Method and kit for detecting HBV cccDNA in single liver cells based on droplet digital PCR

Abstract

The present invention discloses a method and a kit for detecting HBV cccDNA in a single liver cell based on droplet digital PCR. The detection method provided by the present invention comprises the following steps: 1) preparing a single cell suspension by enzymatic hydrolysis; 2) DNase I nuclease digestion; 3) laminar flow washing; 4) ddPCR amplification and detecting the droplet fluorescence signal; according to the droplet fluorescence signal, obtaining the proportion of HBV infected cells in the sample to be tested and / or the viral load of HBV infected cells; the method or kit for detecting HBV cccDNA by digital PCR of the present invention has the following beneficial effects: the sample to be tested is a patient's liver biopsy tissue, and the absolute quantification of HBV cccDNA in the liver can be achieved at the single cell level, and the proportion of infected cells can be obtained; the detection sensitivity is high, the specificity is strong, the reaction system is small, the reaction is rapid, and the throughput is high, which is convenient for clinical large sample detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and relates to a method and a kit for detecting HBV cccDNA in single liver cells based on droplet digital PCR. Background Art

[0002] Hepatitis B caused by hepatitis B virus (HBV) infection is a major threat to global public health. HBV infection is an important factor in the occurrence of liver cancer. 84% of liver cancer in my country is caused by HBV infection. HBV exists in the nucleus of infected liver cells in the form of covalently closed circular DNA (cccDNA) for a long time. It exists in the form of free mini-chromosomes and is extremely stable. cccDNA, as a template for HBV replication and transcription, is the biggest clinical challenge for persistent HBV infection and difficulty in complete cure (Werle-Lapostolle B. et al. 2004. Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy. Gastroenterology [J] 126 (7), 1750-1758. Fanning GC. et al. 2019. Therapeutic strategies for hepatitis B virus infection: towards a cure. Nat Rev Drug Discov [J] 18 (11), 827-844).

[0003] As the core link of HBV life cycle, cccDNA is extremely difficult to detect directly. Alternative serum virological tests have always been the main means of evaluating cccDNA levels. Quantification of HBV DNA in serum is mainly used to evaluate the level of viral replication in HBV-infected patients. It is an important clinical indicator for the selection of indications and judgment of efficacy of antiviral treatment, and is significantly correlated with intrahepatic cccDNA levels. However, even if the HBV DNA level in serum continues to be below the detection limit, cccDNA can still be detected in the patient's liver (Lai, CL et al. 2017. Reduction of covalently closed circular DNA with long-term nucleos(t)ide analogue treatment in chronic hepatitis BJ Hepatol 66, 275-281). In addition, quantification of hepatitis B surface antigen (HBsAg) can also reflect cccDNA activity, and the disappearance of HBsAg is the clinical cure goal of CHB. Studies have shown that long-term antiviral treatment significantly reduces HBsAg and cccDNA levels, accompanied by a significant decrease in the correlation between serum HBsAg and cccDNA. Recent studies have found that serum HBV RNA and hepatitis B core-related antigen (HBcrAg) levels can well reflect the replication and transcription of intrahepatic cccDNA when HBV DNA is below the detection limit. They can be used as alternative indicators to reflect the intrahepatic cccDNA transcription activity in CHB patients after virological suppression or disappearance of HBsAg. However, whether they can be used to guide the determination of clinical antiviral treatment discontinuation endpoints remains to be further explored (Mak, LY et al. 2018. Review article: hepatitis B core-related antigen (HBcrAg): an emerging marker for chronic hepatitis B virus infection. Aliment Pharmacol Ther 47, 43-54).Therefore, in clinical applications, directly detecting the cccDNA level in liver tissue is of great reference value for understanding HBV infection, infectivity, disease progression and prognosis (Allweiss, L. et al. 2023. Quantification of the hepatitis B virus cccDNA: evidence-based guidelines for monitoring the key obstacle of HBVcure. Gut 72, 972-983).

[0004] At present, the detection of cccDNA in liver biopsy tissue is mainly carried out at the tissue level (bulk). The specific method is to extract DNA from a part of liver biopsy tissue, and then use conventional Southern Blot, fluorescence quantitative PCR (qPCR), digital PCR (digital PCR, dPCR) and other methods for determination. In fact, what is obtained is the signal mean of a group of cells. The same signal cannot be distinguished from a small number of cells with high copy number virus infection or a large number of cells with low copy number infection; coupled with the errors in the starting cell amount and experimental operation, it cannot truly reflect the proportion of infected cells in the liver tissue and the viral load in the infected cells. At the same time, due to the DNA loss caused by steps such as DNA extraction, for hepatitis B patients with very low cccDNA levels in the liver (<1 copy / cell), especially functionally cured hepatitis B patients, the amount of DNA that can be obtained from normal liver biopsy tissue is difficult to achieve effective cccDNA detection. Therefore, it is urgent to measure cccDNA in liver biopsy tissue at the single cell level.

[0005] dPCR is an absolute quantitative technology for nucleic acid molecules. It mainly involves limiting dilution of PCR reactants, followed by PCR amplification in different reaction chambers, and finally deriving the starting copy number of the target molecule based on the Poisson distribution principle and the number and proportion of positive droplets. Compared with traditional fluorescent quantitative PCR, digital PCR has low sample requirements and high sensitivity, and can accurately detect low-copy or even single-copy mixed samples. At present, dPCR systems mainly include droplet digital PCR (ddPCR) and chamber-based digital PCR (cdPCR). ddPCR technology uses a droplet generator to generate tens of thousands or even millions of nanoliter or even picoliter single oil-in-water droplets at a time as sample dispersion carriers for digital PCR. Unlike cdPCR technology, which relies on flow channel size to separate the reaction system, ddPCR uses a water-oil two-phase split reaction system. Its flow channel size is large and the final reaction system volume is large, which is very suitable for the processing of single-cell suspensions and shows great advantages in the detection of single-cell specific genes or proteins (Fang W. et al. 2024. Digital PCR for Single-Cell Analysis. Biosensors (Basel). 14 (2): 64). However, there is still no solution for cccDNA detection in liver biopsy tissue at the single-cell level. Therefore, the development of a set of absolute quantitative detection methods for HBV cccDNA in liver tissue based on single-cell ddPCR is extremely important for the precise prevention and treatment of HBV. Summary of the invention

[0006] The main purpose of the present invention is to overcome the shortcomings and deficiencies of existing detection technologies, and to provide a method for detecting the absolute content of HBV cccDNA in liver biopsy tissue of patients with hepatitis B or liver cancer at the single-cell level, and to provide information in two dimensions: the proportion of HBV-infected cells in the patient's liver tissue and the viral load of the infected cells.

