CN111206118A

CN111206118A - Kit for detecting hepatitis B virus

Info

Publication number: CN111206118A
Application number: CN202010128133.2A
Authority: CN
Inventors: 刘一博; 金鑫浩; 任鲁风; 张未来; 于军
Original assignee: Ningbo Yinrui Biomedical Instrument Co ltd
Current assignee: Ningbo Yinrui Biomedical Instrument Co ltd
Priority date: 2020-02-28
Filing date: 2020-02-28
Publication date: 2020-05-29

Abstract

The invention relates to the technical field of biology, and discloses a kit for detecting hepatitis B virus, which comprises a primer probe premix solution, a positive quality control product, a negative quality control product and a reaction premix solution. The upstream and downstream primers of HBV-DNA and the upstream and downstream primer sequences of human GAPDH gene used by the kit are respectively shown as SEQ ID No.1-4, and the fluorescent probe of HBV-DNA and the fluorescent probe sequence of human GAPDH gene are respectively shown as SEQ ID No.5 and SEQ ID No. 6. The kit provided by the invention has the advantages of high sensitivity, stability, porosity and accuracy, low false positive reaction, simple operation and high detection efficiency, and can be widely applied to clinical detection of HBV.

Description

Kit for detecting hepatitis B virus

Technical Field

The invention relates to the technical field of biology, in particular to a kit for detecting hepatitis B virus.

Background

Viral hepatitis can be divided into seven categories: hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis C and hepatitis E, wherein the prevalence rate of hepatitis B is the highest. Hepatitis B Virus (HBV) belongs to the hepadnavirus family, and its genome is a circular structure formed by two helical DNA strands, i.e. double-stranded relaxed circular DNA (rcDNA), which includes a negative strand with fixed length and another positive strand with variable length, and its total length is about 3200 bp. After infection of hepatocytes, the semi-circular DNA strand is extended to form a complete circular, i.e. covalently closed circular DNA (cccDNA). The hepatitis B virus infection of the patient causes the liver to be diseased, and further can cause cirrhosis, liver failure, liver cancer and the like. Although the patients do not have great loss on the health in a short period after being infected with the hepatitis B virus, the patients often develop chronic hepatitis B at the onset, the treatment is difficult, and the prognosis is poor. Statistically, about 3.5 million HBV carriers are currently present worldwide, and about 100 million people die of liver failure, liver cirrhosis or liver cancer due to HBV infection every year. In addition, the hepatitis B virus has very strong infectivity, can be transmitted by blood, mother and baby, sex, close contact and other ways, the infectivity is 100 times of that of the AIDS virus, and the infectivity is also the main reason of the large country with hepatitis B in China.

Currently, the detection of HBV mainly includes antigen-antibody specific reaction detection and nucleic acid detection. The commonly used antigen-antibody specific reaction detection is an enzyme-linked immunosorbent assay (ELISA) method, which is simple, rapid and low in cost. However, the method is not accurate and sensitive enough, and cannot quantify, particularly, false negative sometimes occurs on a low-concentration specimen, and the curative effect cannot be dynamically observed and evaluated without quantitative numerical values. Therefore, the method is mainly used for physical examination screening and the like. A commonly used nucleic acid detection is a detection method based on real-time fluorescent quantitative PCR, which detects HBV-DNA, i.e., HBV-cccDNA, in sample blood. The method can carry out quantitative detection on HBV-DNA, has higher sensitivity than an ELISA method, can dynamically reflect the natural process of diseases and the response condition of antiviral treatment, and can also predict the curative effect. However, the method needs to rely on standard substances with different concentrations to prepare a standard curve for quantification, so that the detection cost is greatly increased, and the detection result is relative quantification which cannot be realized absolutely. Meanwhile, operators are required to have certain professional qualities, and the requirements on the laboratory environment are high. In addition, for samples with very low viral load, false negative results appear in the detection, and the sensitivity is greatly reduced.

The digital PCR (digital PCR) technique is the third generation PCR technique that has been developed in recent years, and generally requires that a sample is diluted to a single molecule level and is evenly distributed into tens of thousands to millions of units for reaction, and the concentration of nucleic acid target molecules is calculated through poisson distribution according to the relative proportion of amplified fluorescent signals in each reaction unit and the volume of the reaction unit. The digital PCR technology does not need to adopt reference genes and standard curves, has high accuracy and good reproducibility, can realize absolute quantitative analysis, and greatly improves the detection sensitivity and the detection rate. At present, the more mature digital PCR platform mainly comprises a droplet type digital PCR of Bole company and a chip type digital PCR of Sammer Fei company. However, the operation steps of the digital PCR instrument are multiple and complex, and the micro-drop type digital PCR is inaccurate in detection of ultra-low content objects due to relative loss of liquid drops and needs an additional quality control system; the chip type digital PCR also needs a plurality of instrument combinations, is expensive and inconvenient to operate, and cannot meet the requirement of convenient clinical use.

