Hypersensitivity quantitative detection kit for detecting hepatitis B virus nucleic acid by adopting fluorescent quantitative PCR (Polymerase Chain Reaction) method

Abstract

The invention relates to a reagent, a kit and a method for quantitative detection of HBV (hepatitis B virus) nucleic acid, specifically, the preparation comprises (a) a first upstream primer with a nucleotide sequence as shown in SEQ ID NO: 1, a first downstream primer with a nucleotide sequence as shown in SEQ ID NO: 2 and a first probe with a nucleotide sequence as shown in SEQ ID NO: 3; and / or (b) a second upstream primer with a nucleotide sequence as shown in SEQ ID NO: 4, a second downstream primer with a nucleotide sequence as shown in SEQ ID NO: 5 and a second probe with a nucleotide sequence as shown in SEQ ID NO: 6. The detection reagent, kit or method disclosed by the invention is good in specificity, high in sensitivity and complete in gene coverage and has an excellent effect on a large-volume sample.

Description

Technical Field

[0001] This invention relates to the field of molecular diagnostics technology, specifically to an ultrasensitive quantitative detection kit for detecting hepatitis B virus nucleic acid using real-time fluorescence PCR. Background Technology

[0002] Hepatitis B virus (HBV) infection is a serious public health problem. There are currently more than 80 million people with chronic HBV infection in my country, accounting for one-third of the global total. Every year, about 300,000 people die from cirrhosis and liver cancer caused by hepatitis B, and about 500,000 to 1 million new cases of hepatitis B are diagnosed. Prevention and control of hepatitis B is an important task that we will face now and for a considerable period of time to come.

[0003] Ten HBV genotypes (A to J) have been identified globally, each of which can be further divided into different subtypes, and recombination between genotypes is also observed. Therefore, developing a vaccine that can cover all genotypes and avoid missed detections due to genotype differences is crucial.

[0004] HBV DNA quantification is a core indicator for assessing viral replication activity and guiding antiviral therapy. Developing more sensitive HBV detection products to avoid missed diagnoses of suspected HBV infection and thus enable early prevention and treatment is crucial. Currently, the detection limit of HBV quantification products is typically ≥20 IU / mL. In patients with negative routine tests, approximately 25%-27% can still detect viral replication with highly sensitive detection. Further improving the detection sensitivity to as low as 2 IU / mL could further reduce the missed diagnoses rate and prevent delays in antiviral treatment, especially for patients with occult HBV infection (OBI), early infection, and low-level viral replication, providing critical diagnostic value.

[0005] Currently available HBV DNA quantification products are mainly based on real-time quantitative PCR. However, on the one hand, their genotyping coverage is poor. For example, most products from domestic manufacturers only cover the AD genotype, and a few cover the AH genotype. This incomplete genotyping coverage carries the risk of missing other subtype infections. On the other hand, testing personnel need to prepare enzymes, buffers, and primers / probes in specific ratios, making the process complex. Serum or plasma samples require extraction using magnetic beads or boiling methods, which are not only cumbersome but also prone to aerosol contamination. Therefore, customers increasingly demand ease of use for HBV DNA quantification.

[0006] Therefore, there is an urgent need in this field for a simple, highly sensitive, comprehensive gene coverage, and effective method and kit for HBV nucleic acid quantification in large-volume samples. Summary of the Invention

[0007] The purpose of this invention is to provide a simple, highly sensitive, comprehensive gene coverage, and effective method for quantitative detection of HBV nucleic acid in large-volume samples.

[0008] Another objective of this invention is to provide an HBV nucleic acid quantitative detection kit with high sensitivity, comprehensive gene coverage, and suitability for large-volume samples.

[0009] In a first aspect, the present invention provides a reagent for detecting hepatitis B virus nucleic acid, comprising:

[0010] (a) a first upstream primer with the nucleotide sequence shown in SEQ ID NO:1, a first downstream primer with the nucleotide sequence shown in SEQ ID NO:2, and a first probe with the nucleotide sequence shown in SEQ ID NO:22; and / or

[0011] (b) A second upstream primer with the nucleotide sequence shown in SEQ ID NO:4, a second downstream primer with the nucleotide sequence shown in SEQ ID NO:5, and a second probe with the nucleotide sequence shown in SEQ ID NO:23.

[0012] In another preferred embodiment, the probe is modified with locked nucleic acid (LNA).

[0013] In another preferred embodiment, the probe is modified with one or two locked nucleic acids.

[0014] In another preferred embodiment, the locked nucleic acid is located at positions 1 to 20, more preferably positions 3 to 18, and even more preferably positions 5 to 17 at the 5' end of the probe.

[0015] In another preferred embodiment, the reagent further includes dNTPs, an enzyme for nucleic acid amplification, and a buffer solution.

[0016] In another preferred embodiment, the enzyme used for nucleic acid amplification is selected from the group consisting of Taq enzyme, UDG enzyme, endonuclease (DpnI, RsaI, XhoI) or combinations thereof.

[0017] In another preferred embodiment, the probe is modified with a fluorescent group and a quenching group.

[0018] In another preferred embodiment, the concentration of the upstream primer in the reagent is 10–500 μmol / L, more preferably 50–300 μmol / L, more preferably 80–200 μmol / L, for example about 100 μmol / L.

[0019] In another preferred embodiment, the concentration of the downstream primer in the reagent is 10–500 μmol / L, more preferably 50–300 μmol / L, more preferably 80–200 μmol / L, for example about 100 μmol / L.

[0020] In another preferred embodiment, the concentration of the probe in the reagent is 10–500 μmol / L, more preferably 50–300 μmol / L, more preferably 80–200 μmol / L, for example about 100 μmol / L.

[0021] In another preferred embodiment, the concentration of dNTPs in the reagent is 10–1000 μmol / L, more preferably 50–500 μmol / L, more preferably 100–300 μmol / L, for example about 200 μmol / L.

[0022] In another preferred embodiment, the concentrations of Taq enzyme, UDG enzyme, and endonuclease (DpnI, RsaI, XhoI) in the reagent are each independently 0.1–10 U / μL, more preferably 0.5–5 U / μL, more preferably 1–3 U / μL, for example about 1.2 U / μL.

[0023] In another preferred embodiment, the buffer solution comprises MgCl2 at a concentration of 6 mmol / L, Tris-HCl (pH 8.5) at a concentration of 120 mmol / L, betaine, trehalose, spermidine, Nonard P-40, glycerol, etc.

