A primer probe combination for detecting HPV16 and HPV18 by droplet digital PCR and application thereof

Abstract

The application provides a primer probe combination for detecting HPV16 and HPV18 by using microdroplet digital PCR and application thereof, and belongs to the technical field of human papilloma virus detection products. The primer probe combination for detecting HPV16 and HPV18 by using microdroplet digital PCR comprises a multi-target primer probe combination for detecting HPV16, and a multi-target primer probe combination for detecting HPV18, wherein the nucleotide sequence of the primer probe combination for detecting HPV16 is shown in SEQ ID NO. 1-27, and the nucleotide sequence of the primer probe combination for detecting HPV18 is shown in SEQ ID NO. 28-54. The primer probe combination has high sensitivity and low detection limit, can accurately detect HPV16 and HPV18 with high specificity and low cost, accurately capture trace ctDNA in early cervical cancer patient samples, significantly improve the early diagnosis rate, and effectively avoid the missed detection risk of single detection.

Description

Technical Field

[0001] This invention belongs to the technical field of human papillomavirus detection products, specifically relating to a primer and probe combination for droplet digital PCR detection of HPV16 and HPV18 and its application. Background Technology

[0002] Cervical cancer is the most common gynecological malignancy, with a significant trend towards younger onset. Epidemiological studies have found that almost all cervical cancer cases are caused by persistent infection with high-risk human papillomavirus (HPV), with HPV types 16 and 18 being the most prevalent pathogenic types. The HPV genome usually exists in the host cell nucleus as a free circular episome. A key molecular event in the development from cervical intraepithelial neoplasia (CIN) to invasive cervical cancer is the transformation of the HPV genome from a free state to an integrated state. This means that viral DNA is linearly inserted into the host cell's chromosome through breakage and recombination. HPV genome integration is essentially a non-homologous recombination event; in this process, the viral genome rarely inserts in its entirety, and some viral gene sequences are often deleted during this process. Once circular DNA breaks at one or more sites, the fragments within it may be completely removed during integration and repair. This includes regions such as late-stage genes L1 and L2, and early-stage regulatory genes E1, E2, E4, E6, and E7, which are affected by random breakage and integration and may be partially or completely deleted. Furthermore, circulating tumor DNA (ctDNA) from HPV-related tumors, including the HPV DNA genome, can be released into the bloodstream. However, ctDNA levels in the blood are extremely low, typically accounting for only 0.1%–10% of total cell-free DNA (cfDNA), and even less than 0.01% in early-stage tumor patients. It also has a short half-life (15 minutes–2 hours), making it easily diluted by large amounts of cfDNA from normal cells, and its fragment length is mostly concentrated between 160–180 bp. Therefore, detecting the HPV viral genome in the blood of patients with HPV-related tumors faces the following significant challenges: First, there is the risk of false negatives. Current PCR detection methods in this field, which rely on single targets (especially L1), can report false negatives due to the loss of this target, thus missing the identification of truly positive samples. In recent years, several studies have used deep sequencing technology to retest cervical cancer samples that tested "HPV negative" by conventional PCR, and found that a significant number of these samples actually contained integrated, actively transcribed HPV E6 / E7 genes, but failed to be detected due to the loss of traditional PCR target regions (such as L1). Second, there is limited sensitivity. Although HPV ctDNA has the advantages of strong targeting and the ability to reflect dynamic changes in tumor burden, its trace and short-fragment characteristics make it difficult for existing quantitative PCR (qPCR) or next-generation sequencing (NGS) detection technologies to achieve efficient and accurate capture and detection.

[0003] In summary, persistent infection with HPV16 and HPV18 is a core pathogenic factor in the development of cervical cancer. However, the trace amounts and short fragments of ctDNA, along with some shortcomings in existing detection technologies, mean that there is currently a lack of a method and related products in this field that can accurately and sensitively detect HPV16 and HPV18 with significant clinical value and industrial application prospects. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a primer-probe combination that is highly sensitive (capable of stably detecting low abundance ctDNA with an abundance of less than 0.01%), has a low detection limit, high specificity, and low cost, and can simultaneously and accurately detect HPV16 and HPV18. This combination can accurately capture trace amounts of ctDNA in early cervical cancer patient samples, significantly improve the early diagnosis rate, and effectively avoid the risk of missed detection by single detection.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a primer and probe combination for detecting HPV16 and HPV18 using droplet digital PCR, including a multi-target primer and probe combination for detecting HPV16 with nucleotide sequences as shown in SEQ ID NO.1~SEQ ID NO.27, and a multi-target primer and probe combination for detecting HPV18 with nucleotide sequences as shown in SEQ ID NO.28~SEQ ID NO.54.

[0006] Preferably, it also includes an internal standard primer-probe combination, the nucleotide sequence of which is shown in SEQ ID NO.55~SEQ ID NO.57.

[0007] Preferably, the 5' end of the probe is modified with a fluorescent group, and the 3' end is modified with a quenching group; the fluorescent group includes VIC, FAM, HEX, ROX, CY5 and / or Cy5.5; the quenching group includes BHQ1, BHQ2, MGB and / or TAMRA.

