 51, 52, 56, 66, and 39, as well as one upstream primer, one downstream primer, and one probe for the RPP30 internal reference gene. Information on primers and probes used for HPV detection and / or HPV typing is provided in Table 1.

[0087] Table 1 Primer and probe information

[0088]

[0089]

[0090] (2) Sample processing

[0091] Blood samples were collected from patients with cervical cancer, and the extracted DNA concentration was measured using a Qubit fluorometer. ctDNA purity was tested using an Agilent 2200 / 4200 Tapestry. The predominant fragment size was 170 to 200 bp, confirming the extraction was qualified. Human papillomavirus nucleic acid detection and genotyping kits (dPCR) were used for testing, with a DNA sample load of >20 ng and a volume of ≤14 μL.

[0092] (3) PCR reaction system configuration

[0093] The components and their contents described in Table 2 were prepared to obtain a PCR reaction system.

[0094] Table 2 PCR reaction system

[0095]

[0096]

[0097] Among them, the upstream primers in Table 2 were the upstream primers of HPV16E6, HPV16E7, HPV18E6, HPV18E7, HPV31E7, HPV33E7, HPV35E7, HPV39E6, HPV45E6, HPV45E7, HPV51E6, HPV52E7, HPV56E6, HPV58E7, HPV59E6, HPV66E6, HPV68E6, and RPP30 in Table 1, and the downstream primers were HPV16E6, HPV16E7, HPV18E6, HPV18E7, HPV31E7, HPV33E7, HPV35E7, HPV39E6, HPV45E6, HPV45E7 The downstream primers were HPV5E6, HPV45E7, HPV51E6, HPV52E7, HPV56E6, HPV58E7, HPV59E6, HPV66E6, HPV68E6, and RPP30, and the probes were the probes of HPV16E6, HPV16E7, HPV18E6, HPV18E7, HPV31E7, HPV33E7, HPV35E7, HPV39E6, HPV45E6, HPV45E7, HPV51E6, HPV52E7, HPV56E6, HPV58E7, HPV59E6, HPV66E6, HPV68E6, and RPP30 in Table 1, respectively. The specific sequences correspond to Table 1.

[0098] (4) dPCR detection

[0099] Place the prepared PCR reaction system on a dPCR analyzer and amplify 1.0E+06 copies / mL of the positive plasmid sample and nuclease-free water according to the PCR amplification protocol: 60°C for 5 minutes for droplet spreading, 95°C for 5 minutes for enzyme activation, 95°C for 10 seconds, 60°C for 30 seconds, for 45 cycles. Select the CY5.5 droplet recognition channel. Select combinations that clearly distinguish between positive and negative droplets, have concentrated clusters of positive droplets, and exhibit minimal Rain. If these combinations do not meet the requirements, optimize the upstream or downstream primers.

[0100] The dPCR instrument can divide the reaction system into about 20,000 droplets and perform independent PCR amplification reactions on each droplet unit.

[0101] During the PCR process, Taq enzyme simultaneously extends the DNA chain and dissociates the specific fluorescent probe bound to the template through its own 5'>3' exonuclease activity, causing the fluorescent reporter group to fluoresce under a specific light source. After PCR amplification cycles, microdroplets containing target nucleic acid molecules will fluoresce, while microdroplets without target nucleic acid molecules will not. The fluorescence signal of each droplet unit is then detected. An LED light source transmits fluorescence of a specified wavelength band through a filter to illuminate the microdroplets in a dedicated flat container. The generated fluorescence signal passes through a paired filter and enters the camera sensor to form an image. The software distinguishes negative and positive droplets based on the fluorescence grayscale value of each droplet and analyzes each sample image using a droplet recognition algorithm. The mutant and wild-type copy numbers of the target gene fragment in the reaction system are calculated using a Poisson distribution to achieve quantitative detection. The formula for the Poisson distribution is shown in Equation 1.

[0102] Number of negative droplets ÷ total number of droplets = e －λ ...Formula 1.

[0103] where λ is the average copy number contained in a single droplet.