[0007] In a first aspect, the present invention provides a method for detecting HBV cccDNA in a single cell of a sample to be tested, comprising the following steps:

[0008] 1) preparing a single cell suspension from the sample to be tested by enzymatic hydrolysis;

[0009] 2) using DNase I nuclease to remove free nucleic acids in the supernatant of the single cell suspension to be tested, to obtain a cell suspension to be tested treated with DNase I nuclease;

[0010] 3) washing the cell suspension to be tested after being treated with DNase I nuclease by laminar flow washing to obtain a washed cell suspension;

[0011] 4) using a ddPCR system to encapsulate the washed cell suspension and the ddPCR amplification system into droplets to form microdroplets; then performing ddPCR amplification on the microdroplets and detecting the microdroplet fluorescence signal; and obtaining the proportion of HBV-infected cells in the sample to be tested and / or the viral load of HBV-infected cells according to the microdroplet fluorescence signal;

[0012] The ddPCR amplification system includes a β-actin primer pair, a β-actin probe, a cccDNA primer pair and a cccDNA probe;

[0013] The cccDNA primer pair is composed of a single-stranded DNA molecule shown in sequence 4 and a single-stranded DNA molecule shown in sequence 5; the nucleotide sequence of the cccDNA probe is sequence 6, and both ends of the probe are connected to a fluorescent group and a quenching group;

[0014] The β-actin primer pair is composed of a single-stranded DNA molecule shown in sequence 1 and a single-stranded DNA molecule shown in sequence 2; the nucleotide sequence of the β-actin probe is sequence 3, and two ends of the probe are connected to another fluorescent group and the quenching group.

[0015] In the embodiment of the present invention, the fluorescent group of the cccDNA probe is FAM, and the quenching group is BHQ1;

[0016] The fluorescent group of the β-actin probe is VIC, and the quenching group is BHQ1;

[0017] In the above method, in step 1), the enzymes used in the enzymatic hydrolysis method include Collagenase I, Dispase II and DNase I.

[0018] The single cell suspension to be tested is prepared from the sample to be tested by the enzymatic method according to the following method: the sample to be tested is enzymatically hydrolyzed with Collagenase I, Dispase II and DNase I to obtain a single cell suspension to be tested;

[0019] In an embodiment of the present invention, the ratio of the Collagenase I, Dispase II and DNase I enzymes and the sample to be tested is 250 μL Collagenase I (20 mg / mL; Sigma, C0130, ≥125 CDU / mg solid): 100 μL Dispase II (100 mg / mL; Sigma, D4693, ≥0.5 units / mg solid): 50 μL DNase I (100 μg / mL; Roche, 10104159001): 0.5 g (not more than 0.5 g).

[0020] The above enzymatic hydrolysis reaction system also includes RPMI1640 containing 10% fetal bovine serum;

[0021] The enzymatic hydrolysis conditions are: 5% CO2, 37°C, 160RPM, 0.5-2h (until the tissue blocks disappear visible to the naked eye).

[0022] In step 2) above, the DNase I nuclease digestion system for removing free nucleic acids includes 4 μL of DNase I nuclease (100 μg / mL; Roche, 10104159001) and 50 μL of the single cell suspension to be tested (200,000 cells); the digestion conditions are: 5% CO2, 37°C, 160RPM, 15 min.

[0023] In step 3) above, the purpose of the laminar flow washing is to remove the residual DNase I in the supernatant of the cell suspension to be tested after the DNase I nuclease treatment. I. Impurities such as nucleases and cell debris;

[0024] Specifically, the cell suspension to be tested after DNase I nuclease treatment was transferred to the wells of a laminar flow washing plate (Curiox Biosystems, 16-DC-CL-10 or 96-DC-CL-05), and settled for 30 minutes at 4°C. The washing solution was PBS (Gibco, 10010023). The laminar flow washing program was: Cycles--28, volume--80 μL, flow rate--12.

[0025] In step 4) above, the washed cell suspension and the ddPCR amplification system are encapsulated into droplets using reagents required for droplet generation (including droplet generation oil and sealant), specifically: 50 μL of droplet generation oil (Yongnuo Bio, S0100010201) is added to the oil well of the droplet generation chip (Yongnuo Bio, S0100010201), the ddPCR reaction system (ddPCR amplification system) is transferred to the sample well of the droplet generation chip, and then 5 μL of sealant (Yongnuo Bio, S0100010201) is added to each sample well, and the chip is placed in a droplet generator (Yongnuo Bio, MicroDrop-100A) to produce droplets to obtain droplets encapsulating single cells and ddPCR reaction solution.

[0026] The ddPCR amplification system includes a β-actin primer pair, a β-actin probe, a cccDNA primer pair and a cccDNA probe, a single-cell droplet digital PCR reaction premix (2X, Yongnuo Bio, S0200022901), a membrane-breaking agent (which may be other reagents for rupturing cell membranes, such as SDS, etc.), 5x KAPA Enhancer 1 (Sigma, KK5517) and dNTP (NEB, N0447S);

[0027] In the ddPCR amplification system, the molar ratio of each primer in the cccDNA primer pair, the cccDNA probe, each primer in the β-actin primer pair, and the β-actin probe is 900:900:300:900:900:300.

[0028] In the above, according to the droplet fluorescence signal, the proportion of HBV-infected cells in the sample to be tested and / or the viral load of HBV-infected cells are analyzed, and the amount of cccDNA and β-actin in a single droplet are quantitatively standardized respectively; further, β-actin is used to calibrate whether the droplet contains cells, and cccDNA is used to calibrate whether there is cccDNA in the cell. And standardize with the housekeeping gene β-actin, and finally achieve absolute quantification of cccDNA in a single cell. Further details are:

[0029] The fluorescence value of each droplet detected by the instrument is compared with the fluorescence value of the negative control. The droplets in the FAM channel that are higher than the fluorescence value of the negative control are cccDNA. + The droplet above the VIC channel is β-actin + Micro droplets.

[0030] Statistics cccDNA + β-actin + Double positive microdroplets and cccDNA - β-actin+ Single positive droplet number.

[0031] According to cccDNA + β-actin + Double positive microdroplets and cccDNA - β-actin + The number of single positive droplets was calculated, the proportion of cells containing cccDNA was calculated, and the cccDNA was further calculated. + The number of cccDNA copies in cells.

[0032] cccDNA + Cell ratio = cccDNA + β-actin + Number of droplets / (cccDNA + β-actin + Droplet number + cccDNA - β-actin + Number of droplets)*100%

[0033] Taking β-actin as the internal reference, its actual copy number defaults to 2 (except for cancer samples):

[0034] Average cccDNA content = (cccDNA copies / 20μL) / (β-actin copies / 20μL)*2;

[0035] cccDNA + Cell cccDNA content = (cccDNA copies / 20μL) / (β-actin copies / 20μL*cccDNA + β-actin + Cell ratio)*2.

[0036] The cccDNA copies / 20 μL and β-actin copies / 20 μL in the above formula are the total cccDNA copy number and β-actin total copy number of all droplets obtained by the droplet detector.

[0037] The proportion of HBV infected cells was determined by cccDNA + Cell ratio expression;

[0038] The viral load of HBV infected cells is the average HBV viral load in a single cell of HBV infected cells. + The cell cccDNA content is reflected.

[0039] The number of single cells in the droplet is no more than 2000, that is, the number of single cells in the ddPCR amplification reaction system is no more than 2000.