[0008] Preferably, HPV16, HPV18 and the internal standard are distinguished based on the color of the fluorescent group modified by the probe.

[0009] This invention also provides the application of the above-mentioned primer-probe combination in the preparation of HPV16 and / or HPV18 detection products.

[0010] Preferably, the detection product includes a droplet digital PCR detection product.

[0011] Preferably, the reaction system of the droplet digital PCR detection product, in 22 μL increments, consists of 4.5 μL of PCR reaction mixture, 2.5 μL of primer-probe mixture, and 15 μL of DNA sample.

[0012] Preferably, the primer-probe mixture has a volume of 2.5 μL, consisting of 2.375 μL of the primer-probe combination and 0.125 μL of nuclease-free water; the primer-probe combination is obtained by mixing equal volumes of primer-probe combinations for each target; in the primer-probe combination for each target, the concentrations of the probe, upstream primer, and downstream primer are 200 μM, and the volume ratio of the probe, upstream primer, and downstream primer is 0.025:0.05:0.05.

[0013] Preferably, the reaction program for the droplet digital PCR detection product is as follows: droplet spreading at 60°C for 5 min; enzyme activation at 95°C for 5 min; denaturation at 95°C for 20 s, annealing and extension at 63°C for 30 s, repeated 5 times; denaturation at 95°C for 20 s, annealing and extension at 60°C for 30 s, repeated 40 times; final extension at 55°C for 20 min.

[0014] The present invention also provides a kit for detecting HPV16 and HPV18 using droplet digital PCR, comprising a primer-probe mixture, a PCR reaction mixture, a positive control, a negative control, and an internal control; the positive control is a plasmid containing target gene fragments of HPV16 and HPV18; the negative control is nuclease-free water; the internal control is a plasmid containing an internal target gene fragment that does not cross-reference with HPV16 and HPV18; the primer-probe mixture comprises the above-mentioned primer-probe combination and nuclease-free water.

[0015] The beneficial effects of this invention are: The primer-probe combination provided by this invention enables simultaneous and accurate detection of multiple targets, including HPV16 and HPV18, effectively avoiding the risk of missed detection in single-target detection. It also boasts high sensitivity, with a minimum detection concentration of 1.5 cp / mL for HPV16 and 1 cp / mL for HPV18, accurately capturing trace amounts of ctDNA in the blood of early-stage cervical cancer patients, significantly improving the early diagnosis rate. Using the primer-probe combination provided by this invention ensures a 100% sample detection rate, with the deviation between the detected concentration and the reference concentration within ±50%.

[0016] When using the primer and probe combination provided by this invention to perform droplet digital PCR detection of HPV16 and HPV18, there is no need to rely on expensive high-throughput sequencing equipment, the detection cost is controllable, the operation process is simple and the detection cycle is short, no professional bioinformatics analysis is required, it is easy to popularize and apply in primary medical institutions, it can promote the downward extension of cervical cancer screening to the grassroots level, help improve the hierarchical diagnosis and treatment system, and alleviate the problem of uneven distribution of medical resources in low- and middle-income areas.

[0017] When using the primer-probe combination provided by this invention, dynamic monitoring of tumor burden in cervical cancer patients can be achieved through minimally invasive blood sampling. The test results are used to assist doctors in monitoring the treatment effect and recurrence rate (MRD) of cervical cancer patients, which helps to accurately assess the treatment effect, provide timely warning of recurrence and metastasis, and provide objective basis for adjusting clinical treatment plans, enabling individualized precision diagnosis and treatment, improving patient prognosis, which is consistent with the clinical application trend of ctDNA as a tumor dynamic monitoring biomarker. Moreover, the primer-probe combination provided by this invention can serve as a highly efficient tool for large-scale cervical cancer screening, quickly identifying high-risk groups, assisting in optimizing vaccination and screening strategies, and providing technical support for the global goal of eliminating cervical cancer, especially for prevention and control work in areas with high incidence of HPV16 and / or HPV18 infection.

[0018] In summary, the primer-probe combination provided by this invention, which combines high sensitivity, high specificity, low cost, and the ability to simultaneously detect the presence of multiple targets of HPV16 and HPV18 in ctDNA samples, has irreplaceable clinical value and broad application prospects for improving the efficiency of early diagnosis of cervical cancer, optimizing disease monitoring strategies, and promoting precision prevention and control of cervical cancer. Attached Figure Description

[0019] Figure 1 The results of detecting plasma samples containing both HPV16 and HPV18 using the primer-probe combination of the present invention are shown in the upper left, where the results of detecting HPV16 are shown in the upper right, the results of detecting HPV18 are shown in the lower left, and the results of detecting the internal control primer-probe are shown in the lower left. Figure 2 To detect the relationship between single and multiple HPV16 load using droplet digital PCR. Detailed Implementation

[0020] This invention provides a primer-probe combination for detecting HPV16 and HPV18 using droplet digital PCR, characterized in that it includes a multi-target primer-probe combination for detecting HPV16 with nucleotide sequences as shown in SEQ ID NO.1~SEQ ID NO.27, and a multi-target primer-probe combination for detecting HPV18 with nucleotide sequences as shown in SEQ ID NO.28~SEQ ID NO.54.