[0024] In a second aspect, the present invention provides a kit for detecting hepatitis B virus nucleic acid, comprising a container and reagents as described in the first aspect of the present invention located within the container.

[0025] In another preferred embodiment, the kit further includes magnetic beads.

[0026] In another preferred embodiment, the kit further includes lysis buffer and / or lysis buffer solution.

[0027] In another preferred embodiment, the kit further includes an elution buffer, preferably 0.1×TE buffer.

[0028] In another preferred embodiment, the lysis buffer comprises guanidine hydrochloride, sodium hydroxide, sodium trimethylolpropane sulfonate, ethanolamine, and sodium dodecyl sulfate; preferably 0.5 mol / L to 2 mol / L guanidine hydrochloride, 0.05 mol / L sodium hydroxide, 1 to 50 mmol / L sodium trimethylolpropane sulfonate, 1 to 10 mmol / L ethanolamine, and 0.01 to 1% sodium dodecyl sulfate; more preferably 1.5 mol / L guanidine hydrochloride, 0.05 mol / L sodium hydroxide, 5 to 30 mmol / L sodium trimethylolpropane sulfonate, 2 to 8 mmol / L ethanolamine, and 0.05 to 5% sodium dodecyl sulfate.

[0029] In another preferred embodiment, the reagents, magnetic beads, lysis buffer, lysis buffer, and elution buffer are located in the same or different containers.

[0030] In a third aspect, the present invention provides a method for detecting hepatitis B virus nucleic acid, comprising the steps of:

[0031] The nucleic acid of the sample to be tested is mixed with the reagents described in the first aspect of the present invention to carry out a nucleic acid amplification reaction, collect and analyze fluorescence signals, thereby determining whether hepatitis B virus nucleic acid exists in the sample, and quantifying the hepatitis B virus nucleic acid in the sample by combining a standard curve.

[0032] In another preferred embodiment, the sample to be tested includes blood, plasma, and / or serum samples.

[0033] In another preferred embodiment, the volume ratio of the sample to the reagent is 1 to 5:1, more preferably 3 to 5:1.

[0034] In another preferred embodiment, the reaction conditions for the nucleic acid amplification reaction are 25–50°C for 2 min; 95°C for 30 s; repeated for 35–45 cycles: 95°C for 0 s, 60°C for 5 s.

[0035] In another preferred embodiment, fluorescence signals are acquired during the cyclic procedure of the amplification reaction.

[0036] In another preferred embodiment, the method includes the steps of:

[0037] (s1) Nucleic acid extraction from the sample to be tested; and

[0038] (s2) The extracted nucleic acid is mixed with the reagents described in the first aspect of the present invention to carry out a nucleic acid amplification reaction, collect fluorescence signals, analyze fluorescence signals, thereby determining whether hepatitis B virus nucleic acid exists in the sample to be tested, and quantifying the hepatitis B virus nucleic acid in the sample by combining a standard curve.

[0039] In another preferred embodiment, step (s1) includes:

[0040] (s1a) Preprocess the blood sample to be tested to obtain a plasma sample or a serum sample; and

[0041] (s1b) The plasma or serum sample is mixed with magnetic beads and lysis buffer, the magnetic beads are collected using a magnetic adsorption column, the nucleic acids on the magnetic beads are eluted using elution buffer, and the eluted nucleic acids are separated.

[0042] In another preferred embodiment, in step (s1a), the amount of the sample to be tested is 1 to 20 mL, more preferably 3 to 15 mL, and even more preferably 5 to 10 mL.

[0043] In another preferred embodiment, in step (s1b), the amount of the plasma sample is 0.1 to 3 mL, more preferably 0.5 to 2 mL, and more preferably 1 mL; the amount of the serum sample is 0.1 to 3 mL, more preferably 0.5 to 2 mL, and more preferably 1 to 2 mL.

[0044] In another preferred embodiment, the magnetic beads have a particle size of 0.1–10 μm, more preferably 0.5–5 μm, and even more preferably 1–2 μm.

[0045] In another preferred embodiment, the magnetic beads are those used in an ultrasonic nucleic acid extractor.

[0046] In another preferred embodiment, in step (s1b), the lysis solution comprises 1.5M guanidine hydrochloride, 0.05M sodium hydroxide, 5-30mM sodium trimethylolpropane sulfonate, 2-8mM ethanolamine and 0.05-5% sodium dodecyl sulfate.

[0047] In another preferred embodiment, the amount of the eluent is 50–200 μL, more preferably 70–150 μL, more preferably 90–130 μL, for example 100 μL.

[0048] In another preferred embodiment, the elution buffer is 0.1×TE buffer or enzyme-free water.

[0049] In another preferred embodiment, the criterion for the method is that the sensitivity test of 1.5 IU / mL is qualified.

[0050] In another preferred embodiment, step (s1a) includes the following steps:

[0051] (s1a1) Let the blood sample to be tested stand at room temperature (10-30℃) for 30-60 minutes, and the serum will spontaneously agglutinate and precipitate; or centrifuge at 1600 rpm for 10 minutes at room temperature to separate the serum, which is then used as the serum sample to be tested.

[0052] (s1a2) Add EDTA anticoagulant to the blood sample to be tested, and immediately gently invert the collection tube 5 to 8 times to mix the venous blood and anticoagulant thoroughly. Let it stand at room temperature for 10 to 30 minutes to separate the plasma; or centrifuge at 1600 rpm for 10 minutes at room temperature to separate the plasma as the plasma sample to be tested.

[0053] (s1a3) Add the separated serum or plasma samples to the lysis buffer for subsequent nucleic acid extraction.

[0054] In another preferred embodiment, the lysis buffer comprises guanidine hydrochloride, sodium hydroxide, sodium trimethylolpropane sulfonate, ethanolamine, and sodium dodecyl sulfate; preferably 0.5-2M guanidine hydrochloride, 0.05M sodium hydroxide, 1-50mM sodium trimethylolpropane sulfonate, 1-10mM ethanolamine, and 0.01-1% sodium dodecyl sulfate; more preferably 1.5M guanidine hydrochloride, 0.05M sodium hydroxide, 5-30mM sodium trimethylolpropane sulfonate, 2-8mM ethanolamine, and 0.05-5% sodium dodecyl sulfate.

[0055] In another preferred embodiment, in step (s1a3), the volume ratio of serum sample or plasma sample to lysis buffer is 10 to 50:1, more preferably 15 to 40:1, more preferably 20 to 30:1, for example 25:1.