[0104] Figure 1 and Figure 2 Results showed that the use of primer probes HPV16E6, HPV16E7, HPV18E6, HPV18E7, HPV31E7, HPV33E7, HPV35E7, HPV39E6, HPV51E6, HPV52E7, HPV56E6, HPV58E7, HPV59E6, HPV66E6, and HPV68E6 clearly differentiated positive and negative droplets, with positive droplets clustered. HPV16E7 demonstrated better discrimination than the primer probe HPV16E6. HPV18E7 also demonstrated better discrimination than the primer probe HPV18E6. However, the use of primer probe HPV45E6 failed to differentiate between positive and negative droplets. Screening with the primer probe HPV45E7 allowed differentiation between positive and negative droplets, but the positive droplet clusters were not clustered.

[0105] In this example, the dosage of the upstream primer, downstream primer, and probe of HPV45E7, which has poor discrimination, was adjusted. That is, the concentrations of the upstream primer, downstream primer, and probe in Table 2 were divided into five groups. In Group 1, the upstream primer of HPV45E7 was 500 nM, the downstream primer was 500 nM, and the probe was 250 nM; in Group 2, the upstream primer of HPV45E7 was 500 nM, the downstream primer was 500 nM, and the probe was 400 nM; in Group 3, the upstream primer of HPV45E7 was 500 nM, the downstream primer was 500 nM, and the probe was 400 nM; The upstream primer of HPV45E7 was 750nM, the downstream primer was 750nM, and the probe was 400nM; the upstream primer of HPV45E7 in group 4 was 500nM, the downstream primer was 500nM, and the probe was 500nM; the upstream primer of HPV45E7 in group 5 was 750nM, the downstream primer was 750nM, and the probe was 500nM. Then, the primer and probe HPV45E7 were detected using the method of steps (1) to (4). The results are shown in FIG. Figure 3 .

[0106] According to the conditions of clear distinction between positive and negative droplets, concentrated positive droplets, and less Rain, Figure 3 The results showed that when the concentrations of the upstream primer, downstream primer and probe of HPV45E7 were 500 nM, 500 nM and 500 nM, the detection using the primer-probe HPV45E7 had a good discrimination.

[0107] The primer and probe information of each HPV type used in the present invention is shown in Table 3.

[0108] Table 3 Primer and probe information for different HPV typing

[0109]

[0110]

[0111] The sequences of the primers and probes in Table 3 are shown in Table 1.

[0112] Example 2

[0113] A primer-probe combination for detecting HPV and / or HPV typing based on a dPCR method, wherein the primer-probe combination is HPV16E7, HPV18E7, HPV31E7, HPV45E7, HPV33E7, HPV35E7, HPV59E6, HPV68E6, HPV58E7, HPV51E6, HPV52E7, HPV56E6, HPV66E6 and HPV39E6, with RPP30 as an internal reference, wherein the specific sequence of the primer-probe combination and RPP30 is shown in Table 1, wherein the HPV1 The 5' end of the probe sequence for HPV6E7, HPV18E7, HPV31E7, HPV45E7, HPV33E7, HPV35E7, HPV59E6, HPV68E6, HPV58E7, HPV51E6, HPV52E7, HPV56E6, HPV66E6, and HPV39E6 was modified with fluorescent labels VIC, ROX, CY5, ATTO425, VIC, ROX, CY5, CY5, ATTO425, VIC, ROX, CY5, CY5, and ATTO425, respectively. The 5' end of the probe sequence for RPP30 was modified with a fluorescent label FAM.

[0114] Example 3

[0115] A kit for detecting HPV and / or HPV typing by dPCR amplification, the kit comprising independently packaged internal reference RPP30, a primer-probe combination, and a PCR reaction mixture. The primer-probe combination is the primer-probe combination described in Example 2, with each primer packaged separately. The volume concentration of the primer-probe combination is: 1.1 μL of a 10 μM upstream primer, 1.1 μL of a 10 μM downstream primer, and 0.55 μL of a 10 μM probe. The PCR reaction mixture comprises 2.2 μL of 10× PCR buffer, 0.5 μL of 10 mM dNTPs, 1.5 μL of 25 mM MgCl2, 0.1 μL of 10 U / μL Taq DNA polymerase, 14 μL of template input, and 0.95 μL of water. The amounts of the upstream primer, downstream primer, and probe in the RPP30 are shown in Table 3.