[0021] In the primer-probe combination described in this invention, an internal standard primer-probe combination is preferably also included, the nucleotide sequence of which is shown in SEQ ID NO. 55~SEQ ID NO. 57. The primer-probe combination provided by this invention targets the E1, E2, E6, E7, L1, and L2 gene sequences of HPV16 and HPV18 types. Conserved regions are screened using MEGA7, and primers are designed within these conserved regions to ensure coverage of all variants of HPV16 and HPV18 types. Furthermore, to improve the accuracy and sensitivity of detection, this invention provides two or three sets of primers-probes targeting different locations on the same gene. Specific details are shown in Table 1.

[0022] Table 1 Primer-Probe Combination Information

[0023] Note: Y in the table represents a degenerate base, which is either C or T.

[0024] The primer-probe combination provided by this invention uses droplet digital PCR (ddPCR) as a platform and targets different genomic fragments of HPV16 and HPV18. Compared with existing detection methods, it has the following significant advantages in terms of detection performance, quantitative accuracy, and ease of operation: I. Comparison with sequencing methods: While sequencing methods offer high throughput, simultaneous analysis of multiple gene variations, and high sensitivity, they suffer from drawbacks such as high detection costs, complex data analysis (requiring interpretation by specialized bioinformaticians), and long detection cycles (2-3 days). Furthermore, they place stringent demands on experimental equipment (high-throughput sequencers) and operators, hindering their widespread adoption in primary healthcare institutions and making them unsuitable for large-scale screening. The droplet-based digital PCR detection system provided by this invention offers low cost, data analysis relying on existing software, a short detection cycle, and high sensitivity and specificity, making it suitable for large-scale screening.

[0025] II. Comparison with traditional qPCR: 1. The primer-probe combination provided by this invention overcomes the limitations of existing detection sensitivity and detection limits: (1) Extremely low detection limit: Compared with the detection limit of 1% for qPCR, >10% for Sanger sequencing, and 0.1~1% for NGS, the detection limit of droplet digital PCR of this invention can reach as low as 0.01% or even lower, and the detection limit of ddPCR for specific targets is reduced to 0.002%, which can effectively detect low concentration samples, weak positive or false negative samples, and is suitable for scenarios such as minimal residual disease monitoring and low abundance gene mutation detection.

[0026] (2) Strong anti-interference ability: The detection system provided by the present invention can tolerate PCR inhibitors, is less affected by amplification efficiency, and has better detection stability than qPCR for complex samples (such as partially purified samples containing inhibitors and clinical plasma samples). It can accurately quantify without additional processing.

[0027] 2. Improved quantitative accuracy and repeatability: (1) Absolute quantification without the need for standards: The detection system provided by this invention is based on the Poisson distribution principle and directly achieves absolute quantification of nucleic acids (DNA / mRNA / miRNA / LncRNA, etc.) through microreaction unit counting, avoiding systematic errors caused by qPCR's reliance on standard curves and indirect quantification, and ensuring strong comparability of batch / laboratory data.

[0028] (2) High consistency of results: High reproducibility can be achieved between different operators and laboratories. The comparison between the Sinaf product and the Qiager platform shows that the quantitative indication error is only 2% (Qager is 9%), and the CV value is as low as 0.1~0.2% (Qager is 1.2%), which solves the problem of deviation in the determination results caused by the difference in droplet volume of the traditional dPCR platform (the difference between some platforms is 1.3~2 times).

[0029] (3) Droplet uniformity and volume accuracy: The droplet volume variation coefficient generated by the "vibration injection technology" is only 1.42%, which is far superior to microfluidic chips (which are affected by environmental factors such as flow rate and temperature and have systematic deviations) and micro-pit / micro-cavity technology (which have uneven volume and large batch-to-batch differences). Moreover, the effective number of droplets reaches 21,000 and the sample utilization rate is 84%, which further ensures quantitative accuracy.

[0030] III. Advantages compared to singleton ddPCR targeting HPV16 and HPV18: 1. Target coverage: The primer-probe combination provided by this invention can achieve simultaneous detection of "multi-type + multi-gene fragments", which is adapted to the characteristics of carcinogenic mechanisms.

[0031] When cervical lesions progress to the cancer stage, a significant proportion of patients will lose some segments of the HPV genome in their cervical specimens, such as L1, L2, E2, E4, and E5.

[0032] (1) Limitations of single ddPCR technology: Single ddPCR can only detect a single gene target of one HPV type (such as HPV16E6 or HPV18E7), and cannot simultaneously cover the two core pathogenic subtypes of HPV16 and 18. It cannot capture the “retained / lost” characteristic fragments in the process of HPV gene integration, thus leading to false negative test results.

