Nucleic acid combination, kit and method for simultaneous quantitative detection of HBV cccDNA and HBV rcDNA

Abstract

The present invention provides a nucleic acid combination, a kit, and a method for the simultaneous quantitative detection of HBV cccDNA and HBV rcDNA, relating to the field of molecular biology technology. The nucleic acid combination comprises: a degenerate primer that spans the gap on the negative strand of the HBV rcDNA, the degenerate primer being as shown in SEQ ID No. 1 or SEQ ID No. 2; and a qPCR primer for amplifying HBV DNA, the qPCR primer being used to amplify the HBV PreC / C gene or the S gene. The present invention utilizes the differences in the molecular structure of cccDNA and rcDNA to design primers that increase the copy number of the cccDNA positive strand template in proportion. qPCR is then performed simultaneously with a sample that has not undergone cccDNA pre-amplification to quantify HBV DNA and compare the results to calculate the initial copy number of cccDNA and rcDNA. The present invention can simply and efficiently achieve accurate and efficient quantification of HBV cccDNA and rcDNA simultaneously.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology technology, and in particular to a nucleic acid combination, a kit and a method for simultaneously and quantitatively detecting HBV cccDNA and HBV rcDNA. Background Art

[0002] Chronic infection with hepatitis B virus (HBV) is accompanied by the production of covalently closed circular DNA (cccDNA) in the nucleus of hepatocytes, causing the body to carry the virus for a long time. Clearing cccDNA is a difficult problem in the antiviral treatment of hepatitis B.

[0003] Currently, there are several methods for detecting cccDNA: (1) Southern blot hybridization: DNA fragments are separated by electrophoresis according to DNA size, transferred to a membrane, hybridized with labeled sequence-specific probes, washed, and finally the labeled DNA bands are detected (Li Chunyan et al., 2013; Cai D et al., 2013); (2) TaqMan probe quantification: Based on the differences in the physical, chemical and biological properties of cccDNA and rcDNA, enzyme cleavage is combined with qPCR technology to perform cross-gap selective PCR amplification, and quantitative detection is performed using TaqMan probes targeting the downstream of the HBV gap chain (Shi Tianshu et al., 2020); (3) Magnetic bead capture hybridization: Functionalized nanoparticles are prepared by modifying silica-coated nanoparticles with streptavidin or short oligonucleotides, and the particles are coupled with biotin-labeled probes. The functionalized nanoparticles are hybridized with fluorescently labeled oligonucleotide targets to form magnetic nanoparticles-SA-biotin-HBV. cccDNA probe complex is placed in the sample solution for hybridization with cccDNA, cccDNA is enriched and quantified by conventional PCR (Guo YC et al., 2015); (4) Rolling circle amplification (RCA) method: RCA primers targeting multiple binding sites are designed using Phi29 DNA polymerase, and phi29 DNA polymerase selectively amplifies cccDNA circular template molecules in RCA (Zhao Xu, 2010; Takkenberg RB et al., 2010); (5) In situ hybridization PCR method: Detect HBV cccDNA in infected hepatocytes. The distribution and location of HBV cccDNA in liver tissue can be observed under a light microscope (Shi Tianshu et al., 2020); (6) Droplet digital PCR (dd PCR) method: Based on the interaction between HBV rcDNA and ccc Based on the structural differences of DNA, plasmid-safe ATP-dependent DNAse (PSAD) is used for enzymatic digestion, primers and probes that span the minus-strand gap are designed, and the sample is processed into droplets so that each sample droplet contains no or one to several target nucleic acids to be tested. After PCR amplification, the absolute amount of DNA is calculated using the Poisson distribution principle (Tian Yuan et al., 2021; Gao YT et al., 2010; Huang JT et al., 2018).However, the above-mentioned existing technologies all have certain defects. For example, the use of Southern blotting for cccDNA detection requires a complex operation process, high cost, low sensitivity, poor controllability, and is difficult to apply in clinical practice; when using TaqMan probes for cccDNA quantification, due to the homology between cccDNA and rcDNA, TaqMan probes are not completely specific for cccDNA detection, and there are large errors; the use of magnetic bead capture hybridization method for cccDNA quantification is complicated, costly, and has limited application; the operation process of using the RCA method for cccDNA quantification is time-consuming; the sample preparation for the visualization detection of cccDNA using in situ hybridization PCR is difficult, requires complex equipment, and has limited application; the operation of using the dd PCR method for cccDNA quantification is complicated, requires a "digestion-purification" process, has large detection errors, and is limited in application. Therefore, it is necessary to develop a new detection method.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of the present invention is to provide a nucleic acid combination, a kit and a method for the simultaneous quantitative detection of HBV cccDNA and HBV rcDNA, which solves the defects of the cccDNA detection methods in the prior art and avoids errors caused by processes such as digestion and purification.