[0040] The above-mentioned ddPCR amplification reaction system is specifically as follows: single-cell droplet digital PCR reaction premix (2X) 10 μL, 20 μM β-actin primer (F+R) 0.9 μL, 20 μM β-actin probe 0.3 μL, 20 μM cccDNA primer (F+R) 0.9 μL, 20 μM cccDNA probe 0.3 μL, membrane permeabilization agent 0.6 μL, 5x KAPA Enhancer1 plus 2.6 μL, 10 mM dNTP plus 0.4 μL, single-cell suspension 4 μL;

[0041] The conditions of the above ddPCR reaction were as follows: membrane permeabilization at 85°C for 60 min, pre-denaturation at 95°C for 10 min, denaturation at 95°C for 30 s, annealing and extension at 60°C for 1 min, for a total of 55 cycles, and the products were stored at 16°C.

[0042] In the above method, the sample to be tested is an in vitro sample, specifically a liver cancer tissue or liver tissue or a cell line containing HBV cccDNA or a cell line suspected of containing HBV cccDNA.

[0043] In the above method, the detection is quantitative detection.

[0044] In a second aspect, the present invention provides a kit for detecting HBV cccDNA in a single cell of a sample to be tested, comprising the following 1), or 1) and 2):

[0045] 1) the cccDNA primer pair, the cccDNA probe, the β-actin primer pair and the β-actin probe described in the first aspect;

[0046] Among them, the molar ratio of the two primers in the cccDNA primer pair, the cccDNA probe, the two primers in the β-actin primer pair and the β-actin probe is 900:900:300:900:900:300.

[0047] 2) DNase I endonuclease.

[0048] The kit described above also includes reagents required for ddPCR amplification;

[0049] Furthermore, the reagents required for the ddPCR amplification include a single-cell droplet digital PCR reaction premix (2X, Yongnuo Bio, S0200022901), a membrane disrupting agent (which can be other reagents for disrupting cell membranes, such as SDS, etc.), 5x KAPA Enhancer 1 (Sigma, KK5517) and dNTP (NEB, N0447S).

[0050] The kit may further include reagents required for droplet generation, specifically including droplet generation oil and a sealant.

[0051] The above-mentioned kit may further include reagents required for the enzymatic hydrolysis method, wherein the reagents required for the enzymatic hydrolysis method include Collagenase I, Dispase II and DNase I.

[0052] The above-mentioned kit may also include a positive quality control sample and a negative quality control sample.

[0053] The positive quality control sample is a DNA sample extracted from HepG2.2.15 cells.

[0054] The negative control sample may include PBS, and may also include PBMC cells from healthy subjects.

[0055] The detection may be a quantitative detection.

[0056] In the above-mentioned kit, the sample to be tested is an in vitro sample, specifically liver cancer tissue or liver tissue or a cell line containing HBV cccDNA or a cell line suspected of containing HBV cccDNA.

[0057] In a third aspect, the present invention provides a use of the kit described in the second aspect or any substance or any combination of substances in the kit in the preparation of a product for diagnosing or assisting in the diagnosis of HBV-infected patients.

[0058] The product described above is a kit.

[0059] The HBV cccDNA quantitative detection ddPCR kit of the present invention is divided into two parts: a detection system and a monitoring system. The detection system includes the above two primer pairs and corresponding probes. The monitoring system includes: (1) positive quality control, a DNA sample extracted from HepG2.2.15 cells. Under normal circumstances, the positive quality control should show positive detection of cccDNA and β-actin as a criterion for judging whether the reagents of the kit are qualified. If the positive quality control does not show a positive signal, it means that there is a problem with the quality of the reagents. It should be noted that in order to avoid nucleic acid contamination, the positive control does not need to be set up for each experiment, and is only used when the reagent performance is regularly tested. It is generally recommended to test it once every two months, or once a new reagent is opened. (2) Negative quality control, including PBS, can also include PBMC single cell suspension of healthy people, and one of the two is set as a negative control during the experiment. For PBMC negative control, under normal circumstances, only β-actin is detected but no HBV cccDNA is detected. If HBV cccDNA positive droplets appear in the negative quality control during droplet scanning, it indicates that there is contamination during the sample addition process. For the PBS negative control, under normal circumstances, no β-actin and HBV cccDNA are detected. If positive droplets appear in the negative control during droplet scanning, it indicates that there is contamination in the reagents or the experimental process.

[0060] The method or kit for detecting HBV cccDNA by digital PCR of the present invention has the following beneficial effects: (1) The sample to be tested is a liver biopsy tissue of a patient, and the absolute quantification of HBV cccDNA in the liver can be achieved at the single-cell level, and the proportion of infected cells can be obtained; (2) The detection sensitivity is high and the specificity is strong, which greatly improves the sensitivity and accuracy of existing detection methods; (3) The reaction system is small (20 μL), and the amount of sample to be tested and ddPCR reagent is small; (4) The reaction is rapid, and the experimental results can be obtained in 5 hours, which is convenient for disease diagnosis; (5) High throughput, which is convenient for large clinical sample detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 The experimental process and results of preparing liver single cell suspension by enzymatic hydrolysis in Example 1. (A) The operation process of enzymatic hydrolysis to dissociate liver tissue; (B) The state of cells after dissociation (bright field); (C) The result of dissociated cells after staining with AOPI.

[0062] Figure 2The results of Example 3 HBV cccDNA and β-actin annealing temperature optimization and ddPCR specific detection of cccDNA are shown in Figure 3. The X-axis is the total number of droplets, the Y-axis is the fluorescence signal intensity, and the horizontal solid line (red line) is the fluorescence signal threshold. Droplets with fluorescence signal intensity greater than the threshold are positive droplets, i.e., green / blue droplets; droplets with fluorescence signal intensity less than the threshold are negative droplets, i.e., gray droplets. (A) The annealing temperature of cccDNA and β-actin primers was optimized using HepG2.2.15 cells. The upper figure shows the optimization result of β-actin primer annealing temperature, and the fluorescence signal threshold of β-actin is 3369; the lower figure shows the optimization result of cccDNA primer annealing temperature; the threshold of cccDNA is 2880. (B) After optimizing the annealing temperature, the results of ddPCR specific detection of cccDNA in DNA extracted from HepG2.2.15, using PBS as a blank control, the upper figure shows the fluorescence signal value of β-actin; the lower figure shows the fluorescence signal value of cccDNA.

[0063] Figure 3 The working principle diagram of single-cell ddPCR in Example 4 and the results of cccDNA content detection in HepG2.2.15 cells. (A) The upper figure shows a schematic diagram of the ddPCR workflow, and the lower figure shows the actual situation of droplets encapsulating single cells. (B) ddPCR performs absolute quantification of cccDNA in HepG2.2.15 single cells. The X-axis is the total number of droplets, the Y-axis is the fluorescence signal intensity, and the horizontal solid line (red line) is the fluorescence signal threshold; droplets with fluorescence signal intensity greater than the threshold are positive droplets, i.e., blue / green droplets; droplets with fluorescence signal intensity less than the threshold are negative droplets, i.e., gray droplets. The upper figure shows the β-actin gene detection results in HepG2.2.15 single cells, and the lower figure shows the cccDNA detection results. (C) A two-dimensional diagram of the β-actin and cccDNA detection results in HepG2.2.15 single cells. In this experiment, PBMC was set as a negative control and PBS was set as a blank control.