[0033] (2) Advantages of the multiplex ddPCR technology of this invention: Synergistic detection of multiple gene fragments: Targeting the core mechanism of HPV integration leading to oncology, target combinations of key oncogenic genes (E6, E7, E1, E2, L1, and L2) are designed simultaneously to form target detection redundancy. This can match the molecular characteristics of "retaining oncogenic genes and losing regulatory genes" during HPV16 and HPV18 integration, providing a complete basis for determining the infection type (integrated / free). Secondly, considering the low concentration of ctDNA, multi-target detection can increase the detection probability. For example, when HPV18 loses the E2 gene due to integration, the multiplex detection kit can confirm the presence of infection through positive signals of L1, E6, and / or E7 genes, avoiding missed detection caused by the loss of a single target in single-target technology, and significantly improving detection sensitivity.

[0034] 2. Detection efficiency and practicality: Adaptable to diverse clinical needs, reducing testing costs: (1) Limitations of single-type technology: If multi-type screening or multi-gene fragment detection is required, single-type technology requires setting up a reaction system for each target / type, consuming a large amount of samples, reagents and detection time. Moreover, multiple reactions are prone to introducing systematic errors due to differences in operation, resulting in poor consistency of results, which makes it difficult to meet the needs of large-scale clinical screening or detection of small samples (such as peripheral blood ctDNA).

[0035] (2) Multiple technical advantages of the present invention: 1) Improved sample utilization: Only one sample (such as cervical exfoliated cells or peripheral blood) is needed to complete the simultaneous detection of multiple types and targets, which significantly reduces the sample requirement. It is especially suitable for detection scenarios with trace samples (such as ctDNA) or precious samples, solving the pain point of high sample consumption in single technology.

[0036] 2) Optimization of detection efficiency and cost: One reaction replaces multiple reactions in single-reaction technology, which greatly shortens the detection cycle, reduces the consumption of reagents such as primers, probes, and enzymes, and reduces the time and economic costs of detection. At the same time, it reduces the number of operation steps, reduces human error, and improves the reproducibility of results.

[0037] In summary, the primer-probe combination and the multiplex detection system for HPV16 and HPV18 ctDNA based on droplet digital PCR provided by this invention have significant clinical value and promising industrial application prospects.

[0038] In the primer-probe combination of the present invention, the 5' end of the probe is preferably modified with a fluorescent group, and the 3' end is preferably modified with a quenching group; the fluorescent group preferably includes VIC, FAM, HEX, ROX, CY5 and / or Cy5.5; the quenching group preferably includes BHQ1, BHQ2, MGB and / or TAMRA. The present invention does not have a specific limitation on the specific source of the fluorescent group and quenching group, and conventional commercially available products in the art can be used. In the present invention, HPV16, HPV18, and the internal standard are distinguished based on the color of the fluorescent group modified by the probe. That is, in the multi-target primer-probe combination for detecting HPV16, the color of the fluorescent group modified by the probe must be different from the color of the fluorescent group modified by the probe in the multi-target primer-probe combination for detecting HPV18 and the internal standard primer-probe combination, and the color of the fluorescent group modified by the probe in the multi-target primer-probe combination for detecting HPV18 must also be different from the color of the fluorescent group modified by the probe in the internal standard primer-probe combination. In multi-target primer-probe combinations for detecting HPV16, primers and probes designed for different HPV16 targets can be modified with the same or different fluorescent groups. If they differ, the specific HPV16 target can be distinguished by color. Similarly, in multi-target primer-probe combinations for detecting HPV18, primers and probes designed for different HPV18 targets can be modified with the same or different fluorescent groups. If they differ, the specific HPV18 target can be distinguished by color.

[0039] This invention also provides the application of the above-mentioned primer-probe combination in the preparation of HPV16 and / or HPV18 detection products. In this invention, the detection product preferably includes a droplet digital PCR detection product, and the type of product preferably includes a kit. When the detection product is a droplet digital PCR detection product, the reaction system of the droplet digital PCR detection product, in 22 μL increments, preferably consists of 4.5 μL of PCR reaction mixture, 2.5 μL of primer-probe mixture, and 15 μL of DNA sample. The volume of the primer-probe mixture, in 2.5 μL increments, preferably consists of 2.375 μL of the primer-probe combination and 0.125 μL of nuclease-free water; the primer-probe combination is obtained by mixing equal volumes of primer-probe combinations for each target; in the primer-probe combination for each target, the concentrations of the probe, upstream primer, and downstream primer are in 200 μM increments, and the volume ratio of the probe, upstream primer, and downstream primer is 0.025:0.05:0.05, as specifically shown in Table 2.

[0040] Table 2. Amounts of each primer / probe and nuclease-free water added to the primer / probe mixture.

[0041] In this invention, the preferred reaction procedure for the droplet digital PCR detection product is shown in Table 3: Table 3. Reaction procedures for droplet digital PCR detection products.

[0042] The present invention also provides a kit for detecting HPV16 and HPV18 by droplet digital PCR, comprising primer and probe mixture, PCR reaction mixture, positive control, negative control and internal control, the specific composition of which is preferably shown in Table 4.

[0043] Table 4. Main components of the droplet digital PCR kit for detecting HPV16 and HPV18.