[0006] The technical solutions provided by the present invention are as follows:

[0007] In one aspect, the present invention provides a nucleic acid combination for simultaneous quantitative detection of HBV cccDNA and HBV rcDNA, comprising:

[0008] A degenerate primer that spans the gap on the negative strand of the HBV rcDNA, wherein the degenerate primer is shown as SEQ ID No. 1 or SEQ ID No. 2;

[0009] and qPCR primers for amplifying HBV DNA, wherein the qPCR primers are used to amplify HBV PreC / C gene or S gene.

[0010] Based on the differences in the molecular structures of HBV cccDNA and rcDNA, this patented invention designs special primers that can span the HBV "gap" (degenerate primers that span the gap on the negative strand of HBV rcDNA). These primers can pre-amplify HBV cccDNA, increasing the copy number of the cccDNA positive strand template in proportion, whereas rcDNA cannot be pre-amplified due to the presence of the "gap." Subsequently, qPCR is performed simultaneously on the pre-amplified samples and the samples that have not undergone cccDNA pre-amplification to quantify HBV DNA and compare them. The initial copy numbers of HBV cccDNA and rcDNA are calculated based on the quantitative equations.

[0011] In one embodiment, when the HBV genotype is C, B, and I, the degenerate primer is shown as SEQ ID No. 1; when the HBV genotype is A to J, the degenerate primer is shown as SEQ ID No. 2.

[0012] Specifically, primers were designed for different genotypes, enabling detection of the domestically prevalent HBV genotypes C, B, and I, as well as the globally prevalent HBV pan-genotypes A to J.

[0013] The gap degenerate primers designed according to HBV genotypes C, B, and I are as follows: BCI: 5'-GGT(C / G)TG(T / C)(T / G)(C / T)ACCAGCACCAT-3' (1796-1815 nt); i.e.: 5'-GGTSTGYKYACCAGCACCAT-3' (also SEQ ID No. 1);

[0014] The gap degenerate primers designed according to HBV genotypes A to J are as follows: AJ: 5'-GGT(C / G)TG(T / C)(T / G)(C / T)ACCA(G / T)CA(C / T)CAT-3' (1796-1815 nt); i.e.: 5'-GGTSTGYKYACCAKCAYCAT-3' (also SEQ ID No. 2).

[0015] In the present invention, the qPCR primers used to amplify HBV DNA are designed for the HBV PreC / C gene and / or the S gene, preferably for the HBV PreC / C gene. In the present invention, the target gene for HBV DNA qPCR detection is the PreC / C or S gene.

[0016] In one embodiment, when performing fluorescent quantitative PCR, primers specific for both can be used to detect them separately by adjusting the cccDNA pre-amplification extension time within a range of 1 to 3 minutes. In a specific embodiment, detection was performed for the HBV PreC / C gene (pre-amplification extension for 1 minute, product 115 bp).

[0017] qPCR primers were designed to amplify the positive strand of HBV DNA targeting the HBV PreC / C gene and combining the conserved regions of the gene for genotypes (B, C, and I) and pan-genotypes (A to J);

[0018] When the HBV genotype is C, B, and I, the upstream and downstream primers of the qPCR are shown as SEQ ID No. 3 and SEQ ID No. 4; when the HBV genotype is A to J, the qPCR primers are shown as SEQ ID No. 3 and SEQ ID No. 5.

[0019] In another aspect, the present invention provides a detection kit for quantitatively detecting HBV cccDNA and rcDNA, the kit comprising the aforementioned nucleic acid combination.