[0064] Figure 4The results of ddPCR detection of cccDNA content in single cells of tumor tissues of clinical liver cancer patients in Example 5. (A) Single-cell ddPCR technology is used to absolutely quantify cccDNA in tumor tissues of liver cancer patients. The X-axis is the total number of droplets, the Y-axis is the fluorescence signal intensity, and the horizontal solid line (red line) is the fluorescence signal threshold; droplets with fluorescence signal intensity greater than the threshold are positive droplets, i.e., green / blue droplets; droplets with fluorescence signal intensity less than the threshold are negative droplets, i.e., gray droplets; the upper figure is the β-actin gene detection result in single tumor cells, and the lower figure is the cccDNA detection result. (B) Two-dimensional graph of β-actin and cccDNA detection results in single tumor cells. In this experiment, PBMC was set as a negative control and PBS as a blank control.

[0065] Figure 5 The results of cccDNA content detection in single cells of liver biopsy tissue of hepatitis B patients in Example 6. (A) Single-cell ddPCR technology is used to absolutely quantify cccDNA in liver tissue of hepatitis B patients. The X-axis is the total number of droplets, the Y-axis is the fluorescence signal intensity, and the horizontal solid line (red line) is the fluorescence signal threshold; droplets with fluorescence signal intensity greater than the threshold are positive droplets, i.e., blue / green droplets; droplets with fluorescence signal intensity less than the threshold are negative droplets, i.e., gray droplets; the upper figure is the β-actin gene detection result in single cells of liver tissue, and the lower figure is the cccDNA detection result. (B) Two-dimensional graph of β-actin and cccDNA detection results in single cells of liver tissue. In this experiment, PBMC was set as a negative control and PBS was set as a blank control.

[0066] Figure 6 Graphs showing the specificity detection results of primers in the ddPCR reaction system in Example 7, (A) is the melting curve of the β-actin primer amplification product, and (B) is the melting curve of the cccDNA primer amplification product. DETAILED DESCRIPTION

[0067] The present invention is further described in detail below in conjunction with specific embodiments, and the embodiments provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The embodiments provided below can be used as a guide for further improvement by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0068] The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified. The quantitative experiments in the following examples are repeated twice unless otherwise specified.

[0069] Example 1: Preparation of liver single cell suspension by enzymatic hydrolysis

[0070] Figure 1 This is the experimental process and results of preparing liver single cell suspension by enzymatic hydrolysis in Example 1.

[0071] 1. Tissue preservation and transportation: The samples (biopsy tissue samples from liver puncture surgery or liver cancer tissue samples removed after liver cancer surgery in the Affiliated Hospital of Yunnan University) were completely immersed in pre-cooled The samples were transported on ice in a Tissue Storage Solution (<8°C, Mitenyl Biotec, 130-100-008) to avoid bubbles and prolonged contact with air.

[0072] 2. Tissue pretreatment: Wash the tissue with pre-cooled culture medium (RPMI1640 + 10% FBS by volume); immerse the tissue in 500 μL culture medium and cut the tissue into pieces of about 2-4 mm with scissors. 3 of small pieces.

[0073] 3. Tissue dissociation: Transfer an appropriate amount (less than 500 mg) of tissue blocks to a T25 cell culture flask, add culture medium to 4.5 mL, add 250 μL Collagenase I (20 mg / mL; Sigma, C0130, ≥125 CDU / mg solid), 100 μL Dispase II (100 mg / mL; Sigma, D4693, ≥0.5 units / mg solid), and 50 μL DNase I (100 μg / mL; Roche, 10104159001), respectively, and dissociate for 0.5-2 h at 5% CO2, 37°C, and 160 RPM until the tissue blocks disappear as seen with the naked eye.

[0074] 4. Cell washing: Filter the cell suspension with a 70μm Cell Strainer. Centrifuge the filtrate at 400g for 5 minutes at 4℃, remove the supernatant, and collect the cell precipitate.

[0075] 5. Red blood cell lysis: Use appropriate amount of eBioscience TM The red blood cells in the above cell pellet were lysed with 1X red blood cell lysis buffer (Invitrogen, 00-4333-57). After terminating the reaction, the mixture was centrifuged at 500 g for 5 min at 4°C, the supernatant was removed, the cell pellet was collected, and the cells were resuspended in an appropriate amount of culture medium (RPMI 1640 + 10% FBS by volume) to obtain a single cell suspension.

[0076] 6. Determination of cell concentration and viability: Use Countstar counter to determine cell concentration and viability by AOPI method.

[0077] Table 1 shows the cell concentration and viability after dissociation of 10 clinical samples

[0078]

[0079]

[0080] Example 2. Design and synthesis of ddPCR primer probes and establishment of methods

[0081] 1. Design and synthesis of ddPCR primer probes

[0082] The primers were designed by the following method: the HBV whole genome sequence was downloaded from the public nucleic acid database. First, the Primer Premier 6.0 software was used to design the cccDNA primer pair and probe across the double gaps DR1 and DR2 of relaxed circular DNA (rcDNA) for the HBV genome sequence. Secondly, the primer pair and probe of β-actin were designed based on the gene sequence of β-actin. Then, the primers and probes were specifically compared by the NCBI-prime Blast online tool (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ). Through experimental screening and verification, primer pairs and probes with strong specificity, sensitivity and amplification efficiency were obtained.

[0083] To improve the specificity of HBV cccDNA amplification, a cccDNA primer pair spanning the double gap of rcDNA and the corresponding fluorescent probe were designed as follows:

[0084] cccDNA-F: 5'-CTTCTCATCTGCCGGACC-3'

[0085] cccDNA-R: 5'-AGTATGCCTCAAGGTCGGTCGT-3'

[0086] cccDNA-probe: 5'-fluorophore-CTCTGCACGTCGCATGGAGAC-quencher-3';

[0087] The housekeeping gene preferably selects the β-actin gene. The primer pair and probe of β-actin are used to quantitatively detect the content of β-actin, and the sequence is:

[0088] β-actin-F: 5'-GCTAAGTCCTGCCCTCATTTC-3'

[0089] β-actin-R: 5'-ATCTTCATTGTGCTGGGTGCC-3'

[0090] β-actin-probe: 5'-fluorescent group-TGACGTGGACATCCGCAAAGAC-quencher group-3'.

[0091] Therefore, the nucleotide sequences of the primers and probes after selecting appropriate fluorescent groups and quenching groups are shown in Table 2 below:

[0092] Table 2 shows the primer and probe sequences

[0093]

[0094] 2. Establishment of cccDNA detection method

[0095] 1. Collect cells

[0096] If the sample to be tested is a cell, the cell is cultured, and when the coverage of the cells to be tested in the T25 cell culture flask reaches 80%, the original culture medium is discarded, 1 mL of PBS is added for washing once, and then 1 mL of 0.25% Trypsin-EDTA Solution (Gibco, 25200056) is added, and the T25 cell culture flask is placed in a 37°C CO2 cell incubator for digestion for 3 min, and 2 mL of FBS-containing culture medium (RPMI 1640 + 10% FBS by volume) is added to terminate the digestion, and the cells are dispersed by gently blowing with a pipette tip to collect the single cell suspension.