[0044] The positive control was prepared as follows: E1, E2, E6, E7, L1, and L2 gene fragments of HPV16 and HPV18 were each inserted into their respective pUC57 plasmid vectors. After transformation into *E. coli* for amplification culture, a large amount of DNA was extracted from the fragmented bacterial cells, and the supernatant was purified by column chromatography. The positive control described in this invention refers to a mixture of multiple recombinant plasmids, each carrying a target gene.

[0045] When using the primer-probe combination and kit provided by this invention for droplet digital PCR detection of the sample to be tested, the preferred steps include: extracting nucleic acid from the sample to be tested, performing a droplet digital PCR reaction in a digital PCR analyzer using the primer-probe combination described above, and then interpreting the results. In this invention, the sample to be tested preferably includes plasma samples or cervical exfoliated cells. When the sample to be tested is a plasma sample, the collected plasma sample is stored in a plasma preservation solution bottle protected from light (<30 days at room temperature, <180 days at 4℃ / -20℃). In this invention, when performing nucleic acid extraction, a suitable nucleic acid extraction kit is selected according to the sample type, and nucleic acid extraction is performed according to the instructions. After nucleic acid extraction, it is preferred to use a Qubit fluorometer to detect the concentration of ctDNA. The ctDNA loading volume is >20ng (maximum loading volume 14μL); the purity of ctDNA is detected using an Agilent 2200 / 4200 Tapestation device. If the main fragment distribution is 170bp~200bp, the extraction is considered successful. The extracted ctDNA is stored at -20℃ for less than 3 months, with a permissible freeze-thaw cycle of ≤5 times. It is transported on dry ice for ≤110 hours. In this invention, the digital PCR analyzer preferably consists of a temperature control module, a droplet generation module, an optical module, a motion control module, a power supply module, and software (control and data processing software). The main functions of each module are as follows: Temperature control module: controls temperature rise and fall, providing a suitable temperature environment for droplet generation and nucleic acid amplification; Droplet generation module: prepares microdroplets; Optical module: excites, conducts, converts, and collects fluorescence signals according to the program settings; Motion control module:, with the assistance of the software, controls the movement of each component through transmission parts; Power supply module: provides the electrical energy required for the operation of various modules within the device; Software (control and data processing software): data processing software pre-installed on the product's control computer, providing a user interface. In this invention, the main function of the digital PCR analyzer is to divide the prepared reaction system into tens of thousands of microdroplets. After PCR amplification cycles, the fluorescence signal of each droplet is detected, and 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. The preferred technical parameters of the digital PCR analyzer used in this invention are shown in Table 5.

[0046] Table 5 Technical parameters of the digital PCR analyzer

[0047] In this invention, a digital PCR analyzer can divide the reaction system into approximately 20,000 microdroplets and perform independent PCR amplification on each microdroplet unit. During PCR, while the Taq enzyme extends the DNA chain, it can dissociate from its own 5'→3' exonuclease activity and bind to a specific fluorescent probe that binds to the template, causing the fluorescent reporter group to fluoresce under a specific light source. After PCR amplification cycles, microdroplets with target nucleic acid molecules will fluoresce, while microdroplets without target nucleic acid molecules will not fluoresce, and the fluorescence signal of each droplet unit is then detected. An LED light source emits excitation light of a specified wavelength through a filter, illuminating the microdroplets in a dedicated plate container; the fluorescence signal generated by the microdroplets enters the camera sensor after passing through a paired filter, ultimately forming an image. The software distinguishes between negative and positive droplets based on the fluorescence grayscale value of each droplet, and uses a droplet recognition algorithm to analyze each sample image. 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.

[0048] When using the primer-probe combination and kit provided by this invention for droplet digital PCR detection of HPV16 and HPV18, the positive and negative concordance rate is as high as 100%. The detection limit for HPV16 is 1.5 cp / mL, and the detection limit for HPV18 is 1 cp / mL. The precision is ≤10%, and the specificity is high. There is no cross-reactivity with HPV genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. It can tolerate 200 ng of wild-type human genomic DNA.

[0049] The technical solutions provided by the present invention will be 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.

[0050] Unless otherwise specified, the following embodiments are all conventional methods.

[0051] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0052] The design concept of the following embodiments is as follows: Simulated plasma samples are constructed based on plasmid standards, and a detection system is built: the simulated plasma is serially diluted, with a minimum detection concentration of 1.5 cp / mL for HPV16 and 1 cp / mL for HPV18; the system is adjusted to ensure a 100% detection rate for all samples, and the deviation between the detection concentration and the reference concentration is within ±50%. Based on cervical samples (with known HPV types), the constructed ddPCR method is used for detection, and the consistency of HPV type detection is analyzed; cross-sectional clinical blood samples are collected from patients with cervical cancer or precancerous lesions, and the HPV ctDNA content in plasma is detected using the HPV16 and HPV18 kits provided by this invention, with sequencing as the gold standard to verify the accuracy of plasma HPV16 / 18 ctDNA detection.