[0020] In one embodiment, in the nucleic acid combination, the working concentration of the degenerate primers is 0.2-0.3 μM, more preferably 0.2 μM; the working concentration of the qPCR primers is 0.4-0.5 μM, more preferably 0.5 μM.

[0021] In a specific embodiment, the working concentration of the cccDNA pre-amplification primers BCI (SEQ ID No. 1) and AJ (SEQ ID No. 2) used is 0.2 μM, which can ensure the effective amplification of cccDNA and avoid affecting the subsequent HBV DNA quantification; the working concentration of the HBV DNA qPCR primers is 0.5 μM, which can ensure the efficient amplification of HBV DNA and avoid the inhibition of the amplification efficiency by BCI and AJ primers.

[0022] In one embodiment, the detection kit further comprises an HBV DNA template and a fluorescent quantitative PCR reaction solution. In one embodiment, the detection kit further comprises sterile deionized water.

[0023] In one embodiment, the HBV DNA template is from a biological sample or body fluid.

[0024] In another aspect, the present invention also provides a method for quantitatively detecting HBV cccDNA and HBV rcDNA for the purpose of non-disease diagnosis and / or treatment, the method comprising performing fluorescent quantitative PCR on the test sample using the aforementioned nucleic acid combination or the aforementioned detection kit.

[0025] In one embodiment, the method comprises:

[0026] (a) pre-amplifying HBV cccDNA using the degenerate primers shown in SEQ ID No. 1 or SEQ ID No. 2;

[0027] (b) Quantitative detection of HBV cccDNA and rcDNA using upstream and downstream primers of qPCR;

[0028] (c) Based on the quantitative equations, the initial copy numbers of HBV cccDNA and HBV rcDNA were calculated by calculating the increase in DNA template before and after HBV cccDNA pre-amplification and comparing the copy numbers of HBV total DNA obtained by qPCR.

[0029] In one embodiment, the initial copy numbers of cccDNA and rcDNA are calculated according to the following equations:

[0030]

[0031] c: initial HBV cccDNA copy number of the specimen;

[0032] r: the initial HBV rcDNA copy number of the specimen;

[0033] N0: quantitative HBV DNA copy number before cccDNA pre-amplification;

[0034] N n : Quantitative HBV DNA copy number after n cycles of cccDNA pre-amplification (n represents the number of cycles).

[0035] In one embodiment, the positive strand copy number of the HBV cccDNA is increased by 3 to 18 times, preferably by 6 to 12 times, through pre-amplification.

[0036] In a specific embodiment, HBV cccDNA can be pre-amplified for 6, 9, and 12 cycles simultaneously, and the average of the three qPCR results is taken to improve the accuracy of HBV cccDNA and rcDNA quantification.

[0037] In one embodiment, the qPCR quantitative method is absolute quantification, and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or mitochondrial cytochrome c oxidase subunit 1 (COX1) gene can be selected as an internal reference for normalization.

[0038] In one embodiment, the conditions of the fluorescent quantitative PCR are: pre-denaturation at 93-96°C for 20-30s; denaturation at 92-96°C for 5-10s, annealing at 58-62°C for 25-30s, and extension at 70-75°C for 10-30s, for 40-50 cycles.

[0039] In a preferred embodiment, the conditions of the fluorescent quantitative PCR are: pre-denaturation at 95°C for 30s; denaturation at 95°C for 5s, annealing at 60°C for 30s, extension at 72°C for 30s, for 40-50 cycles, fluorescence detection and melting curve analysis.

[0040] Beneficial effects:

[0041] The present invention designs nucleic acid combinations for different HBV genotype detection, which has a wide range of applications;

[0042] The method of the present invention is simple to operate, avoids errors caused by processes such as digestion and purification, and can perform "two-step" qPCR amplification in a single tube to simultaneously achieve accurate and efficient quantification of HBV cccDNA and rcDNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0044] Figure 1 Schematic diagram of the location of the primers used in the present invention for pre-amplification of HBV cccDNA in the HBV genome and the amplification principle, where A and B represent HBV rcDNA and cccDNA, respectively; the BCI / AJ primers can amplify cccDNA but cannot amplify rcDNA across the "GC" gap;