[0097] If the sample to be tested is a liver biopsy tissue sample or a liver cancer tissue sample, a single cell suspension is prepared according to the method of Example 1.

[0098] After obtaining a single cell suspension, the cells can be washed twice with PBS, and then 500 μL PBS is added to resuspend the cells to obtain a single cell suspension and perform the following steps. Alternatively, the following steps can be performed directly:

[0099] The cell viability and concentration of the cell suspension to be tested were measured in CountStar.

[0100] 2. Treat the single cells to be tested with DNase I nuclease:

[0101] Take 50 μL of the above-mentioned cell suspension to be tested (200,000 cells) and place it in a new 1.5 mL EP tube, add 4 μL of DNase I nuclease (100 μg / mL; Roche, 10104159001), use a pipette to mix the DNase I nuclease and the 54 μL system of the cell suspension to be tested, place the EP tube in an incubator at 37°C, 160 RPM, digest for 30 minutes, and obtain the cell suspension to be tested after being treated with DNase I nuclease.

[0102] 3. Laminar flow washing: Use laminar flow washing to remove impurities such as residual DNase I nuclease and cell debris in the supernatant of the cell suspension to be tested, as follows:

[0103] The cell suspension to be tested after DNase I nuclease treatment was transferred to the wells of a laminar flow washing plate (Curiox Biosystems, 16-DC-CL-10 or 96-DC-CL-05), and allowed to settle for 30 min at 4°C. The washing solution was PBS (Gibco, 10010023). The laminar flow washing program was: Cycles--28, volume--80 μL, flow rate--12.

[0104] 4. Cell suspension recovery and cell counting: After laminar flow washing is completed, 25 μL of cell suspension remains in the well. Add 25 μL of new PBS, gently blow to resuspend the cells, transfer them to a 1.5 mL centrifuge tube, and finally adjust the volume to 100 μL to obtain a single cell suspension after washing.

[0105] The cell viability and concentration of the washed single cell suspension were measured in CountStar and the concentration was adjusted to ≤500 cells / μL.

[0106] 5. Preparation of ddPCR reaction system

[0107] According to the system shown in Table 3 below: The washed single cell suspension obtained in step 4 above is used to prepare a ddPCR reaction system according to the system shown in Table 3 below, wherein the number of single cells does not exceed 2000 single cells.

[0108] Table 3 shows the ddPCR reaction system

[0109]

[0110]

[0111] The single cell suspension in Table 3 was replaced with PBS (Gibco, 10010023) as a negative control.

[0112] 6. Preparation of microdroplets

[0113] Add 50 μL of droplet generation oil (Yongnuo Bio, S0100010201) to the oil well of the droplet generation chip (Yongnuo Bio, S0100010201), transfer the ddPCR reaction system prepared in step 5 above into the sample well of the droplet generation chip, then add 5 μL of sealant (Yongnuo Bio, S0100010201) to each sample well, place the chip in a droplet generator (Yongnuo Bio, MicroDrop-100A) to produce droplets, and obtain droplets encapsulating single cells and ddPCR reaction solution.

[0114] 7. ddPCR amplification

[0115] The prepared droplets were transferred to a 96-well PCR reaction plate using a low-adsorption pipette tip and heat-sealed with a matching aluminum film (190°C, 5 sec). ddPCR amplification was performed in a PCR instrument. To ensure the stability of each droplet, the heating rate of the sample well was set to no higher than 2°C / sec. The ddPCR amplification reaction program is shown in Table 4 below:

[0116] Table 4 shows the ddPCR amplification procedure

[0117]

[0118] 8. Droplet detection and result calculation and analysis

[0119] After ddPCR amplification, the reaction plate was placed in a matching microdroplet detector (Yongnuo Bio, MicroDrop-100B) for detection of FAM and VIC channels, and cccDNA was analyzed and calculated using the Poisson distribution principle. + and β-actin + Droplet number and copy number of both.

[0120] The details are as follows:

[0121] The amplified product was scanned for droplet analysis, and the number of droplets was detected by the copy number detection method. The total number of droplets prepared in each well must exceed 50,000. The negative control product did not have positive droplets, indicating that the experiment was successful. The fluorescence value of each droplet detected by the instrument was compared with the fluorescence value of the negative control (here, the blank control PBS). The droplets in the FAM channel that were higher than the fluorescence value of the negative control were cccDNA+ droplets, and the droplets in the VIC channel that were higher than the fluorescence value of the negative control were β-actin+ droplets.

[0122] Statistics cccDNA + β-actin + Double positive microdroplets and cccDNA - β-actin + Single positive droplet number.

[0123] According to cccDNA + β-actin + Double positive microdroplets and cccDNA - β-actin + The number of single positive droplets was calculated, the proportion of cells containing cccDNA was calculated, and the cccDNA was further calculated. + The number of cccDNA copies in cells.

[0124] cccDNA + Cell ratio = cccDNA+ β-actin + Number of droplets / (cccDNA + β-actin + Droplet number + cccDNA - β-actin + Number of droplets)*100%

[0125] Taking β-actin as the internal reference, normal human cells are diploid:

[0126] Average cccDNA content = (cccDNA copies / 20μL) / (β-actin copies / 20μL)*2;

[0127] cccDNA + Cell cccDNA content = (cccDNA copies / 20μL) / (β-actin copies / 20

[0128] μL*cccDNA + β-actin + Cell ratio)*2.

[0129] The cccDNA copies / 20 μL and β-actin copies / 20 μL in the above formula are the total cccDNA copy number and β-actin total copy number of all droplets obtained by the droplet detector.

[0130] 3. cccDNA detection kit

[0131] The single-cell HBV cccDNA detection ddPCR kit of the present invention comprises a system required for HBV cccDNA quantitative detection;

[0132] The system required for the quantitative detection of HBV cccDNA includes a cccDNA primer pair and a cccDNA probe, and a housekeeping gene β-actin primer pair and a β-actin probe;

[0133] The system required for the quantitative detection of HBV cccDNA also includes: DNase I nuclease (Roche, 10104159001), and reagents required for ddPCR amplification;

[0134] The reagents required for ddPCR amplification are specifically single-cell droplet digital PCR reaction premix (2X, Yongnuo Bio, S0200022901), membrane permeabilization agent (which can be other types of membrane permeabilization agents, such as SDS, etc.), 5xX KAPA Enhancer 1 (Sigma, KK5517), and dNTP (NEB, N0447S).

[0135] The kit also includes reagents required for droplet generation (Yongnuo Bio, S0200022901), specifically including droplet generation oil and sealant.

[0136] The above-mentioned kit also includes a system required for dissociating liver tissue into single cells, and the above-mentioned system required for dissociating liver tissue into single cells specifically includes Collagenase I, Dispase II and DNase I.

[0137] The above-mentioned kit may also include cccDNA negative quality control products and positive quality control products;

[0138] Negative quality control products include PBS (Gibco, 10010023) and PBMC from healthy individuals.

[0139] The positive quality control product was DNA extracted from HepG2.2.15 cells containing cccDNA.

[0140] Example 3. Optimization of ddPCR primer probe annealing temperature and detection of cccDNA positive quality control products

[0141] 1. Optimization of ddPCR primer probe annealing temperature

[0142] HepG2.2.15 cells were used as samples for primer-probe annealing temperature optimization.