[0053] Example 1 A primer-probe combination for detecting HPV16 and HPV18 using droplet digital PCR is shown in Table 1. In the multi-target primer-probe combination for detecting HPV16, the 5' end of each probe is modified with the fluorescent group VIC, and the 3' end is modified with the quenching group MGB. In the multi-target primer-probe combination for detecting HPV18, the 5' end of each probe is modified with the fluorescent group ROX, and the 3' end is modified with the quenching group MGB. In the internal standard primer-probe combination, the 5' end of each probe is modified with the fluorescent group FAM, and the 3' end is modified with the quenching group MGB.

[0054] Example 2 A kit for detecting HPV16 and HPV18 using droplet digital PCR has the following composition as shown in Table 4.

[0055] Example 3 A method for detecting HPV16 and HPV18 using droplet digital PCR, comprising the following steps: (1) Nucleic acid extraction was performed on the sample to be tested. After the nucleic acid extraction was completed, the concentration of the nucleic acid was detected by Qubit fluorescence meter. When the concentration was >20ng, it indicated that the sample could be loaded. The purity of ctDNA was detected by Agilent 2200 / 4200 Tapestation. The main fragment distribution was 170bp~200bp. The extraction was confirmed to be qualified and the subsequent steps were carried out. (2) The nucleic acid obtained by using the primer and probe combination of Example 1 was subjected to droplet digital PCR reaction in the digital PCR analyzer shown in Table 5. The reaction system was based on 22 μL and consisted of 4.5 μL of PCR reaction mixture (the specific composition of the PCR reaction mixture is shown in Table 4), 2.5 μL of primer and probe mixture and 15 μL of DNA sample. The specific composition of the primer and probe mixture is shown in Table 2 and the reaction procedure is shown in Table 3. (3) Then interpret the results: Qualitative detection: If more than one fluorescence signal of the corresponding color of VIC is captured and the fluorescence signal of the corresponding color of FAM is captured, it indicates that the sample to be tested contains HPV16; if more than or equal to one fluorescence signal of the corresponding color of ROX is captured and the fluorescence signal of the corresponding color of FAM is captured, it indicates that the sample to be tested contains HPV18. Quantitative detection: The system's built-in software uses a droplet recognition algorithm to analyze the image of each sample. 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.

[0056] Example 4 The method described in Example 3 was used to detect a plasma sample known to contain both HPV types 16 and 18. The nucleic acid extraction kit used was a nucleic acid extraction reagent manufactured by Suzhou Sinolife Medical Technology Co., Ltd., catalog number: DT042050C. The results are as follows: Figure 1 As shown, the top left (VIC channel) shows the results of primer probe detection of HPV16 (red dots are positive droplets, indicating that HPV16 was detected); the top right (ROX channel) shows the results of primer probe detection of HPV18 (green dots are positive droplets, indicating that HPV18 was detected); and the bottom left (FAM channel) shows the results of internal control primer probe detection (blue dots are positive droplets, indicating that the experimental system was effective).

[0057] Comparative Example 1 The difference from Example 3 is that the primer-probe combination in step (2) is replaced with the following primer-probe combination: HPV16 upstream primer: 5'-CACGTAGAGAAACCCAGCTGT-3' (SEQ ID NO.58), HPV16 downstream primer: 5'-GATCAGTTGTCTCTGGTTGCAAA-3' (SEQ ID NO.59), HPV16 probe: 5'-VIC-TCATGCATGGAGATACACCTACA-MGB-3' (SEQ ID NO.60); HPV18 upstream primer: 5'-ACCTTCTATGTCACGAGCAA-3' (SEQ ID NO.61), HPV18 downstream primer: 5'-GGGCTGGTAAATGTTGATGAT-3' (SEQ ID NO.62), HPV18 probe: 5'-ROX-TAAGCGACTCAGAGGAAGAAAACGATG-MGB-3' (SEQ ID NO.62). IDNO.63); the primer-probe combination in the primer-probe mixture is obtained by mixing equal volumes of primer-probe combinations for each target; in the primer-probe combination for each target, the concentrations of probe, upstream primer and downstream primer are based on 100 μM, and the volume ratio of probe, upstream primer and downstream primer is 0.05:0.11:0.11; the rest are the same as in Example 3.

[0058] Example 5 The performance advantages of the multi-target droplet digital PCR method for detecting HPV16 and HPV18 of the present invention (Example 3) compared with the single-target detection method (Comparative Example 1) in low-abundance nucleic acid detection were verified.

[0059] Fragmented HPV16 and HPV18 nucleic acid samples were used, and different concentration gradients were set up. Under the same experimental conditions, the methods of Example 3 (multi-target detection system) and Comparative Example 1 (single-target detection system) were used for detection. The results are shown in Table 6.

[0060] Table 6 shows the detection results of the method in Example 3 (multi-target detection system) and the method in Comparative Example 1 (single-target detection system).

[0061] The results show that the multi-target synergistic detection provided by the present invention can significantly improve the ctDNA signal capture probability and achieve a 5-9 fold increase in detection signal, thereby effectively reducing the risk of detection instability caused by single-target detection due to target sequence deletion or low copy number.