[0045] Figure 2qPCR amplification curves for HBV DNA quantification using cccDNA pre-amplified in Sample 1 of the present invention, where A and B are the amplification curves for 0 cycles of pre-amplification using BCI and AJ primers (dark blue and purple) and the HBV DNA control qPCR (cyan and light brown), respectively; C and D are the amplification curves for 9 cycles of pre-amplification using BCI and AJ primers (yellow and sky blue) and the HBV DNA control qPCR (cyan and light brown), respectively;

[0046] Figure 3 qPCR amplification curves for HBV DNA quantification using pre-amplified cccDNA of sample 2 of the present invention; A and B are the amplification curves for 0 cycles of pre-amplification using BCI and AJ primers (dark blue and light blue) and the HBV DNA control qPCR (brown and dark brown), respectively; C and D are the amplification curves for 9 cycles of pre-amplification using BCI and AJ primers (blue and pink) and the HBV DNA control qPCR (brown and dark brown), respectively. DETAILED DESCRIPTION

[0047] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0048] Example 1. Experimental design and experimental procedures

[0049] (1) Grouping for HBV genotype detection

[0050] Testing is performed based on the domestically prevalent HBV genotypes C, B, and I, as well as the globally prevalent HBV pan-genotypes A to J.

[0051] (2) Primer design

[0052] First, pre-amplification primers that can span the GC gap of the negative strand of HBV cccDNA are designed. The location of the pre-amplification primers for HBV cccDNA in the HBV genome and the amplification effect are as follows: Figure 1 shown.

[0053] Among them, the gap degenerate primers designed according to HBV genotypes C, B and I are as follows:

[0054] BCI: 5'-GGT(C / G)TG(T / C)(T / G)(C / T)ACCAGCACCAT-3'(1796-1815nt)

[0055] That is: 5'-GGTSTGYKYACCAGCACCAT-3' (SEQ ID No. 1);

[0056] The gap degenerate primers designed according to HBV genotypes A to J are as follows:

[0057] AJ: 5'-GGT(C / G)TG(T / C)(T / G)(C / T)ACCA(G / T)CA(C / T)CAT-3'(1796-1815nt)

[0058] Namely: 5'-GGTSTGYKYACCAKCAYCAT-3' (SEQ ID No. 2).

[0059] Secondly, qPCR primers for amplifying HBV DNA were designed.

[0060] qPCR primers were designed to amplify the positive strand of HBV DNA using the HBV PreC / C gene and the conserved regions of this gene across genotypes (B, C, and I) and pan-genotypes (A to J). The primer sequences are as follows, and the amplified product is 115 bp.

[0061] A-J genotype upstream primer (CF): 5'-ACTTTTTCACCTCTGCCTAA-3' (1819-1838 nt) (SEQ ID No. 3);

[0062] Downstream primer (CR1) for genotypes B, C, and I: 5′-AGCTCCAAATTCTTTATA-3′ (1933–1916 nt) (SEQ ID No. 4);

[0063] A~J genotype downstream degenerate primer (CR2):

[0064] 5'-AGC(T / G)CCAAATTCTTTATA-3'(1933~1916nt)

[0065] That is: 5'-AGCKCCAAATTCTTTATA-3' (1933-1916 nt) (SEQ ID No. 5).

[0066] (3) qPCR steps for simultaneous quantification of HBV cccDNA and rcDNA

[0067] Step 1 PCR: Pre-amplification of HBV cccDNA.

[0068] HBV cccDNA was pre-amplified 3 to 18 times using BCI and AJ primers that can span the "GC" gap of the HBV DNA negative strand, increasing the copy number of the HBV cccDNA positive strand by 3 to 18 times.

[0069] The HBV cccDNA pre-amplification reaction solution is as follows, with a total volume of 5 μL.

[0070]

[0071] The reaction conditions for pre-amplification of HBV cccDNA are as follows:

[0072] 95℃, 5min;

[0073]

[0074] Step 2: qPCR: quantify the copy number of HBV DNA positive strand before and after pre-amplification of HBV cccDNA.

[0075] The HBV DNA quantitative reaction solution is as follows, with a total volume of 40 μL.

[0076]

[0077] HBV DNA Control qPCR: Quantify the copy number of total HBV DNA. The reaction mixture is as follows, with a total volume of 40 μL.