[0143] 1. Collect cells: same as Example 2.

[0144] 2. Treating the single cell to be tested with DNase I nuclease: the same as in Example 2.

[0145] 3. Laminar flow washing: same as Example 2.

[0146] 4. Cell suspension recovery and cell counting: same as Example 2.

[0147] 5. Preparation of ddPCR reaction system: same as Example 2.

[0148] 6. Preparation of microdroplets: same as Example 2.

[0149] 7. ddPCR amplification:

[0150] Use a low-adsorption pipette tip to transfer the droplets prepared in step 6 to a matching 96-well PCR reaction plate, and heat seal it with a matching aluminum film (190°C, 5sec), and perform ddPCR amplification in a PCR instrument. To ensure the stability of each droplet, the heating rate of the sample well is set to no more than 2°C / sec. The ddPCR amplification reaction program is shown in Table 5 below:

[0151] Table 5 shows the ddPCR amplification procedure

[0152]

[0153] 8. Droplet detection and result calculation and analysis

[0154] After ddPCR amplification, the reaction plate was placed in a matching droplet detector for detection of FAM and VIC channels. The cccDNA was analyzed and calculated according to the method of Example 2 using the Poisson distribution principle. + and β-actin + Number of droplets.

[0155] The results are as follows Figure 2 As shown in A, the upper figure is the optimization result of β-actin primer annealing temperature, and the threshold of β-actin fluorescence signal is 3369; the lower figure is the optimization result of cccDNA primer annealing temperature; the threshold of cccDNA is 2880. It can be seen that the positive and negative boundary of the fluorescence signal of β-actin and cccDNA is the most obvious at 55.3℃. Therefore, the optimal annealing temperature is 55.3℃, and the general value is 55℃.

[0156] 2. cccDNA positive quality control product detection

[0157] DNA extracted from HepG2.2.15 cells was used as a positive quality control.

[0158] 1. Collect cells

[0159] When the coverage rate of HepG2.2.15 cells in the T25 cell culture flask reached 80%, the original culture medium was discarded, 1 mL of PBS was added for washing once, and then 1 mL of 0.25% Trypsin-EDTA Solution (Gibco, 25200056) was added, and the T25 cell culture flask was placed in a 37°C CO2 cell incubator for digestion for 3 min. 2 mL of FBS-containing culture medium (RPMI 1640 + 10% FBS by volume) was added to terminate the digestion, and the cells were dispersed by gently blowing with a pipette tip, and the cell suspension was collected in a 1.5 mL EP tube, centrifuged at 500 g for 5 min, and the supernatant was discarded.

[0160] 2. DNA Extraction

[0161] DNA was extracted using the “Blood / Cell / Tissue Genomic DNA Extraction Kit (Spin Column Type) (DP304)” produced by Tiangen Biochemical Technology Co., Ltd.

[0162] 3. Prepare the ddPCR reaction system according to the system shown in Table 6 below.

[0163] Table 6 shows the ddPCR reaction system

[0164]

[0165] 4. Preparation of microdroplets: Same as Example 2.

[0166] 5. ddPCR amplification:

[0167] Use a low-adsorption pipette tip to transfer the droplets prepared in step 2 to a dedicated 96-well PCR reaction plate, and heat seal it with a matching aluminum film (190°C, 5sec), and perform ddPCR amplification in a PCR instrument. To ensure the stability of each droplet, the heating rate of the sample well is set to no more than 2°C / sec. The ddPCR amplification reaction program is shown in Table 7 below:

[0168] Table 7 shows the ddPCR amplification procedure

[0169]

[0170] 6. Droplet detection and result calculation and analysis

[0171] After ddPCR amplification, the reaction plate was placed in a matching droplet detector for detection of FAM and VIC channels. The cccDNA was analyzed and calculated according to the method of Example 2 using the Poisson distribution principle. + and β-actin + Droplet number and copy number.

[0172] The results are shown in Table 8 and Figure 2 As shown in B.

[0173] Table 8 shows the results of cccDNA detection in HepG2.2.15 DNA

[0174]

[0175] Note: β-actin is used as the internal reference, and its actual copy number defaults to 2, cccDNA content = (cccDNA copies / 20μL) / (β-actin copies / 20μL)*2. Both cccDNA copies / 20μL and β-actin copies / 20μL are obtained by instrument reading.

[0176] Example 4: Single-cell ddPCR specific detection of HBV cccDNA positive cell ratio and cccDNA content in HepG2.2.15 cells

[0177] 1. Collect cells: When the coverage rate of the cells to be tested in the T25 cell culture flask reaches 80%, discard the original culture medium, add 1 mL PBS to wash once, then add 1 mL 0.25% Trypsin-EDTA Solution (Gibco, 25200056), place the T25 cell culture flask in a 37°C CO2 cell incubator for digestion for 3 min, add 2 mL of FBS-containing culture medium to terminate digestion, gently blow with a pipette tip to disperse the cells, collect the cell suspension in a 1.5 mL EP tube, 500g, centrifuge for 5 min, and discard the supernatant.

[0178] The cells were washed twice with PBS, and finally 500 μL of PBS was added to resuspend the cells to obtain HepG2.2.15 cell suspension.

[0179] The cell viability and concentration of HepG2.2.15 cell suspension were measured in CountStar.

[0180] The above-mentioned cells to be tested are as follows: HepG2.2.15 sample 1, HepG2.2.15 sample 2, 20% PBMC+80% HepG.2.15, 40% PBMC+60% HepG.2.15, 50% PBMC+50% HepG.2.15, 60% PBMC+40% HepG.2.15, 80% PBMC+20% HepG.2.15.

[0181] PBMC was used as negative control and PBS was used as blank control.

[0182] HepG2.2.15 sample 1 and HepG2.2.15 sample 2 are two experiments of HepG2.2.15 cell line.

[0183] 20% PBMC+80% HepG.2.15 is prepared by mixing the above HepG2.2.15 cell suspension and PBMC cell suspension at a cell number percentage of 80% and 20%.

[0184] The buffer of each of the above cell suspensions is PBS.

[0185] 40% PBMC+60% HepG.2.15 is prepared by mixing the above HepG2.2.15 cell suspension and PBMC cell suspension at a cell number percentage of 60% and 40%.

[0186] 50% PBMC+50% HepG.2.15 is prepared by mixing the above HepG2.2.15 cell suspension and PBMC cell suspension at a cell number percentage of 50% and 50%.

[0187] 60% PBMC+40% HepG.2.15 is prepared by mixing the above HepG2.2.15 cell suspension and PBMC cell suspension at cell number percentages of 40% and 60%.

[0188] 80% PBMC+20% HepG.2.15 is prepared by mixing the above HepG2.2.15 cell suspension and PBMC cell suspension at cell number percentages of 20% and 80%.

[0189] 2. Treat the single cells to be tested with DNase I nuclease: Take 50 μL of HepG2.2.15 cell suspension (200,000 cells) and place it in a new 1.5 mL EP tube, add 4 μL of DNase I nuclease (100 μg / mL; Roche, 10104159001), mix the DNase I nuclease and the HepG2.2.15 single cell suspension with a pipette tip, place the EP tube in an incubator at 37°C, 160 RPM, digest for 30 minutes, and obtain the HepG2.2.15 cell suspension treated with DNase I nuclease.