[0056] In another preferred embodiment, the method is in vitro.

[0057] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0058] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0059] Figure 1 The sensitivity test results of the method of the present invention are shown.

[0060] Figure 2 The linear range of the method of the present invention is shown when using standard samples for verification.

[0061] Figure 3 The results of detecting hepatitis B virus nucleic acid of different genotypes using the method of the present invention are shown.

[0062] Figure 4 The results show a comparison between the method of the present invention and commercially available products.

[0063] Figure 5 The repeatability test results of the method of the present invention are shown.

[0064] Figure 6 The linear range of the method for validating the present invention using virus samples is shown.

[0065] Figure 7 The results of detecting hepatitis B virus nucleic acid of different genotypes using the method of the present invention are shown. Detailed Implementation

[0066] Through extensive and in-depth research, and after numerous experiments and screenings, the inventors unexpectedly discovered for the first time an HBV nucleic acid quantitative detection kit and method with high specificity, high sensitivity, comprehensive gene coverage, and suitability for large-volume samples. Specifically, through extensive screening, the inventors ultimately obtained primers and probes capable of covering all genotypes of HBV AJ (including A: A1–A7, B: B1–B9, C: C1–C16, D: D1–D10, F: F1–F4, I: I1–I2, and J). By modifying the probes with locked nucleic acids, the background value of the probes is lowered and the Tm value is higher, further improving specificity. The introduction of endonucleases (DpnI, RsaI, XhoI) into the reaction system can fragment HBV DNA, further improving the amplification efficiency of target genes and significantly increasing sensitivity. The kit of this invention uses a fully premixed system, single-tube single-use, and requires no aliquoting. The method of this invention uses a rapid thermal cycling program, shortening the amplification program to 16 minutes. Based on these, this invention was completed.

[0067] the term

[0068] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0069] As used herein, the term “comprising” or its variations such as “including” or “comprising” are understood to include the said element or component without excluding other elements or other components.

[0070] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0071] As used herein, unless otherwise stated, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).

[0072] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0073] The reagent of the present invention

[0074] The reagent of the present invention is used to detect hepatitis B virus nucleic acid, comprising (a) a first upstream primer with a nucleotide sequence as shown in SEQ ID NO:1, a first downstream primer with a nucleotide sequence as shown in SEQ ID NO:2, and a first probe with a nucleotide sequence as shown in SEQ ID NO:3; and / or

[0075] (b) A second upstream primer with the nucleotide sequence shown in SEQ ID NO:4, a second downstream primer with the nucleotide sequence shown in SEQ ID NO:5, and a second probe with the nucleotide sequence shown in SEQ ID NO:6.

[0076] In a preferred embodiment, the probe is modified with locked nucleic acids. In another preferred embodiment, the probe is modified with one or two locked nucleic acids. Modifying the middle site of the probe with locked nucleic acids ensures a lower probe background value, further improving the specificity of the reagents of the present invention.

[0077] In a preferred embodiment, the reagents comprise the primers and probes shown in the table below:

[0078]

[0079] In a preferred embodiment, the reagent further includes dNTPs, an enzyme for nucleic acid amplification, and a buffer. In a preferred embodiment, the enzyme for nucleic acid amplification is selected from the group consisting of Taq enzyme, UDG enzyme, endonuclease (DpnI, RsaI, XhoI), or combinations thereof, i.e., an enzyme mix. In a preferred embodiment, the reagent further includes an endonuclease, preferably a nucleic acid endonuclease, wherein the endonuclease is any one of DpnI, RsaI, XhoI, or a combination thereof. The reagent of the present invention is a fully premixed system, single-tube single-use, requiring no aliquoting.

[0080] The primers and probes of this invention can simultaneously detect all HBV AJ genotypes, providing broad coverage and significantly improving the detection coverage and sensitivity of the reagents. The primers and probes of this invention target the S and X genes of the HBV genome. The primers and probes of this invention are modified with locked nucleic acids, increasing the Tm value of the primers. A fluorescent group is modified in the middle of the probe, and the 3' end of the probe is phosphorylated, enabling multiplex fluorescence detection.

[0081] The reagent kit of the present invention

[0082] The kit of the present invention is used to detect hepatitis B virus nucleic acid, comprising a container and the reagent of the present invention located in the container.

[0083] The method of the present invention

[0084] The method of the present invention is a method for detecting hepatitis B virus nucleic acid, comprising the following steps:

[0085] The nucleic acid of the sample to be tested is mixed with the reagents of the present invention to carry out a nucleic acid amplification reaction, and the fluorescence signal is collected and analyzed to determine whether hepatitis B virus nucleic acid is present in the sample. The hepatitis B virus nucleic acid in the sample is then quantified by combining a standard curve.

[0086] The method of the present invention is applicable to plasma and / or serum samples.

[0087] In a preferred embodiment, the nucleic acid amplification reaction conditions are 25–50°C for 2 min; 95°C for 30 s; repeated for 35–45 cycles: 95°C for 0 s, 60°C for 5 s. The method of this invention employs a rapid thermal cycling procedure, shortening the nucleic acid amplification step to 16 min.

[0088] In a preferred embodiment, step (s1) includes:

[0089] (s1a) Preprocess the sample to be tested to obtain a plasma sample or a serum sample; and

[0090] (s1b) The plasma or serum sample is mixed with magnetic beads and lysis buffer, the magnetic beads are collected using a magnetic adsorption column, the nucleic acids on the magnetic beads are eluted using elution buffer, and the eluted nucleic acids are separated.

[0091] This invention employs an ultrasonic nucleic acid extractor for sample processing, avoiding the high-salt lysis method of conventional magnetic bead extraction, resulting in purer extracted nucleic acids. Elution is performed using 0.1×TE buffer, introducing even less salt. The method utilizes ultrasonic cell disruption, followed by magnetic bead adsorption of nucleic acids under low-salt conditions. The magnetic beads are then collected, and sample extraction is completed in just 3 minutes.

[0092] An exemplary method for detecting hepatitis B virus nucleic acid includes:

[0093] Step 1: Sample preprocessing;

[0094] Step 2: Nucleic acid extraction and enrichment;

[0095] Step 3: Preparation of reaction solution;

[0096] Step 4: Multiplex real-time fluorescence PCR detection;

[0097] Step 1 includes:

[0098] Step 101: Following laboratory methods, prepare the collected whole blood samples into plasma / serum as required for later use;

[0099] Step 102: Store plasma / serum samples in a refrigerator at a temperature between -20℃ and 8℃ for later use.