[0116] Example 4

[0117] The method for dPCR amplification and detection of HPV and / or HPV typing in this embodiment comprises the following steps:

[0118] (1) Collection and extraction of exfoliated cell samples

[0119] Collect cervical exfoliated cell specimens positive for HPV16, 18, 31, 45, 33, 35, 58, 59, 68, 51, 52, 56, 66, and 39, transfer them to preservation solution, and store them at room temperature. After DNA extraction, store them in a -80°C ultra-low temperature freezer.

[0120] (2) dPCR amplification

[0121] A PCR reaction system was prepared using the PCR reaction mixture and the upstream primer, downstream primer, and probe amounts in the primer-probe combination described in Example 3, and PCR amplification was performed using the primer-probe combination using a dPCR instrument. The PCR amplification procedure is described in Example 1, and the primer-probe sequences are shown in Table 1.

[0122] The test results are expressed as nucleic acid extract concentration copies / mL:

[0123] Note: 1. Calculation of copy number: Copy number = instrument measurement result (copies / μL) × generated droplet volume (nL, read on the instrument) × number of effective droplets (read on the instrument measurement result) / 1000.

[0124] 2. Actual sample volume (μL) = template sample volume × generated droplet volume (nL, read on the instrument) × number of effective droplets (read on the instrument measurement result) / (1000 × 22 μL).

[0125] 3. Nucleic acid extract concentration (copies / mL) = copy number × 1000 / actual sample volume.

[0126] Example 5

[0127] Based on the similarity and interference between primers, target gene concentrations were set at 1.25E+05 copies / mL and 2.5E+03 copies / mL, respectively. The HPV68 and HPV58 target gene concentrations were 1.25E+05 copies / mL and 1.25E+04 copies / mL, respectively. Five combinations were grouped according to HPV typing for multiple validation. Using the method described in Example 4, combinations with clear differentiation between positive and negative droplets, concentrated clusters of positive droplets, and minimal Rain were selected. The grouping of the five combinations is shown in Table 4.

[0128] Table 4 Grouping of 5 combinations

[0129]

[0130] Figures 4 to 8 The results showed that target gene-positive droplets in tubes 4 and 5 were stratified, and some target gene-positive droplets were diffuse, such as HPV51, HPV59, and HPV35 in tube 4, and the internal reference gene and HPV56 in tube 5. Therefore, tubes 4 and 5 were eliminated, and the primer-probe combination of tubes 1, 2, and 3 was used for subsequent verification.

[0131] Example 6

[0132] A primer-probe combination for detecting HPV and / or HPV typing based on a dPCR method, wherein the primer-probe combination is primer-probe combination 1, primer-probe combination 2, or primer-probe combination 3; the primer-probe combination 1 is HPV16E7, HPV18E7, HPV31E7, and HPV45E7; the primer-probe combination 2 is HPV33E7, HPV35E7, HPV59E6, HPV68E6, and HPV58E7; the primer-probe combination 3 is HPV51E6, HPV52E7, HPV56E6, HPV66E6, and HPV39E6, with RPP30 as an internal reference, wherein the primer-probe combination 1 is HPV16E7, HPV18E7, HPV31E7, and HPV45E7; the primer-probe combination 2 is HPV33E7, HPV35E7, HPV59E6, HPV68E6, and HPV58E7; the primer-probe combination 3 is HPV51E6, HPV52E7, HPV56E6, HPV66E6, and HPV39E6, The specific sequences of the needle combinations and RPP30 are shown in Table 1, wherein the 5'-ends of the probe sequences of HPV16E7, HPV18E7, HPV31E7, HPV45E7, HPV33E7, HPV35E7, HPV59E6, HPV68E6, HPV58E7, HPV51E6, HPV52E7, HPV56E6, HPV66E6, and HPV39E6 are modified with fluorescent labels VIC, ROX, CY5, ATTO425, VIC, ROX, CY5, CY5, ATTO425, VIC, ROX, CY5, CY5, and ATTO425, respectively. The 5'-end of the probe sequence of RPP30 is modified with a fluorescent label FAM.