[0190] 3. Laminar flow washing: Use laminar flow washing to remove residual DNase I nuclease and DNA in the supernatant of HepG2.2.15 cell suspension, as follows:

[0191] The HepG2.2.15 cell suspension treated with DNase I nuclease was transferred to the wells of a laminar flow washing plate (Curiox Biosystems, 16-DC-CL-10 or 96-DC-CL-05), and allowed to settle for 30 min at 4°C. The washing solution was PBS (Gibco, 10010023). The laminar flow washing program was: Cycles--28, volume--80 μL, flow rate--12.

[0192] 4. Cell suspension recovery and cell counting: After laminar flow washing, 25 μL of cell suspension remains in the well. Add 25 μL of new PBS, gently blow to resuspend the cells, transfer them to a 1.5 mL centrifuge tube, and finally adjust the volume to 100 μL to obtain the washed HepG2.2.15 single cell suspension.

[0193] The cell viability and concentration of the washed HepG2.2.15 single cell suspension were measured in CountStar and the concentration was adjusted to ≤500 cells / μL.

[0194] 5. ddPCR reaction: According to the method of Example 2, the washed single cell suspension obtained in 4 was used to prepare a ddPCR reaction system.

[0195] 6. Preparation of microdroplets: same as Example 2.

[0196] 7. ddPCR amplification: same as Example 2.

[0197] 8. Droplet detection and result calculation and analysis: After ddPCR amplification, the reaction plate is placed in the matching droplet detector for detection of FAM and VIC channels. The Poisson distribution principle is used to analyze and calculate cccDNA + and β-actin + Droplet number and copy number of both.

[0198] As in Example 2, according to cccDNA + β-actin + Double positive microdroplets and cccDNA - β-actin + The number of single positive droplets was calculated, the proportion of cells containing cccDNA was calculated, and the cccDNA was further calculated. + The number of cccDNA copies in cells.

[0199] The results are as follows Figure 3 As shown in Table 9 and Table 10, target detection can be achieved.

[0200] Table 9 shows the cccDNA in HepG2.2.15 cell line + Cell ratio test results

[0201]

[0202] Note: cccDNA + Cell ratio = cccDNA + β-actin + Number of droplets / (cccDNA + β-actin + Droplet number + cccDNA - β-actin + Number of droplets)*100%

[0203] Table 10 shows cccDNA in HepG2.2.15 cell line + Results of cccDNA loading in cells

[0204]

[0205] Note: β-actin is used as the internal reference, and its actual copy number defaults to 2:

[0206] Average cccDNA content = (cccDNA copies / 20μL) / (β-actin copies / 20μL)*2; cccDNA +Cell cccDNA content = (cccDNA copies / 20μL) / (β-actin copies / 20μL*cccDNA + β-actin + Cell ratio)*2

[0207] Example 5: Single-cell ddPCR specific detection of HBV cccDNA positive cell proportion and cccDNA in tumor tissue of liver cancer patients + Viral load in cells

[0208] 1. Preparation of tumor single cell suspension

[0209] Preparation of liver tumor tissue single cell suspension: The liver tumor tissue to be tested is used to prepare a liver tumor tissue single cell suspension according to the method of Example 1 to obtain a tumor single cell suspension.

[0210] The cell suspension was collected in a 1.5 mL EP tube, centrifuged at 500 g for 5 min, and the supernatant was discarded.

[0211] The cells were washed twice with PBS, and finally 500 μL PBS was added to resuspend the cells. The cell viability and concentration of the tumor single cell suspension were measured in CountStar.

[0212] The liver tumor tissues to be tested are from liver cancer patient 1, liver cancer patient 2 and liver cancer patient 3.

[0213] PBMC was used as negative control, PBMC1 and PBMC2 were from two healthy individuals, PBS was used as blank control, and blank control 1 and 2 were from 2 experiments respectively.

[0214] 2. Treating the tumor single cells to be tested with DNase I nuclease: According to the method of Example 2, a cell suspension treated with DNase I nuclease is obtained.

[0215] 3. Laminar flow washing: follow the method of Example 2.

[0216] 4. Cell suspension recovery and cell counting: According to the method of Example 2, a single cell suspension of the tumor to be tested is obtained after washing.

[0217] The cell viability and concentration of the washed single-cell suspension of the tumor to be tested were measured in CountStar, and the concentration was adjusted to ≤500 cells / μL.

[0218] 5. Prepare ddPCR reaction system: According to the system shown in Example 2, prepare the washed tumor single cell suspension obtained in 4 to obtain a ddPCR reaction system.

[0219] 6. Preparation of microdroplets: same as Example 2.

[0220] 7. ddPCR amplification: same as Example 2.

[0221] 8. Droplet detection and result calculation and analysis: After ddPCR amplification, the reaction plate is placed in the matching droplet detector for detection of FAM and VIC channels. The Poisson distribution principle is used to analyze and calculate cccDNA + β-actin + Positive droplets and cccDNA - β-actin + Single positive droplet number and its copy number. According to cccDNA + β-actin + Double positive microdroplets and cccDNA - β-actin + The number of single positive droplets was calculated, the proportion of cells containing cccDNA was calculated, and the cccDNA was further calculated. + The number of cccDNA copies in cells.

[0222] The results are as follows Figure 4 As shown in Table 11 and Table 12, target detection can be achieved.

[0223] Table 11 shows the cccDNA in tumor tissues of clinical liver cancer patients + Cell ratio test results

[0224]

[0225]

[0226] Note: cccDNA + The method for calculating the cell ratio is shown in Example 4.

[0227] Table 12 shows cccDNA in liver tissue of clinical liver cancer patients + Results of cccDNA loading in cells

[0228]

[0229] Note: β-actin is used as the internal reference here, and its actual copy number defaults to 2. Since tumor samples may have genomic copy number variations, the cccDNA copy number calculated here is a relative copy number and cannot be considered the true value. + The method for calculating the cellular cccDNA content is shown in Example 4.

[0230] Example 6: Single-cell ddPCR specific detection of HBV cccDNA positive cell ratio and cccDNA in liver tissue of hepatitis B patients +Viral load in cells

[0231] 1. Preparation of single cell suspension of liver tissue

[0232] Preparation of single cell suspension of liver tissue: Prepare single cell suspension of liver transected tissue according to the method of Example 1 to obtain single cell suspension of liver transected tissue.

[0233] The cell suspension was collected in a 1.5 mL EP tube, centrifuged at 500 g for 5 min, and the supernatant was discarded.

[0234] Add 500 μL PBS to resuspend the cells, and measure the cell viability and concentration of the liver tissue single cell suspension in CountStar.

[0235] The liver tissues to be tested are from hepatitis patient 1, hepatitis patient 2, hepatitis patient 3 and hepatitis patient 4.

[0236] PBMC was used as negative control, PBMC1 and PBMC2 were from two healthy individuals, PBS was used as blank control, and blank control 1 and 2 were from 2 experiments respectively.