[0100] Step 103: Take 1-2 mL of plasma / serum sample and transfer it to a new centrifuge tube;

[0101] Step 2 includes:

[0102] Step 201: Add the magnetic bead solution and lysis buffer to the centrifuge tube and vortex to mix.

[0103] Step 202: Set the ultrasonic instrument to: amplitude 70%, frequency: 36.82kHz, running time 150s. After the operation is completed, attach the magnetic bead.

[0104] Step 203: Separate the magnetic beads from the solution and discard the solution;

[0105] Step 204: Elute the magnetic beads with 100 μL of elution buffer;

[0106] Step 3 includes:

[0107] Step 301: Add 10 μL of the fully premixed HBV reaction solution (containing dNTPs / enzyme mix / primers and probes) to the PCR tube;

[0108] Step 302: Add 40 μL of the extracted nucleic acid to form 50 μL of HBV reaction solution and mix gently.

[0109] Step 303: Place the PCR tube containing the HBV reaction solution into a real-time fluorescence PCR instrument for subsequent multiplex real-time fluorescence PCR detection.

[0110] Step 4 includes:

[0111] Step 401: Set the reaction conditions for multiplex real-time fluorescence PCR, including an initial temperature of 25-50℃ for 2 min; 95℃ for 30 s; followed by 40 cycles: 95℃ for 0 s, 60℃ for 5 s;

[0112] Step 402: Collect fluorescence signal data at 60℃ for 5 seconds;

[0113] Step 403: Analyze the fluorescence growth curve and determine the detection results.

[0114] The main advantages of this invention include:

[0115] 1. The kit of the present invention can simultaneously detect all genotypes of hepatitis B virus type AJ, with broad coverage.

[0116] 2. The kit of this invention has good specificity and high sensitivity, enabling accurate detection of low viral load (1.5 IU / mL) with a wide quantitative range of 2 IU / mL to 5 × 10⁻⁵. 9 IU / mL.

[0117] 3. The method of the present invention uses a fully premixed liquid system, and single-tube single-person testing is performed without the need for repackaging, which greatly simplifies the operation process.

[0118] 4. The method of the present invention uses a rapid thermal cycling procedure for detection, reducing 40 cycles to 16 minutes, thereby achieving rapid detection of samples and meeting the requirements for detection efficiency in clinical applications.

[0119] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0120] General Method

[0121] This invention provides a method for quantitative detection of hepatitis B virus nucleic acid based on multiple primer probes, comprising:

[0122] Step 1: Sample preprocessing;

[0123] Step 2: Nucleic acid extraction and enrichment;

[0124] Step 3: Preparation of reaction solution;

[0125] Step 4: Multiplex real-time fluorescence PCR detection.

[0126] Step 1 includes:

[0127] Step 101: Following laboratory methods, prepare the collected whole blood samples into plasma / serum for later use.

[0128] Step 102: Store plasma / serum samples in a refrigerator at a temperature between -20℃ and 8℃ for later use.

[0129] Step 103: Take 1-2 mL of plasma / serum sample and transfer it to a new centrifuge tube.

[0130] Step 2 includes:

[0131] Step 201: Add the magnetic bead solution and lysis buffer to the centrifuge tube and vortex to mix.

[0132] Step 202: Set the ultrasonic instrument to: amplitude 70%, frequency: 36.82kHz, running time 150s. After the operation is completed, attach the magnetic bead.

[0133] Step 203: Separate the magnetic beads from the solution and discard the solution;

[0134] Step 204: Elute the magnetic beads with 100 μL of elution buffer (0.1×TE buffer).

[0135] Step 3 includes:

[0136] Step 301: Add 10 μL of the fully premixed HBV reaction solution (containing dNTPs / enzyme mix / primers and probes) to the PCR tube;

[0137] Step 302: Add 40 μL of the extracted nucleic acid to form 50 μL of HBV reaction solution and mix gently.

[0138] Step 303: Place the PCR tube containing the HBV reaction solution into a real-time fluorescence PCR instrument for subsequent multiplex real-time fluorescence PCR detection.

[0139] Step 4 includes:

[0140] Step 401: Set the reaction conditions for multiplex real-time fluorescence PCR, including an initial temperature of 25-50℃ for 2 min; 95℃ for 30 s; followed by 40 cycles: 95℃ for 0 s, 60℃ for 5 s;

[0141] Step 402: Collect fluorescence signal data at 60℃ for 5 seconds;

[0142] Step 403: Analyze the fluorescence growth curve and determine the detection results.

[0143] The multiple primer probe system includes:

[0144] Step 501: Design two pairs of specific primers and probes, targeting the S gene and X gene of the HBV genome, respectively;

[0145] Step 502: Introduce LNA modification on primers and probes to increase the Tm value of primers;

[0146] Step 503: Optimize the sequence design of upstream primers and downstream probes to reduce the complexity of the reaction system;

[0147] Step 504: Label the middle of the sequence with a fluorescent group and phosphorylate the 3' end of the probe to achieve multiplex fluorescence detection.

[0148] Example 1 Primer and probe sequence optimization experiment

[0149] Step 1, Sample Preprocessing:

[0150] Step 101: Collect clinically positive samples of common domestic hepatitis B genotypes B, C, and D, one sample each of international reference hepatitis B virus types A, E, F, and G, and pseudovirus type HJ, covering a total of 10 genotypes of HBV, including A, B, C, D, E, F, G, H, I, and J.

[0151] Step 102: Serially dilute the clinical sample, HBV international standard, and pseudovirus to 1.0 × 10⁻⁶ using a negative sample matrix. 4 IU / mL;

[0152] Step 2, Nucleic acid extraction and enrichment:

[0153] Step 201: Dilute the above to 1.0 × 10⁻⁶. 4 Nucleic acid extraction was performed on clinical samples, international standards, and pseudoviruses at a concentration of IU / mL.

[0154] Step 3, Preparation of reaction solution:

[0155] Step 301: Prepare a 5× real-time PCR premix solution, including: upstream primer, downstream primer, fluorescent probe, high-efficiency Taq enzyme, UDG enzyme, and DpnI restriction enzyme buffer. Pipette 10 μL of the 5× real-time PCR premix solution into a PCR reaction tube.