[0062] Example 6 The specificity of the detection method of Example 3 of the present invention for HPV16 and HPV18 and the absence of cross-reactivity with other HPV types were verified.

[0063] The method described in Example 3 was used to detect negative control samples, mixed HPV samples of non-HPV16 and non-HPV18 types, and total cell-free DNA (cfDNA) extracted from negative plasma.

[0064] Twenty negative samples were tested, including eight NTC tests, eight NC tests, and four tests of mixtures of 12 HPV types (the 12 HPV mixtures consisted of a mixture of whole-genome nucleic acids from HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68). All samples were fragmented to simulate cfDNA. NC consisted of cfDNA extracted from negative plasma, and NTC consisted of nuclease-free water. A control group, labeled PC group, was used, consisting of a mixture of fragmented whole-genome nucleic acids from HPV16 and HPV18. The results are shown in Table 7.

[0065] Table 7 shows the results of testing 20 negative samples.

[0066] Meanwhile, the cfDNA samples extracted from 40 negative plasma (NC) samples were tested using the method in Example 3, and the results are shown in Table 8.

[0067] Table 8. Results of cfDNA testing in 40 negative plasma samples

[0068] The results show that the detection method of the present invention has good analytical specificity and can effectively avoid cross-reaction with other HPV types or human genomic DNA.

[0069] Example 7 Limit of Detection (LOD) Validation Objective: To determine the lowest stable detectable concentration of the droplet digital PCR detection method of the present invention (Example 3) in plasma samples.

[0070] Experimental materials and methods: Fragmented HPV16 and HPV18 nucleic acid samples were prepared into plasma samples of 1, 1.5, 2 and 3 copies / mL (cp / mL) after digital PCR determination. The samples were extracted 6 times using the Sinaf Free DNA Extraction Kit and the detection in Example 3 was repeated 6 times (2 mL plasma sample, 30 μL elution, 15 μL detection).

[0071] The results are shown in Table 9. It can be seen that the detection method of Example 3 of the present invention has a minimum detection limit of 1.5 copies / mL and 1.0 copies / mL for HPV16 and HPV18 ctDNA, respectively, which meets the requirements for trace detection of ctDNA.

[0072] Table 9. Limit of Detection (LOD) Verification Results

[0073] Example 8 Precision verification of detection Objective: To verify the quantitative repeatability and stability of the detection system of the present invention (Example 3) at different concentration levels.

[0074] Experimental materials and methods: High, medium and low concentration plasma samples were selected for repeated testing, and the coefficient of variation (CV) was calculated.

[0075] Experimental results: 1. Repeated validation was performed on HPV16 samples with different concentrations. A total of 6 tests were conducted on each sample. The absolute value of the logarithmic value of the sample concentration was calculated, and the CV was examined. The results are shown in Table 10.

[0076] Table 10. Replication validation results of HPV16 samples at different concentrations

[0077] Note: The conversion formula in the table is: [Detection value C (cp / μL) × reaction volume (22μL) / sample volume (15μL)] × 1000 × concentration factor.

[0078] 2. Repeated validation was performed on HPV18 samples with different concentrations. A total of 6 tests were conducted on each sample. The absolute value of the logarithmic value of the sample concentration was calculated, and the CV was examined. The results are shown in Table 11.

[0079] Table 11 Replication validation results of HPV18 samples at different concentrations

[0080] Note: The conversion formula in the table is: [Detection value C (cp / μL) × reaction volume (22μL) / sample volume (15μL)] × 1000 × concentration factor.

[0081] The results above show that the CV values ​​of the high and medium concentration values ​​are all <5% and the CV values ​​of the low concentration values ​​are all <10% in different concentration ranges, which meet the requirements for quantitative detection precision. This indicates that the detection method of the present invention has good repeatability and stability.

[0082] Example 9 Verification of detection accuracy Objective: To verify the consistency between the detection system of the present invention (Example 3) and clinical test results.

[0083] Experimental materials and methods: 37 cervical swab samples with known HPV typing results were used for multi-target droplet digital PCR detection in Example 3.

[0084] The test results are shown in Table 12. Table 12 Detection results of 37 cervical swab samples

[0085] HPV genotyping was performed on the 37 cervical swab samples using qPCR. Specifically, a commercially available, registered test kit (“High-Risk Human Papillomavirus Nucleic Acid (Genotyping) Detection Kit (PCR-Fluorescent Probe Method)”; Medical Device Registration Certificate No.: 20163401295) was used, and the test was conducted according to the product instructions. The qPCR results were compared with those in Table 12, and the comparison results are shown in Table 13.

[0086] Table 13 Comparison Results

[0087] The results show that the detection results of this invention are 100% consistent with clinical results, demonstrating high detection accuracy.

[0088] Example 10 Clinical negative plasma sample testing Objective: To verify the interpretation stability of the detection system of the present invention (Example 3) in clinical negative samples.