[0133] Example 7

[0134] A kit for detecting HPV and / or HPV typing by dPCR amplification, the kit comprising independently packaged internal reference RPP30, a primer-probe combination, and a PCR reaction mixture. The primer-probe combination is the primer-probe combination described in Example 6, with each primer packaged separately. The volume concentration of the primer-probe combination is: 1.1 μL of a 10 μM upstream primer, 1.1 μL of a 10 μM downstream primer, and 0.55 μL of a 10 μM probe. The PCR reaction mixture comprises 2.2 μL of 10× PCR buffer, 0.5 μL of 10 mM dNTPs, 1.5 μL of 25 mM MgCl2, 0.1 μL of 10 U / μL Taq DNA polymerase, 14 μL of template input, and 0.95 μL of water. The amounts of the upstream primer, downstream primer, and probe in the RPP30 are shown in Table 3.

[0135] Example 8

[0136] The method for dPCR amplification and detection of HPV and / or HPV typing in this embodiment comprises the following steps:

[0137] (1) Collection of exfoliated cells and DNA extraction

[0138] Cervical exfoliated cells from individuals positive for HPV types 16, 18, 31, 45, 33, 35, 58, 59, 68, 51, 52, 56, 66, and 39 were collected and transferred to a preservation solution. DNA was extracted from the samples.

[0139] (2) dPCR amplification

[0140] A PCR reaction system was prepared using the PCR reaction mixture described in Example 7 and the upstream primer, downstream primer, and probe from the primer-probe combination described in Example 6. PCR amplification was performed using the primer-probe combination described in Example 6 using a dPCR instrument. The PCR amplification procedure was described in Example 1. After PCR amplification, if the microdroplets produced fluorescence, the sample was positive; if the microdroplets did not produce fluorescence, the sample was negative.

[0141] Example 9

[0142] The primer-probe combination 1, primer-probe combination 2, and primer-probe combination 3 described in Example 6 were verified for accuracy using the method of Example 8 using a fixed-value quality control product purchased from Jingliang. The results are shown in Tables 5 to 7.

[0143] Table 5 Accuracy test results of primer-probe combination 1

[0144]

[0145] Table 6 Accuracy test results of primer-probe combination 2

[0146]

[0147]

[0148] Table 7 Accuracy test results of primer probe combination 3

[0149]

[0150] The results in Tables 5 to 7 show that the quality control products of the measured values ​​of primer-probe combination 1 to primer-probe combination 3 all meet the quality control product standards, and the detection accuracy of the present invention is high.

[0151] Example 10

[0152] HPV16, 18, 31, 45, 33, 35, 58, 59, 68, 51, 52, 56, 66, and 39 quality control products at a concentration of 1.0E+06 copies / mL and 20 ng of fragmented genomic DNA were used to verify the specificity of primer-probe combination 1, primer-probe combination 2, and primer-probe combination 3 described in Example 6 using the method of Example 8.

[0153] Table 8 Specificity detection results of primer-probe combinations

[0154] sample Primer probe combination 1 Primer probe combination 2 Primer probe combination 3 HPV16 + - - HPV18 + - - HPV31 + - - HPV45 + - - HPV33 - + - HPV35 - + - HPV59 - + - HPV68 - + - HPV58 - + - HPV51 - - + HPV52 - - + HPV56 - - + HPV66 - - + HPV39 - - + 20 ng fragmented genomic DNA - - -

[0155] Note: “+” indicates detected, “-” indicates not detected.

[0156] The results in Table 8 show that there is no cross-reaction between the types and genomic DNA, indicating that the primer-probe combination of the present invention has high specificity.

[0157] Example 11

[0158] The detection limit was tested using the method of Example 8. A mixed sample of HPV16, 18, 31, and 45 was diluted to 5 copies / reaction and tested 15 times. The results are shown in Table 9. A mixed sample of HPV33, 35, and 59 and a mixed sample of HPV68 and 58 were diluted to 5 copies / reaction and tested 15 times. The results are shown in Table 10. A mixed sample of HPV51, 52, and 56 was diluted to 5 copies / reaction, and a sample of HPV66 and 39 was diluted to 5 copies / reaction and tested 15 times. The results are shown in Table 11.