[0156] Step 302: Take 40 μL of elution buffer and mix it with the above 10 μL of 5× real-time PCR premixed liquid system, vortex to mix well, and prepare 50 μL of HBV reaction solution;

[0157] Step 303: Transfer the above PCR reaction tubes to a real-time PCR instrument for testing.

[0158] Step 4: Multiplex real-time fluorescence PCR detection:

[0159] Step 401: Set the reaction program for the real-time PCR instrument:

[0160] 1) React at 50℃ for 2 minutes, 1 cycle;

[0161] 2) Pre-denaturation at 95℃ for 30 seconds, 1 cycle;

[0162] 3) Denaturation at 95℃ for 0 seconds, fluorescence collected simultaneously at 60℃ for 5 seconds, 40 cycles;

[0163] Step 403: Obtain fluorescence signal data and reagent curve results;

[0164] Step 404: Result analysis and determination of sample results.

[0165] The different primer and probe sequences are as follows:

[0166]

[0167]

[0168] The results are as follows:

[0169] category Type A Type B Type C Type D Type E Type F G type H type Type I J-type Detection rate Group 1 Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive 10 / 10 Group 2 Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive 10 / 10 Group 3 Positive Positive Positive Positive Positive Positive Positive Negative Positive Positive 9 / 10 Group 4 Positive Positive Positive Positive Positive Positive Positive Positive Positive Negative 9 / 10 Group 5 Positive Positive Positive Positive Positive Positive Positive Positive Negative Positive 9 / 10 Group 6 Positive Positive Positive Positive Positive Positive Negative Positive Positive Positive 9 / 10

[0170] Group 1 consists of SEQ ID NO:1-3, Group 2 consists of SEQ ID NO:4-6, Group 3 consists of SEQ ID NO:7-9, Group 4 consists of SEQ ID NO:10-12, Group 5 consists of SEQ ID NO:13-15, Group 6 consists of SEQ ID NO:16-18, and the exogenous internal reference for each group is SEQ ID NO:19-21. Based on the detection results (negative or positive) of different HBV genotype samples from Groups 1-6, Groups 1 and 2 show better results.

[0171] Example 2: Nucleic Acid Modification Site Exploration Experiment

[0172] Step 1, Sample Preprocessing:

[0173] Step 101: Take the national standard for hepatitis B virus nucleic acid (China National Institutes for Food and Drug Control, batch number: 300022-201601, 0.5ml / vial, concentration 1.0E+08IU / ml);

[0174] Step 102: Dilute the standard to 50 IU / mL, 10 IU / mL, 5 IU / mL, and 1.5 IU / mL using negative sample matrix, and label them as L1-L4;

[0175] Step 2, Nucleic acid extraction and enrichment:

[0176] Step 201: Extract nucleic acids from the serially diluted standards described above;

[0177] Step 3, Preparation of reaction solution:

[0178] Step 301: Prepare a 5× real-time PCR fully premixed liquid system, including: upstream primer, downstream primer, fluorescent probe, high-efficiency Taq enzyme, UDG enzyme, and endonuclease (DpnI) buffer. Pipette 10 μL of the 5× real-time PCR fully premixed liquid system into a PCR reaction tube.

[0179] Step 302: Take 40 μL of elution buffer and mix it with the above 10 μL of 5× real-time PCR premixed liquid system, vortex to mix well, and prepare 50 μL of HBV reaction solution;

[0180] Step 303: Transfer the above PCR reaction tubes to a real-time PCR instrument for testing.

[0181] Step 4: Multiplex real-time fluorescence PCR detection:

[0182] Step 401: Set the reaction program for the real-time PCR instrument:

[0183] 1) React at 50℃ for 2 minutes, 1 cycle;

[0184] 2) Pre-denaturation at 95℃ for 30 seconds, 1 cycle;

[0185] 3) Denaturation at 95℃ for 0 seconds, fluorescence collected simultaneously at 60℃ for 5 seconds, 40 cycles;

[0186] Step 403: Obtain fluorescence signal data and reagent curve results;

[0187] Step 404: Result analysis and determination of sample results.

[0188] The nucleic acid-modified probe sequence is as follows:

[0189]

[0190] The results are as follows:

[0191] category L1 L2 L3 L4 control group Positive Positive Positive Negative experimental group Positive Positive Positive Positive

[0192] The primers and probes for the control group were SEQ ID NO:1-3 and SEQ ID NO:4-6, while the primers for the experimental group were SEQ ID NO:1-2 and SEQ ID NO:4-5, and the probes were locked nucleic acid modified SEQ ID NO:22 and SEQ ID NO:23. The detection results show that the locked nucleic acid modification has better detection sensitivity.

[0193] Example 3: Detection of HBV DNA in plasma samples

[0194] Step 1, Sample Preprocessing:

[0195] Step 101: Draw 5 ml of blood from the patient and place it in a test tube containing EDTA anticoagulant;

[0196] Step 102: Centrifuge at 12000g for 15 minutes, and collect the supernatant into a new centrifuge tube, which is plasma.

[0197] Step 2, Nucleic acid extraction and enrichment:

[0198] Step 201: Transfer 1 mL of supernatant (plasma) to a nucleic acid enrichment tube containing magnetic beads and lysis buffer from an ultrasonic nucleic acid extractor. The magnetic beads have a particle size of 1-2 μm.

[0199] Step 202: Collect the magnetic beads using a magnetic adsorption column and discard the supernatant;

[0200] Step 203: Add 100 μL of elution buffer to elute the nucleic acids on the magnetic beads;

[0201] Step 204: Use a magnetic adsorption column to adsorb magnetic beads, collect the eluent, and measure the absorbance value. The A260 / A280 value should be between 1.8 and 2.0, which can be used as the nucleic acid to be tested.

[0202] Step 3, Preparation of reaction solution:

[0203] Step 301: Prepare a 5× real-time PCR fully premixed liquid system (i.e., fully premixed HBV reaction solution), including: upstream primer, downstream primer, fluorescent probe, high-efficiency Taq enzyme, endonuclease (DpnI) UDG enzyme, buffer, etc. Pipette 10 μL of the 5× real-time PCR fully premixed liquid system into a PCR reaction tube.

[0204] Step 302: Take 40 μL of elution buffer and mix it with the above 10 μL of 5× real-time PCR premixed liquid system, vortex to mix well, and prepare 50 μL of HBV reaction solution;

[0205] Step 303: Transfer the above PCR reaction tubes to a real-time PCR instrument for testing.