[0237] 2. Treating the single liver tissue cells to be tested with DNase I nuclease: According to the method of Example 2, a cell suspension treated with DNase I nuclease was obtained.

[0238] 3. Laminar flow washing: follow the method of Example 2.

[0239] 4. Cell suspension recovery and cell counting: According to the method of Example 2, a single cell suspension of the liver tissue to be tested is obtained after washing.

[0240] The cell viability and concentration of the washed liver tumor single cell suspension were measured in CountStar, and the concentration was adjusted to ≤500 cells / μL.

[0241] 5. Prepare ddPCR reaction system: prepare the washed single cell suspension of liver tissue to be tested obtained in 4 according to the system shown in Example 2 to obtain a ddPCR reaction system.

[0242] 6. Preparation of microdroplets: same as Example 2.

[0243] 7. ddPCR amplification: same as Example 2.

[0244] 8. Droplet detection and result calculation and analysis: After ddPCR amplification, the reaction plate is placed in the matching droplet detector for detection of FAM and VIC channels. The Poisson distribution principle is used to analyze and calculate cccDNA + and β-actin + The number of droplets and the number of copies of both. According to cccDNA+ β-actin + Double positive microdroplets and cccDNA - β-actin + The number of single positive droplets was calculated, the proportion of cells containing cccDNA was calculated, and the cccDNA was further calculated. + The number of cccDNA copies in cells.

[0245] The results are as follows Figure 5 As shown in Table 13 and Table 14, target detection can be achieved.

[0246] Table 13 shows the cccDNA in liver tissue of clinical hepatitis B patients + Cell ratio test results

[0247]

[0248] Note: cccDNA + The method for calculating the cell ratio is shown in Example 4.

[0249] Table 14 shows cccDNA in liver tissue of clinical hepatitis B patients + Results of cccDNA loading in cells

[0250]

[0251]

[0252] Note: Average cccDNA content and cccDNA + The method for calculating the cellular cccDNA content is shown in Example 4.

[0253] Example 7: Specificity detection of primers in ddPCR reaction system

[0254] The HBV genome sequence was downloaded from the public nucleic acid database. First, 20 pairs of cccDNA primer pairs and probes were designed based on the HBV genome sequence by using Primer Premier 6.0 software, spanning the double gaps DR1 and DR2 of rcDNA. Second, 20 pairs of β-actin primer pairs and probes were designed based on the gene sequence of β-actin. Subsequently, the designed primer pairs and probes were specifically compared using the NCBI-prime Blast online tool, and finally 2 pairs of β-actin primer pairs and probes with good specificity and 3 pairs of cccDNA primer pairs and probes with good specificity were screened. The primer pairs and probe sequences are shown in Table 15.

[0255] Table 15 shows the primer pairs and probe sequences

[0256]

[0257] In order to further verify the specificity of the primers, the amplification efficiency and the uniqueness of the amplified products, the DNA extracted from HepG2.2.15 cells was used as a template for verification by real-time quantitative PCR (qPCR). The CT values ​​of qPCR are shown in Table 16, and the melting curves of the amplified products are shown in Figure 6 .

[0258] Table 16 shows the qPCR test results

[0259]

[0260] The Tm value of the melting curve is an important parameter in the melting curve. The Tm value of the melting curve of the target gene is determined by the length of its product and the GC content. The longer the length of the qPCR product of the target gene and the higher the GC content, the larger the Tm value. Different qPCR products have different Tm values ​​due to their different lengths and GC contents. If the primers used for qPCR amplification are not specific, non-specific amplification products will appear. The qPCR products obtained in the experiment are composed of the target product and the non-specific product. Because the lengths and GC contents of the two are different, two Tm values, i.e., two peaks, will appear on the melting curve. If the melting curve presents a single peak but is not sharp, it may be that the Tm value of the non-specific amplification product is close to the Tm value of the target product, resulting in a wide peak in the melting curve. Therefore, by judging whether the peak shape of the melting curve of the product is a single narrow peak, it can be determined whether the primer is specific.

[0261] Figure 6 A is the melting curve of β-actin1 and β-actin2 amplified HepG2.2.15 cell DNA; Figure 6 B is the melting curve of HepG2.2.15 cell DNA amplified by cccDNA1, cccDNA2, and cccDNA3. It can be seen that the melting curves of the products amplified by the β-actin2 and cccDNA3 primer pairs are both single narrow peaks, indicating that the products of the two have good specificity. Combined with the CT value results of the two (β-actin2 is close to β-actin1; the CT value of cccDNA3 is less than that of the other two pairs of primers, indicating that its amplification efficiency is high), it is finally determined that the β-actin2 and cccDNA3 primer pairs are optimal, so β-actin2 (primers in Example 2) and cccDNA3 primer pairs (primers in Example 2) are selected as primers for subsequent experiments.

[0262] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for quantitatively detecting HBV cccDNA in a single cell of a sample to be tested, the method being a method for non-disease diagnosis and treatment purposes, comprising the following steps: 1) Using enzymatic hydrolysis to prepare a single cell suspension from the sample to be tested; 2) using DNase I nuclease to remove free nucleic acids in the supernatant of the single cell suspension to be tested, to obtain a cell suspension to be tested treated with DNase I nuclease; 3) washing the cell suspension to be tested after being treated with DNase I nuclease by laminar flow washing to obtain a washed cell suspension; 4) using a ddPCR system to encapsulate the washed cell suspension and the ddPCR amplification system into droplets to form microdroplets; then performing ddPCR amplification on the microdroplets and detecting the microdroplet fluorescence signal; and obtaining the proportion of HBV-infected cells in the sample to be tested and / or the viral load of HBV-infected cells according to the microdroplet fluorescence signal; The ddPCR amplification system includes a β-actin primer pair, a β-actin probe, a cccDNA primer pair and a cccDNA probe; The cccDNA primer pair is composed of a single-stranded DNA molecule shown in sequence 4 and a single-stranded DNA molecule shown in sequence 5; the nucleotide sequence of the cccDNA probe is sequence 6, and both ends of the probe are connected to a fluorescent group and a quenching group; The β-actin primer pair is composed of a single-stranded DNA molecule shown in sequence 1 and a single-stranded DNA molecule shown in sequence 2; the nucleotide sequence of the β-actin probe is sequence 3, and two ends of the probe are connected to another fluorescent group and the quenching group; In step 1), the enzymes used in the enzymatic hydrolysis method include Collagenase I, Dispase II and DNase I; The sample to be tested is a cell line containing HBV cccDNA or a cell line suspected of containing HBV cccDNA.

2. A kit for detecting HBV cccDNA in a single cell of a sample to be tested, comprising the following 1)-3): 1) The cccDNA primer pair, the cccDNA probe, the β-actin primer pair, and the β-actin probe of claim 1; 2) Reagents required for ddPCR amplification; 3) Reagents required for the enzymatic hydrolysis method, which include Collagenase I, Dispase II and DNaseI.

3. The kit according to claim 2, characterized in that: The sample to be tested is liver cancer tissue or liver tissue or a cell line containing HBV cccDNA or a cell line suspected of containing HBV cccDNA.

4. Use of the kit according to claim 2 or 3 in the preparation of products for diagnosing or assisting in the diagnosis of HBV-infected patients.

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