[0159] Table 9 Detection limit test results of primer probe combination 1

[0160]

[0161] Table 10 Detection limit test results of primer probe combination 2

[0162]

[0163] Table 11 Detection limit test results of primer probe combination 2

[0164]

[0165]

[0166] The results in Tables 9 to 11 show that the detection rate for each type was 100% for 5 copies / reaction, with the deviation between the test concentration (copies / reaction) and the reference concentration (copies / reaction) less than ±50%. The detection limit for HPV16 / 18 / 31 / 45 / 33 / 35 / 59 / 68 / 58 / 51 / 52 / 56 / 66 / 39 reached 5 copies / reaction.

[0167] Example 12

[0168] The consistency evaluation was performed with reference to the method in Example 8 and a commercial kit. The comparison kit was: High-risk human papillomavirus nucleic acid (typing) detection kit (National Medical Device Registration No.: 20163401295). The detection principle is fluorescent quantitative PCR. A cycle threshold of ≤39 is positive. It can identify 15 HR-HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68). 402 clinical specimens were included (some specimens had multiple infections of different types of HPV). The same sample was tested using the above two methods. The results are shown in Table 12.

[0169] Table 12 Comparison results of the consistency between the detection method of the present invention and the HPV QPCR detection method

[0170] HPV type Number of QPCR positive cases Number of dPCR positive cases Kappa value 95% CI HPV16 55 53 0.872 0.801～0.943 HPV18 19 18 0.915 0.819～1.000 HPV31 25 23 0.867 0.763～0.971 HPV33 31 29 0.956 0.858～1.000 HPV35 16 14 0.862 0.729～0.995 HPV39 24 25 0.935 0.862～1.000 HPV45 13 19 0.805 0.654～0.956 HPV51 26 26 0.918 0.838～0.998 HPV52 58 52 0.853 0.779～0.927 HPV56 / 66 64 59 0.837 0.763～0.911 HPV58 54 54 0.829 0.747～0.911 HPV59 / 68 47 45 0.877 0.803～0.951

[0171] The results in Table 12 show that the two detection methods have excellent consistency in all 14 types.

[0172] Example 13

[0173] Detection of HPV types in plasma ctDNA using different HPV16, 18, 31, and 45 gene primer and probe combinations

[0174] The control group was: a primer-probe combination 1-1 for detecting HPV and / or HPV typing based on the dPCR method, wherein the primer-probe combination was primer-probe combination 1-1, and primer-probe combination 1-1 was HPV16E7-1, HPV18E7-1, HPV31E7-1, and HPV45E7-1. The specific primer sequences are shown in Table 13. The 5' ends of the probe sequences of HPV16E7-1, HPV18E7-1, HPV31E7-1, and HPV45E7-1 were modified with fluorescent labels such as VIC, ROX, CY5, and ATTO425, respectively.

[0175] The test group consisted of a primer-probe combination 1 for detecting HPV and / or HPV typing based on the dPCR method, wherein the primer-probe combination 1 was HPV16E7, HPV18E7, HPV31E7, and HPV45E7. The specific primer sequences are shown in Table 1, wherein the 5' ends of the probe sequences of HPV16E7, HPV18E7, HPV31E7, and HPV45E7 were modified with fluorescent markers such as VIC, ROX, CY5, and ATTO425, respectively.

[0176] Table 13 Sequence information of primer-probe combination 1-1

[0177]

[0178]

[0179] The method for detecting HPV or HPV typing in a plasma ctDNA sample using the primer-probe combination 1-1 or primer-probe combination 1 comprises the following steps:

[0180] (1) Simulated sample preparation and extraction

[0181] HPV16, 18, 31, and 45, quality control products purchased from Jingliang, were ultrasonically fragmented into approximately 170bp fragments. These fragments were then diluted with normal human plasma to prepare high, medium, and low concentration samples, respectively, to simulate plasma ctDNA samples. ctDNA extraction was then completed.