[0206] Step 4: Multiplex real-time fluorescence PCR detection:

[0207] Step 401: Set the reaction program for the real-time PCR instrument:

[0208] 1) React at 50℃ for 2 minutes, 1 cycle;

[0209] 2) Pre-denaturation at 95℃ for 30 seconds, 1 cycle;

[0210] 3) Denaturation at 95℃ for 0 seconds, fluorescence collected simultaneously at 60℃ for 5 seconds, 40 cycles;

[0211] Step 403: Obtain fluorescence signal data and reagent curve results;

[0212] Step 404: Result analysis and determination of sample results.

[0213] The plasma sample tested positive for hepatitis B virus nucleic acid.

[0214] Example 4: Detection of HBV DNA in serum samples

[0215] Step 1, Sample Preprocessing:

[0216] Step 101: Draw 10mL of blood from the patient and place it in a blood collection tube;

[0217] Step 102: Place the blood collection tube at room temperature for 30-60 minutes. The supernatant that separates out is the serum.

[0218] Step 103: Centrifuge the blood collection tube at 2500 rpm for 3 minutes and collect the supernatant, which is the serum.

[0219] Step 2, Nucleic acid extraction and enrichment:

[0220] Step 201: Transfer 1-2 mL of serum to a nucleic acid enrichment tube containing a sonic nucleic acid extractor equipped with magnetic beads and lysis buffer. The magnetic beads have a particle size of 1-2 μm.

[0221] Step 202: Collect the magnetic beads using a magnetic adsorption column and discard the supernatant;

[0222] Step 203: Add 100 μL of elution buffer to elute the nucleic acids on the magnetic beads;

[0223] Step 204: Use a magnetic adsorption column to adsorb magnetic beads, collect the eluent, and measure the absorbance value. The A260 / A280 value should be between 1.8 and 2.0, which can be used as the nucleic acid to be tested.

[0224] Step 3, Preparation of reaction solution:

[0225] Step 301: Prepare a 5× real-time PCR fully premixed liquid system, including: upstream primer, downstream primer, fluorescent probe, high-efficiency Taq enzyme, UDG enzyme, and RsaI restriction enzyme buffer. Pipette 10 μL of the 5× real-time PCR fully premixed liquid system into a PCR reaction tube.

[0226] Step 302: Take 40 μL of elution buffer and mix it with the above 10 μL 5× real-time PCR premixed liquid system, vortex to mix well, and prepare 50 μL of HBV reaction solution.

[0227] Step 303: Transfer the above PCR reaction tubes to a real-time PCR instrument for testing.

[0228] Step 4: Multiplex real-time fluorescence PCR detection:

[0229] Step 401: Set the reaction program for the real-time PCR instrument:

[0230] 1) React at 50℃ for 2 minutes, 1 cycle;

[0231] 2) Pre-denaturation at 95℃ for 30 seconds, 1 cycle;

[0232] 3) Denaturation at 95℃ for 0 seconds, fluorescence collected simultaneously at 60℃ for 5 seconds, 40 cycles;

[0233] Step 403: Obtain fluorescence signal data and reagent curve results;

[0234] Step 404: Result analysis and determination of sample results.

[0235] The serum sample test result was positive for hepatitis B virus nucleic acid.

[0236] Example 5: Reagent kit detection sensitivity verification

[0237] This example verifies the sensitivity of 1.5 IU / mL, and the specific steps are as follows:

[0238] Step 1, Sample Preprocessing:

[0239] Step 101: Take the national standard of hepatitis B virus nucleic acid (China National Institutes for Food and Drug Control, batch number: 300022-201601, 0.5ml / vial, concentration 1.0E+08IU / ml) and quantitative references S1-S4 of known concentrations;

[0240] Step 102: Dilute the standard to 50 IU / mL, 10 IU / mL, 5 IU / mL, and 1.5 IU / mL using negative sample matrix, and label them as L1-L4;

[0241] Step 2, Nucleic acid extraction and enrichment:

[0242] Step 201: Extract nucleic acids from the serially diluted standards described above;

[0243] Step 3, Preparation of reaction solution:

[0244] Step 301: Prepare a 5× real-time PCR fully premixed liquid system, including: upstream primer, downstream primer, fluorescent probe, high-efficiency Taq enzyme, UDG enzyme, and RsaI restriction enzyme buffer. Pipette 10 μL of the 5× real-time PCR fully premixed liquid system into a PCR reaction tube.

[0245] Step 302: Take 40 μL of elution buffer and mix it with the above 10 μL 5× real-time PCR premixed liquid system, vortex to mix well, and prepare 50 μL of HBV reaction solution.

[0246] Step 303: Transfer the above PCR reaction tubes to a real-time PCR instrument for testing.

[0247] Step 4: Multiplex real-time fluorescence PCR detection:

[0248] Step 401: Set the reaction program for the real-time PCR instrument:

[0249] 4) React at 50℃ for 2 minutes, 1 cycle;

[0250] 5) Pre-denaturation at 95℃ for 30 seconds, 1 cycle;

[0251] 6) Denaturation at 95℃ for 0 seconds, fluorescence collected simultaneously at 60℃ for 5 seconds, 40 cycles;

[0252] Step 403: Obtain fluorescence signal data and reagent curve results;

[0253] Step 404: Result analysis. Use quantitative references S1-S4 to plot a standard curve and determine the quantitative results of the sample.

[0254] The results are as follows Figure 1 As shown in Table 1, the national standard for hepatitis B virus nucleic acid was serially diluted to 50 IU / mL, 10 IU / mL, 5 IU / mL and 1.5 IU / mL, labeled as L1-L4. All of L1-L4 were detectable and the absolute deviation of the logarithmic value of the detection concentration did not exceed 0.5 logarithmic orders of magnitude of the theoretical concentration. The sensitivity of this method at 1.5 IU / mL was verified as qualified.

[0255] Table 1

[0256]

[0257]

[0258] Example 6: Validation of the linear range of the reagent kit

[0259] Step 1, Sample Preprocessing:

[0260] Step 101: Take the national standard of hepatitis B virus nucleic acid (China National Institutes for Food and Drug Control, batch number: 300022-201601, 0.5ml / vial, concentration 1.0E+08IU / ml, marked as X1) and quantitative reference materials S1-S4 of known concentration Y;

[0261] Step 102: Serially dilute the standard to 1.0 × 10⁻⁶ using a negative sample matrix. 7 1.0×10 6 1.0×10 5 1.0×10 4 1.0×10 3 1.0×10 2 10, 5, 2 IU / mL, labeled as X2-X10;

[0262] Step 2, Nucleic acid extraction and enrichment:

[0263] Step 201: Extract nucleic acids from the serially diluted standards described above;

[0264] Step 3, Preparation of reaction solution:

[0265] Step 301: Prepare a 5× real-time PCR fully premixed liquid system, including: upstream primer, downstream primer, fluorescent probe, high-efficiency Taq enzyme, UDG enzyme, and XhoI restriction enzyme buffer. Pipette 10 μL of the 5× real-time PCR fully premixed liquid system into a PCR reaction tube.