The existing research for detecting hepatitis B virus nucleic acid based on digital PCR technology is less, syphilis and the like (2018) disclose primers and probes for absolute quantitative detection of hepatitis B virus based on chip-type digital PCR in the establishment and application of the absolute quantitative detection method of hepatitis B virus based on chip-type digital PCR technology, and optimize amplification conditions and a reaction system, wherein the detection limit of chip-type digital PCR is 3.94 multiplied by 10⁴copies/mL, detection range is 3.94 × 10⁴copies/mL-2.82×10⁷copies/mL, relative standard deviation of Repeatability (RSD) 2.89% -13.37%. Chinese patent CN108441578A discloses a micro-drop digital PCR detection method of hepatitis B virus cccDNA and a kit thereof, wherein the kit comprises a pair of specific primers and a fluorescent probe, and hepatitis B virus cccDNA positive control, negative control and the like, and 10 can be detected⁴Samples of copies/mL. Currently available base numbersThe PCR detection of hepatitis B virus has few types of reagents and still has some defects, including difficulty in effectively detecting samples with extremely low DNA content, low sensitivity and the like.

Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and the present invention provides a kit for detecting hepatitis B virus. The kit can effectively detect HBV-DNA (namely HBV-cccDNA) in blood, serum or saliva, and has the advantages of convenient operation and accurate and reliable result.

Disclosure of Invention

The invention aims to provide a kit for detecting hepatitis B virus. The kit can effectively detect the detection object with extremely low concentration, and has high accuracy, sensitivity, porosity and stability of the detection result.

The invention provides a fluorescent probe for detecting hepatitis B virus, which comprises a fluorescent probe for detecting HBV-DNA and a fluorescent probe for detecting human GAPDH gene;

the nucleotide sequence of the fluorescent probe of the HBV-DNA is shown as SEQ ID NO. 5;

the nucleotide sequence of the fluorescent probe of the human GAPDH gene is shown as SEQ ID NO. 6.

Preferably, the 5 th and 10 th bases of the 5' end of the fluorescent probe of HBV-DNA are locked nucleic acid modified bases; the 3 rd and 8 th base positions of the 5' end of the fluorescent probe of the human GAPDH gene are locked with nucleic acid modified bases.

The structural formula of the locked nucleic acid is shown as the formula (1):

preferably, the 5 'end of the fluorescent probe contains a fluorescent reporter group FAM and/or CY3, and the 3' end of the fluorescent probe contains a fluorescent quencher group BHQ-1 and/or BHQ-2;

further preferably, the 5 'end of the fluorescent probe of HBV-DNA contains a fluorescence reporter group FAM, and the 3' end of the fluorescent probe of HBV-DNA contains a fluorescence quencher group BHQ-1; the 5 'end of the fluorescent probe of the human GAPDH gene contains a fluorescence reporter group CY3, and the 3' end of the fluorescent probe of the human GAPDH gene contains a fluorescence quenching group BHQ-2.

The invention also provides a kit for detecting hepatitis B virus, which comprises primer probe premix;

the primer probe premix comprises a primer pair for detecting HBV-DNA, a primer pair of human GAPDH gene, the fluorescent probe of HBV-DNA and the fluorescent probe of human GAPDH gene;

the nucleotide sequences of the upstream primer and the downstream primer of the HBV-DNA primer pair are shown as SEQ ID NO 1 and SEQ ID NO 2;

the nucleotide sequences of the upstream primer and the downstream primer of the primer pair of the human GAPDH gene are shown as SEQ ID NO. 3 and SEQ ID NO. 4.

Preferably, the 5 'end of the fluorescent probe of HBV-DNA and/or human GAPDH gene contains a fluorescence reporter group FAM and/or CY3, and the 3' end of the fluorescent probe contains a fluorescence quencher group BHQ-1 and/or BHQ-2.

Preferably, the concentration ratio of the primer pair for detecting HBV-DNA, the primer pair for detecting human GAPDH gene, the fluorescent probe for HBV-DNA and the fluorescent probe for detecting human GAPDH gene in the primer probe premix is 2:2:0.5-2: 0.5-2.

Further preferably, the concentration ratio of the primer pair for detecting HBV-DNA, the primer pair for detecting human GAPDH gene, the fluorescent probe for HBV-DNA and the fluorescent probe for detecting human GAPDH gene in the primer probe premix is 2:2:1: 1.