[0182] Among them, the high, medium and low concentrations of HPV16 were 4.03E+05, 4.03E+04 and 4.03E+02 copies / mL respectively; the high, medium and low concentrations of HPV18 were 4.90E+05, 4.90E+04 and 4.90E+02 copies / mL respectively; the high, medium and low concentrations of HPV31 were 5.38E+05, 5.38E+04 and 5.38E+02 copies / mL respectively; the high, medium and low concentrations of HPV45 were 4.87E+05, 4.87E+04 and 4.87E+02 copies / mL respectively.

[0183] (2) dPCR amplification

[0184] A PCR reaction system was prepared: 1.1 μL of a 10 μM upstream primer, 1.1 μL of a 10 μM downstream primer, 0.55 μL of a 10 μM probe, 2.2 μL of 10× PCR buffer, 0.5 μL of 10 mM dNTPs, 1.5 μL of 25 mM MgCl₂, 0.1 μL of 10 U / μL Taq DNA polymerase, 14 μL of template input, and 0.95 μL of water. PCR amplification was performed using a dPCR instrument with primer-probe combination 1-1 or primer-probe combination 1, respectively. The PCR amplification procedure is described in Example 1. Fluorescence detection channels were VIC, CY5, ROX, and ATT0425. The detection results are shown in Tables 14-15.

[0185] Table 14 Detection results of primer-probe combination 1 for simulated plasma

[0186]

[0187] Table 15 Detection results of primer-probe combination 1-1 for simulated plasma

[0188]

[0189] Figures 9 to 16The results in Table 14 and Table 15 show that the primer probe sequence used in primer probe combination 1 can well detect free ctDNA

[0190] The primer-probe combination 1-1 had a low detection value and failed to detect low-concentration samples, making it unsuitable for plasma ctDNA detection.

[0191] Example 14

[0192] This example uses the dPCR amplification method for detecting HPV and / or HPV typing of the present invention and a high-risk human papillomavirus nucleic acid (typing) detection kit (National Medical Device Registration No.: 20163401295) for comparative detection.

[0193] (1) Simulated sample preparation and extraction

[0194] HPV16, 18, 31, 45, 33, 35, 58, 59, 68, 51, 52, 56, 66, and 39, quality control materials purchased from Jingliang, were used. Ultrasound was used to fragment samples of approximately 170 bp in size and diluted with normal human plasma to simulate clinical samples. ctDNA was extracted.

[0195] (2) dPCR amplification

[0196] A PCR reaction system was prepared using the PCR reaction mixture described in Example 7 and the upstream primer, downstream primer, and probe from the primer-probe combination described in Example 6. PCR amplification was performed using the primer-probe combination described in Example 6 using a dPCR instrument. The PCR amplification procedure was described in Example 1. Fluorescence detection channels were FAM, VIC, CY5, ROX, and ATT0425.

[0197] Fluorescence quantitative PCR amplification: Four HPV PCR reaction solutions were added to the enzyme mixture, thoroughly mixed to form a PCR mixture, and centrifuged briefly for later use. The PCR amplification program was 50°C for 2 minutes for UNG enzyme reaction, 94°C for 5 minutes for enzyme activation, and 45 cycles of 94°C for 15 seconds and 57°C for 30 seconds. Fluorescence detection channels were FAM, HEX, CY5, ROX, and CY5.

[0198] Table 16 dPCR detection results of simulated plasma

[0199]

[0200] Table 17 QPCR test results of simulated plasma

[0201]

[0202]

[0203] Note: NoCt in the table means not detected.

[0204] Figures 17 to 44 The results in Tables 16 and 17 show that dPCR detected two low-concentration simulated plasma ctDNA samples for each target with a 100% detection rate. However, in QPCR, some targets were not detected or were below the positive judgment value (Ct>39 for negative). For example, HPV68 was not detected at both concentrations, and one of the two replicates for HPV16, HPV31, HPV45, and HPV66 (5.00E+02 copies / mL) was not detected. This indicates that the present invention has a significant advantage in detecting HPV and its typing using dPCR in plasma free ctDNA samples.