[0266] Step 302: Take 40 μL of elution buffer and mix it with the above 10 Ml 5× real-time PCR premixed liquid system, vortex to mix well, and prepare 50 μL of HBV reaction solution;

[0267] Step 303: Transfer the above PCR reaction tubes to a real-time PCR instrument for testing.

[0268] Step 4: Multiplex real-time fluorescence PCR detection:

[0269] Step 401: Set the reaction program for the real-time PCR instrument:

[0270] 1) React at 50℃ for 2 minutes, 1 cycle;

[0271] 2) Pre-denaturation at 95℃ for 30 seconds, 1 cycle;

[0272] 3) Denaturation at 95℃ for 0 seconds, fluorescence collected simultaneously at 60℃ for 5 seconds, 40 cycles;

[0273] Step 403: Obtain fluorescence signal data and reagent curve results;

[0274] Step 404: Result analysis. Use quantitative references S1-S4 to plot a quantitative standard curve and determine the quantitative result of the sample.

[0275] The results are as follows Figure 2 As shown in Table 2, the national standard for hepatitis B virus nucleic acid was serially diluted to 1.0 × 10⁻⁶. 8 1.0×10 7 1.0×10 6 1.0×10 5 1.0×10 4 1.0×10 3 1.0×10 2 The concentrations of 10, 5, and 2 IU / mL, labeled as X1-X10, were all detectable with a correlation coefficient of 0.999 > 0.980, indicating that the linear range of this method was validated.

[0276] Table 2

[0277]

[0278]

[0279] Example 7: Reagent kit detection for AJ genotype verification

[0280] This embodiment uses hepatitis B virus nucleic acid of different genotypes to verify the detection effect. The specific steps are as follows:

[0281] Step 1, Sample Preprocessing:

[0282] Step 101: Collect one clinically positive sample of common hepatitis B types B, C, and D in China, and one sample of each of the international reference types of hepatitis B virus A, E, F, G, and HJ pseudoviruses, covering a total of 10 genotypes of HBV, including A, B, C, D, E, F, G, H, I, and J.

[0283] Step 102: Serially dilute the clinical samples, international standards, and pseudoviruses to 1.0 × 10⁻⁶ using a negative sample matrix. 4 IU / mL;

[0284] Step 2, Nucleic acid extraction and enrichment:

[0285] Step 201: Dilute the above to 1.0 × 10⁻⁶. 4 Nucleic acid extraction was performed on clinical samples, international standards, and pseudoviruses at a concentration of IU / mL.

[0286] Step 3, Preparation of reaction solution:

[0287] Step 301: Prepare a 5× real-time PCR fully premixed liquid system, including: upstream primer, downstream primer, fluorescent probe, high-efficiency Taq enzyme, UDG enzyme, and XhoI restriction enzyme buffer. Pipette 10 μL of the 5× real-time PCR fully premixed liquid system into a PCR reaction tube.

[0288] Step 302: Take 40 μL of elution buffer and mix it with the above 10 μL of 5× real-time PCR premixed liquid system, vortex to mix well, and prepare 50 μL of HBV reaction solution;

[0289] Step 303: Transfer the above PCR reaction tubes to a real-time PCR instrument for testing.

[0290] Step 4: Multiplex real-time fluorescence PCR detection:

[0291] Step 401: Set the reaction program for the real-time PCR instrument:

[0292] 4) React at 50℃ for 2 minutes, 1 cycle;

[0293] 5) Pre-denaturation at 95℃ for 30 seconds, 1 cycle;

[0294] 6) Denaturation at 95℃ for 0 seconds, fluorescence collected simultaneously at 60℃ for 5 seconds, 40 cycles;

[0295] Step 403: Obtain fluorescence signal data and reagent curve results;

[0296] Step 404: Result analysis and determination of sample results.

[0297] The results are as follows Figure 3 As shown in Table 3, by validating clinically positive hepatitis B samples covering 10 genotypes of HBV (A, B, C, D, E, F, G, H, I, and J), the international reference standard for hepatitis B virus and pseudoviruses were analyzed to obtain 1.0 × 10⁻⁶ samples. 4 At a concentration of IU / mL, all subtypes could be detected normally, and the absolute deviation of the logarithmic value of the detected concentration did not exceed 0.5 logarithmic orders of magnitude of the theoretical concentration. The coverage of different subtypes of this method was verified as qualified.

[0298] Table 3

[0299] HBV genotype CT value Quantitative concentration IU / mL Concentration logarithm absolute deviation Type A 25.42 4.59E+03 3.66 0.34 Type B 25.14 5.58E+03 3.75 0.25 Type C 24.87 6.77E+03 3.83 0.17 Type D 24.95 6.40E+03 3.81 0.19 Type E 24.91 6.59E+03 3.82 0.18 Type F 24.56 8.46E+03 3.93 0.07 G type 25.36 4.79E+03 3.68 0.32 H type 24.36 9.73E+03 3.99 0.01 Type I 25.52 4.26E+03 3.63 0.37 J-type 25.01 7.52E+03 3.88 0.12

[0300] Example 8: Comparison of the reagent kit with clinical samples from commercially available products

[0301] The method of this invention is compared with the results of clinical sample testing using commercially available products. The specific steps are as follows:

[0302] Step 1, Sample Preprocessing:

[0303] Step 101: Take one clinically positive hepatitis B sample of different concentrations (high, medium, and low) and label them as Y1-Y3;

[0304] Step 2, Nucleic acid extraction and enrichment:

[0305] Step 201: Extract nucleic acid from the above clinical samples;

[0306] Step 3, Preparation of reaction solution:

[0307] Step 301: Prepare a fully premixed liquid system for 5× real-time PCR, including: upstream primer, downstream primer, fluorescent probe, high-efficiency Taq enzyme, UDG enzyme, and endonuclease (DpnI, RsaI, XhoI mixed) buffer. Pipette 10 μL of the 5× real-time PCR fully premixed liquid system into a PCR reaction tube.