Preferably, the kit further comprises a reaction premix, and the reaction premix comprises but is not limited to: dATP, dCTP, dGTP, dUTP, BSA (bovine serum albumin), hot start Taq enzyme, uracil-DNA-glycosylase (UDG enzyme), ROX and single molecule amplification enhancers; the monomolecular amplification enhancer comprises 0.2 percent of TritonX-100 and 0.1-0.3U of thermostable pyrophosphatase by volume ratio.

Further preferably, the reaction premix has dATP, dCTP, dGTP and dUTP concentration of 0.4mM, BSA concentration of 10. mu.g/. mu.L, ROX concentration of 100nM, UDG enzyme concentration of 1U, and hot start Taq enzyme concentration of 0.5-2U.

Preferably, the kit also comprises a positive quality control substance and a negative quality control substance;

the positive quality control product contains 10000 copies/mu L HBV-DNA; the negative quality control product is 10 ng/. mu.L human normal liver cell line DNA.

Specifically, the positive quality control product is prepared by mixing a plasmid containing 20000 copies/. mu.L HBV-DNA fragments and an equal volume of 10 ng/. mu.L human normal liver cell line DNA.

The invention also provides application of the kit in detecting HBV-DNA on a chip-type digital PCR platform.

Compared with the prior art, the invention has the advantages that:

the invention provides a kit for detecting hepatitis B virus, which can be used for a chip-type digital PCR system. The invention carries out locked nucleic acid modification on the fluorescent probe, and optimizes the content of each component in the kit. The standard substance with the final concentration as low as 1 copy/mu L can be effectively distinguished, the technical requirement of 100IU/mL in clinic can be met, and the sensitivity is improved to 10 IU/mL. The kit has the advantages of obviously improved detection sensitivity, stability, porosity and accuracy, effectively reduced reaction false positive, simple operation and high detection efficiency, and can be widely applied to clinical detection of HBV.

Detailed Description

The following description of the embodiments is only intended to aid in the understanding of the method of the invention and its core ideas. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention. The following description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The examples do not show the specific techniques or conditions, and the techniques or conditions are described in the literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruker et al, Huang Petang et al) or according to the product instructions. The digital PCR instrument used in the present invention is the nucleic acid amplification instrument of patent CN 201911061352.7.

Example 1: synthesis of primer probe for detecting hepatitis B virus

A primer pair for detecting HBV-DNA, a primer pair for human GAPDH gene, a fluorescent probe for HBV-DNA and a fluorescent probe for human GAPDH gene were designed and synthesized based on the hepatitis B virus gene (NC-003977.2), as follows:

the nucleotide sequences of the upstream primer and the downstream primer of the primer pair of the HBV-DNA are shown as SEQ ID NO.1 and SEQ ID NO. 2;

the nucleotide sequences of the upstream and downstream primers of the primer pair of human GAPDH gene are shown as SEQ ID NO. 3 and SEQ ID NO. 4.

the nucleotide sequence of the fluorescent probe of human GAPDH gene is shown in SEQ ID NO 6.

The 5 th and 10 th bases of the 5' end of the fluorescent probe of the HBV-DNA are locked nucleic acid modified bases; the 3 rd and 8 th base positions of the 5' end of the fluorescent probe of the human GAPDH gene lock nucleic acid modified bases.

Both primers and probes were synthesized by Shanghai Biotech and purified using HPLC grade. The 5 ' end of the fluorescent probe of HBV-DNA contains a fluorescent reporter group FAM, the 3 ' end contains a fluorescent quenching group BHQ-1, and the 5 th and 10 th basic groups at the 5 ' end are locked nucleic acid modified basic groups; the 5 ' end of the fluorescent probe of the human GAPDH gene contains a fluorescent reporter group CY3, the 3 ' end contains a fluorescent quenching group BHQ-2, and the 3 rd and 8 th base positions of the 5 ' end are locked with nucleic acid modified bases. The fluorescent reporter group and the quencher group can be reasonably selected according to a specific platform.

Example 2: kit for detecting hepatitis B virus

The kit comprises a primer probe premix, a positive quality control product, a negative quality control product and a reaction premix.

Primer probe premix: the concentrations of the HBV-DNA upstream and downstream primers synthesized in example 1 and the human GAPDH gene upstream and downstream primers were 2. mu.M; the concentrations of the HBV-DNA fluorescent probe and the human GAPDH gene fluorescent probe are both 1 mu M, and primer probe premix liquid is prepared by mixing.

Positive quality control product: (1) artificially synthesizing a target fragment containing HBV-DNA with the length of 500bp, connecting and loading the target fragment into a plasmid pUC57 (Promega), transferring into escherichia coli, screening positive bacteria for cloning, extracting and purifying the plasmid, cutting the plasmid into linear by using restriction enzyme, preparing a plasmid containing the HBV-DNA fragment of 20000 copies/mu L, and diluting the plasmid with physiological saline to the concentration of 10 ng/mu L;

(2) culturing a human normal liver cell line L-02 cell line, extracting DNA, quantifying by using the Qubit 3.0, and diluting to the concentration of 10 ng/. mu.L by using physiological saline as a background;

(3) and (3) mixing the plasmid with the concentration of 10 ng/mu L obtained in the step (1) and the background DNA obtained in the step (2) in equal volume to prepare a mixed solution containing 10000 copies/mu L of HBV-DNA, namely the positive quality control product.