[0089] Experimental Materials and Methods: Cell-free DNA (cfDNA) extracted from 22 clinically negative plasma samples was detected using the method described in Example 3. Control groups were established: a first control group (PC) consisted of a mixture of fragmented HPV16 and HPV18 whole-genome nucleic acids; and a second control group (NC) consisted of nuclease-free water.

[0090] The results are shown in Table 14, indicating that the HPV16 and HPV18 tests were negative in the 22 negative samples, meeting the interpretation criteria.

[0091] Table 14. Detection results of cfDNA in 22 clinically negative samples.

[0092] Example 11 Consistency analysis of multiplex and singlex detection results Objective: To compare the quantitative consistency of the multiplex droplet digital PCR detection system (Example 3) and the single droplet digital PCR detection system (Comparative Example 1) in real samples.

[0093] Experimental materials and methods: 125 plasma samples from the same batch were tested using the detection systems of Example 3 (multiplex) and Comparative Example 1 (singleplex), and correlation analysis was performed.

[0094] The results of Example 3 (multiplex droplet digital PCR detection) were compared with those of Comparative Example 1 (single-multiplex droplet digital PCR detection) to evaluate consistency. The HPV16 type of the same sample was analyzed using both methods. Simultaneously, the consistency of the quantitative trends of the two detection methods was compared, and the results are shown in Table 15. Figure 2 As shown.

[0095] Table 15 Analysis of HPV16 types detected in the same samples using two different methods.

[0096] Depend on Figure 2 It can be seen that the regression equation for HPV16 viral load in singlet ddPCR and multiplet ddPCR is y=7.3435x-3.1657, R0 2 =0.997, R 2 A value close to 1 indicates a clear linear correlation between the viral load detected by the two methods. The viral load of HPV16 detected by multiplex ddPCR is 7.3435 times that of single-dip ddPCR. This is because multiplex ddPCR detects multiple targets simultaneously, which can more comprehensively capture the nucleic acid signals of all HPV in the sample, and the quantitative results are closer to the true HPV viral load. In contrast, single-dip ddPCR only detects a single target, which may underestimate the viral load due to the low copy number of the target.

[0097] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A primer-probe combination for droplet digital PCR detection of HPV16 and HPV18, characterized in that, This includes multi-target primer-probe combinations for detecting HPV16 with nucleotide sequences as shown in SEQ ID NO.1~SEQ ID NO.27, and multi-target primer-probe combinations for detecting HPV18 with nucleotide sequences as shown in SEQ ID NO.28~SEQ ID NO.

54.

2. The primer-probe combination according to claim 1, characterized in that, It also includes an internal standard primer-probe combination, the nucleotide sequence of which is shown in SEQ ID NO.55~SEQ ID NO.

57.

3. The primer-probe combination according to claim 2, characterized in that, The probe is modified with a fluorescent group at its 5' end and a quenching group at its 3' end; the fluorescent group includes VIC, FAM, HEX, ROX, CY5 and / or Cy5.5; the quenching group includes BHQ1, BHQ2, MGB and / or TAMRA.

4. The primer-probe combination according to claim 3, characterized in that, HPV16, HPV18, and the internal standard are distinguished based on the color of the fluorescent group modified by the probe.

5. The use of the primer-probe combination according to any one of claims 1 to 4 in the preparation of HPV16 and / or HPV18 detection products.

6. The application according to claim 5, characterized in that, The testing products include droplet digital PCR testing products.

7. The application according to claim 6, characterized in that, The reaction system of the droplet digital PCR detection product, in 22 μL increments, consists of 4.5 μL of PCR reaction mixture, 2.5 μL of primer-probe mixture, and 15 μL of DNA sample.

8. The application according to claim 7, characterized in that, The primer-probe mixture has a volume of 2.5 μL, consisting of 2.375 μL of the primer-probe combination and 0.125 μL of nuclease-free water. The primer-probe combination is obtained by mixing equal volumes of primer-probe combinations for each target site. In the primer-probe combination for each target site, the concentrations of the probe, upstream primer, and downstream primer are each 200 μM, and the volume ratio of the probe, upstream primer, and downstream primer is 0.025:0.05:0.

05.

9. The application according to claim 6, characterized in that, The reaction procedure for the droplet digital PCR detection product is as follows: droplet spreading at 60℃ for 5 min; enzyme activation at 95℃ for 5 min; denaturation at 95℃ for 20 s, annealing and extension at 63℃ for 30 s, repeated 5 times; denaturation at 95℃ for 20 s, annealing and extension at 60℃ for 30 s, repeated 40 times; final extension at 55℃ for 20 min.

10. A kit for detecting HPV16 and HPV18 using droplet digital PCR, characterized in that, The mixture includes a primer-probe mixture, a PCR reaction mixture, a positive control, a negative control, and an internal control; the positive control is a plasmid containing HPV16 and HPV18 target gene fragments; the negative control is nuclease-free water; the internal control is a plasmid containing an internal target gene fragment and having no cross-linking with HPV16 and HPV18; the primer-probe mixture includes the primer-probe combination as described in any one of claims 1 to 4 and nuclease-free water.

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