[0308] Step 302: Take 40 μL of elution buffer and mix it with the above 10 μL of 5× real-time PCR premixed liquid system, vortex to mix well, and prepare 50 μL of HBV reaction solution;

[0309] Step 303: Transfer the above PCR reaction tubes to a real-time PCR instrument for testing.

[0310] Step 4: Multiplex real-time fluorescence PCR detection:

[0311] Step 401: Set the reaction program for the real-time PCR instrument:

[0312] 1) React at 50℃ for 2 minutes, 1 cycle;

[0313] 2) Pre-denaturation at 95℃ for 30 seconds, 1 cycle;

[0314] 3) Denaturation at 95℃ for 0 seconds, fluorescence collected simultaneously at 60℃ for 5 seconds, 40 cycles;

[0315] Step 403: Obtain fluorescence signal data and reagent curve results;

[0316] Step 404: Result analysis and determination of sample results.

[0317] The results are as follows Figure 4 As shown, this method detected all clinically positive hepatitis B samples with high, medium, and low concentrations, while competing products missed detection at low concentrations. This method showed better detection results compared to competing products.

[0318] Example 9: Reproducibility Validation of the Reagent Kit

[0319] The experimental procedure was as described in Example 7. Two samples were diluted to 1.5 IU / mL according to their concentrations, and the tests were repeated 20 times.

[0320] The results are as follows Figure 5 As shown in Table 4, the detection rate was 20 / 20 and the absolute deviation of the logarithmic value of the detected concentration did not exceed 0.5 logarithmic orders of magnitude of the theoretical concentration, thus the sensitivity of 1.5 IU / mL was qualified.

[0321] Table 4

[0322]

[0323]

[0324] Example 10

[0325] One high-concentration sample and one high-concentration pseudovirus sample were successively diluted to 5×10⁻⁶. 9 IU / mL, 5×10 8 IU / mL, 5×10 7 IU / mL, 5×10 6 IU / mL, 5×10 5 IU / mL, 5×10 4 IU / mL, 5×10 3 IU / mL, 5×10 2 IU / mL, 5×10 1 Gradient quantification was performed using IU / mL, 5 IU / mL, and 2 IU / mL. The experimental procedure is as described in Example 3.

[0326] The results are as follows Figure 6 As shown in Table 5, high-concentration samples and high-concentration pseudovirus samples were serially diluted to 5 × 10⁻⁶. 9 IU / mL, 5×10 8 IU / mL, 5×10 7 IU / mL, 5×10 6 IU / mL, 5×10 5 IU / mL, 5×10 4 IU / mL, 5×10 3 IU / mL, 5×10 2 IU / mL, 5×10 1 IU / mL, 5IU / mL and 2IU / mL, 2~5×10 9 The IU / mL concentration range was detectable and the correlation coefficient was ≥0.980, indicating that the linear range verification was successful.

[0327] Table 5

[0328]

[0329] Example 11

[0330] The test samples included international standard samples of types A, B, C, D, E, F, G, H, and I, and pseudovirus J. The viral concentration in the samples was 10. 3 IU / mL and 2IU / mL. The experimental procedure is as described in Example 3.

[0331] The results are as follows Figure 7 As shown in Table 6, international standard samples of types A, B, C, D, E, F, G, H, and I, and pseudovirus J were tested at 10... 3 At concentrations of 1U / mL and 2IU / mL, all subtypes could be detected normally, and the absolute deviation of the logarithmic value of the detected concentration did not exceed 0.5 logarithmic orders of magnitude of the theoretical concentration. The sensitivity verification of different subtypes was qualified.

[0332] Table 6

[0333]

[0334]

[0335] Example 12

[0336] Fifty clinical samples were used as test samples, of which at least 35 were positive samples. The method of the present invention and commercially available products were used for testing. The method of the present invention is as described in Example 3.

[0337] The results are shown in Table 7.

[0338] Table 7

[0339]

[0340]

[0341]

[0342] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A reagent for detecting hepatitis B virus nucleic acid, characterized in that, include (a) a first upstream primer with the nucleotide sequence shown in SEQ ID NO:1, a first downstream primer with the nucleotide sequence shown in SEQ ID NO:2, and a first probe with the nucleotide sequence shown in SEQ ID NO:22; and / or (b) A second upstream primer with the nucleotide sequence shown in SEQ ID NO:4, a second downstream primer with the nucleotide sequence shown in SEQ ID NO:5, and a second probe with the nucleotide sequence shown in SEQ ID NO:

23.

2. The reagent as described in claim 1, characterized in that, The probe is modified with locked nucleic acid.

3. The reagent as described in claim 2, characterized in that, The probe has one or two locked nucleic acid modifications.

4. The reagent as described in claim 2, characterized in that, The locked nucleic acid is located at positions 1 to 20, preferably positions 3 to 18, and more preferably positions 5 to 17 at the 5' end of the probe.

5. A kit for detecting hepatitis B virus nucleic acid, characterized in that, Includes a container and the reagent as described in claim 1 located within the container.

6. A method for detecting hepatitis B virus nucleic acid, characterized in that, Including the following steps: The nucleic acid of the sample to be tested is mixed with the reagent described in claim 1 to perform a nucleic acid amplification reaction, and the fluorescence signal is collected and analyzed to determine whether hepatitis B virus nucleic acid is present in the sample. The hepatitis B virus nucleic acid in the sample is then quantified using a standard curve.

7. The method as described in claim 6, characterized in that, The samples to be tested include blood, plasma, and / or serum samples.

8. The method as described in claim 6, characterized in that, The volume ratio of the sample to the reagent is 1 to 5:1, preferably 2 to 5:1, and more preferably 3 to 5:

1.

9. The method as described in claim 6, characterized in that, The reaction conditions for the nucleic acid amplification reaction are 25-50℃ for 2 min; 95℃ for 30 s; repeated for 35-45 cycles: 95℃ for 0 s, 60℃ for 5 s.

10. The method as described in claim 6, characterized in that, Including the following steps: (s1) Nucleic acid extraction from the sample to be tested; and (s2) The extracted nucleic acid is mixed with the reagent described in claim 1 to carry out a nucleic acid amplification reaction, the fluorescence signal is collected and analyzed to determine whether hepatitis B virus nucleic acid exists in the sample to be tested, and the hepatitis B virus nucleic acid in the sample is quantified by combining the standard curve.

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