[0205] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A primer-probe combination for detecting HPV and / or HPV typing based on dPCR, characterized in that: Comprising primer probe combination 1, primer probe combination 2 and primer probe combination 3; The primer-probe combination 1 includes probes for detecting HPV16E7, HPV18E7, HPV31E7 and HPV45E7, upstream primers and downstream primers; The primer-probe combination 2 includes probes for detecting HPV33E7, HPV35E7, HPV59E6, HPV68E6 and HPV58E7, upstream primers and downstream primers; The primer-probe combination 3 includes probes for detecting HPV51E6, HPV52E7, HPV56E6, HPV66E6 and HPV39E6, upstream primers and downstream primers; The nucleotide sequences of the HPV16E7 probe, upstream primer, and downstream primer are shown in SEQ ID NOs. 4 to 6; The nucleotide sequences of the HPV18E7 probe, upstream primer, and downstream primer are shown in SEQ ID NOs. 10 to 12; The nucleotide sequences of the HPV31E7 probe, upstream primer, and downstream primer are shown in SEQ ID NOs. 13 to 15; The nucleotide sequences of the HPV45E7 probe, upstream primer, and downstream primer are shown in SEQ ID NOs. 25 to 27; The nucleotide sequences of the HPV33E7 probe, upstream primer, and downstream primer are shown in SEQ ID NOs. 16 to 18; The nucleotide sequences of the HPV35E7 probe, upstream primer, and downstream primer are shown in SEQ ID NOs. 19 to 21; The nucleotide sequences of the HPV59E6 probe, upstream primer, and downstream primer are shown in SEQ ID NOs. 43 to 45; The nucleotide sequences of the HPV68E6 probe, upstream primer, and downstream primer are shown in SEQ ID NOs. 49 to 51; The nucleotide sequences of the HPV58E7 probe, upstream primer, and downstream primer are shown in SEQ ID NOs. 40 to 42; The nucleotide sequences of the HPV51E6 probe, upstream primer, and downstream primer are shown in SEQ ID NOs. 31 to 33; The nucleotide sequences of the HPV52E7 probe, upstream primer, and downstream primer are shown in SEQ ID NOs. 34 to 36; The nucleotide sequences of the HPV56E6 probe, upstream primer, and downstream primer are shown in SEQ ID NOs. 37 to 39; The nucleotide sequences of the HPV66E6 probe, upstream primer, and downstream primer are shown in SEQ ID NOs. 46 to 48; The nucleotide sequences of the HPV39E6 probe, upstream primer and downstream primer are shown in SEQ ID NOs. 22 to 24.

2. The primer-probe combination according to claim 1, characterized in that The 5' end of the probe is modified with a fluorescent group.

3. The primer-probe combination according to claim 2, characterized in that The fluorescent groups modified at the 5' end of the nucleotide sequences of the probes of HPV16E7, HPV18E7, HPV31E7, HPV45E7, HPV33E7, HPV35E7, HPV59E6, HPV68E6, HPV58E7, HPV51E6, HPV52E7, HPV56E6, HPV66E6 and HPV39E6 are VIC, ROX, CY5, ATTO425, VIC, ROX, CY5, CY5, ATTO425, VIC, ROX, CY5, CY5 and ATTO425, respectively.

4. Use of the primer-probe combination according to any one of claims 1 to 3 in the preparation of a product for detecting HPV and / or HPV typing based on dPCR.

5. The use according to claim 4, characterized in that The sample detected by the primer-probe combination includes any one of a tissue sample, an exfoliated cell sample and a plasma sample of a patient with HPV-related lesions.

6. A kit for detecting HPV and / or HPV typing based on dPCR, characterized in that: The method comprises the primer-probe combination according to any one of claims 1 to 3.

7. The kit according to claim 6, characterized in that The kit also includes an internal reference gene and a PCR reaction mixture; the internal reference gene includes RPP30, and the nucleotide sequences of the RPP30 probe, upstream primer, and downstream primer are shown in SEQ ID NOs. 52 to 54; the PCR reaction mixture includes the following components at final concentrations: 1× PCR buffer, 50 to 400 μM dNTPs, 1 to 2.5 mM MgCl2, 0.1 U to 5 U Taq DNA polymerase, 400 to 750 nM upstream primers and downstream primers, and 150 to 500 nM probe.

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