[0078]

[0079] HBV DNA qPCR conditions are as follows:

[0080]

[0081] (4) Calculation method for simultaneous quantification of HBV cccDNA and rcDNA

[0082] By calculating the increase in DNA template before and after HBV cccDNA pre-amplification and based on the copy number of total HBV DNA obtained by control qPCR, the initial copy numbers of cccDNA and rcDNA were calculated using the following equations.

[0083]

[0084] c: initial HBV cccDNA copy number of the specimen;

[0085] r: the initial HBV rcDNA copy number of the specimen;

[0086] N0: quantitative HBV DNA copy number before cccDNA pre-amplification;

[0087] N n : Quantitative HBV DNA copy number after n cycles of cccDNA pre-amplification.

[0088] Example 2. Experimental results and evaluation

[0089] After 6, 9, and 12 cycles of pre-amplification with BCI primers, the relative cccDNA contents in samples 1 and 2 were 15.08-18.47% and 61.82-69.23%, respectively, demonstrating good consistency. However, the relative quantification results for cccDNA at 3, 15, and 18 pre-amplification cycles showed significant discrepancies. For samples with varying cccDNA content, 6 to 12 cycles of pre-amplification with BCI primers were appropriate; similarly, the AJ primers also achieved the desired amplification results.

[0090] Compared with the HBV DNA control qPCR, the use of BCI and AJ primers to pre-amplify cccDNA had no effect on the subsequent qPCR amplification efficiency. The HBV DNA qPCR amplification curves of samples 1 and 2 after 9 cycles of pre-amplification with BCI and AJ primers are shown in Figure 2. Figure 1 and 2 The consistency of HBV DNA quantification results after 6 to 12 cycles of pre-amplification with BCI primers is shown in Table 1.

[0091] Table 1. Consistency analysis of HBV cccDNA and rcDNA qPCR detection results (sample 1)

[0092]

[0093]

[0094] *DNA copies are the qPCR results after cccDNA pre-amplification.

[0095] Table 2. Consistency analysis of HBV cccDNA and rcDNA qPCR detection results (sample 2)

[0096]

[0097] *DNA copies are the qPCR results after cccDNA pre-amplification.

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for quantitatively detecting HBV cccDNA and HBV rcDNA for the purpose of non-disease diagnosis and / or treatment, characterized in that: include: (a) Pre-amplifying HBV cccDNA using the degenerate primers shown in SEQ ID No. 1; The pre-amplification reaction solution for HBV cccDNA is as follows, with a total volume of 5 μL: 2 × TB Green Fast qPCR Mix2.5 μL; HBV DNA template 1.0 μL; 0.1 μL of 10 μM degenerate primer of SEQ ID No. 1; ddH2O 1.4 μL; The reaction conditions for pre-amplification of HBV cccDNA are as follows: (b) Simultaneous quantitative detection of HBV cccDNA and rcDNA using upstream and downstream primers of qPCR; HBV DNA qPCR conditions are as follows: The HBV DNA quantification reaction solution was as follows, with a total volume of 40 μL; 2× TB Green Fast qPCR Mix17.5 μL; First step PCR reaction solution 5.0 μL; 0.8 μL of 25 μM upstream primer; 0.8 μL of 25 μM downstream primer; ddH2O 15.9 μL; HBV DNA control qPCR: quantify the copy number of total HBV DNA; the reaction solution is as follows, total volume is 40 μL; 2× TB Green Fast qPCR Mix20.0 μL; HBV DNA template 1.0 μL; 0.8 μL of 25 μM upstream primer; 0.8 μL of 25 μM downstream primer; ddH2O 17.4 μL; The HBV genotypes are C, B, and I, and the upstream and downstream primers are shown in SEQ ID No. 3 and SEQ ID No. 4; (c) Calculate the initial copy numbers of HBV cccDNA and HBV rcDNA based on the quantitative equations by calculating the increase in DNA template before and after HBV cccDNA pre-amplification and comparing the copy numbers of HBV total DNA obtained by qPCR; The quantitative equations are as follows: ； c: initial HBV cccDNA copy number of the specimen; r: the initial HBV rcDNA copy number of the specimen; N0: quantitative HBV DNA copy number before cccDNA pre-amplification; Nn: HBV DNA quantitative copy number after n cycles of cccDNA pre-amplification.

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