Negative quality control product: culturing human normal liver cell line L-02 cell line, extracting DNA, using Qubit 3.0 to make quantification, diluting to 10 ng/microliter concentration, and obtaining negative quality control product.

The reaction premix comprises: dATP, dCTP, dGTP and dUTP concentrations are all 0.4mM, BSA concentration is 10 mug/muL, ROX concentration is 100nM, UDG enzyme concentration is 1U, hot start Taq enzyme concentration is 1U and single molecule amplification enhancer; the single-molecule amplification enhancer comprises 0.2 percent by volume of TritonX-100 and 0.2U of thermostable pyrophosphatase.

Example 3: method for detecting hepatitis B virus

(1) Preparation of digital PCR reaction system: mixing a DNA template to be detected with a primer probe premix solution and a reaction premix solution in the kit of the embodiment 2 according to a reaction system in the table 1, uniformly mixing the mixture for 15s by using soft vortex, and collecting the solution to the bottom of a test tube by instantaneous centrifugation to obtain a digital PCR reaction system.

Table 1: reaction system

(2) And (2) loading the reaction system obtained in the step (1) onto a PCR chip to form a micro-reaction unit. And (3) carrying out reaction by using a digital PCR instrument according to the PCR reaction conditions in the table 2 to obtain an amplification product.

Table 2: PCR reaction conditions

(3) Collecting the fluorescence signals of the amplification products obtained in the step (2), and selecting FAM and CY3 as the channels for fluorescence detection. And judging and reading the effective fluorescence positive points of the two channels, analyzing and detecting the content of the DNA template of the hepatitis B virus.

Example 4: detection of HBV-DNA standards

The preparation of HBV-DNA standard substance comprises the following steps:

(1) artificially synthesizing target fragment containing HBV-DNA with length of 500bp, connecting and loading it into plasmid pUC57 (Promega), transferring it into colibacillus, screening positive bacteria, cloning, extracting and purifying plasmid, and usingThe plasmid was cut into linear fragments by restriction endonuclease and prepared to have a copy number concentration of 2X 10^1-7copies/. mu.L of plasmid solution of HBV-DNA target fragment, diluted with physiological saline to a concentration of 10 ng/. mu.L;

(3) mixing the plasmid solution obtained in step (1) at a concentration of 10 ng/. mu.L with the background DNA obtained in step (2) at the same volume to prepare a mixture containing 1X 10^1-7copies/. mu.L of HBV-DNA standard.

The HBV-DNA standards of 7 different concentration gradients obtained above, the negative quality control in the kit described in example 2 and the blank (i.e., physiological saline) were tested by the method in example 3. The results of the experiment are shown in table 3.

Table 3: test results of the standards

The result shows that the kit provided by the invention can effectively detect a sample with the final concentration of DNA of 1 ng/mu L, can effectively distinguish a standard substance with the final concentration as low as 1 copy/mu L, and has the advantages of obviously improved hole entry rate, high accuracy and no false positive reaction, wherein the hole entry rate is 98.2%.

Example 5: kit for detecting hepatitis B virus

The kit is only different from the kit described in the embodiment 2 in that the concentrations of the HBV-DNA fluorescent probe and the human GAPDH gene fluorescent probe in the primer probe premix are both 0.5 mu M; the concentration of hot start Taq enzyme in the reaction premix is 0.5U; the heat-stable pyrophosphatase in the monomolecular amplification enhancer was 0.1U. The kit is used for detecting the standard substance in the example 4, the detection method is the method described in the example 3, and the detection results are shown in table 4.

Table 4: test results of the standards

The results show that: the kit provided by the invention can effectively detect a sample with the final concentration of DNA of 1 ng/muL, can effectively distinguish a standard substance with the final concentration as low as 1 copy/muL, and has the advantages of obviously improved hole entry rate, 96.3% hole entry rate, high accuracy and no false positive reaction.

Example 6: kit for detecting hepatitis B virus

The kit is only different from the kit described in the embodiment 2 in that the concentrations of the HBV-DNA fluorescent probe and the human GAPDH gene fluorescent probe in the primer probe premix are both 2 mu M; the concentration of hot start Taq enzyme in the reaction premix is 2U; the heat-stable pyrophosphatase in the monomolecular amplification enhancer was 0.3U. The kit was used to detect the standards in example 4 by the method described in example 3, and the results are shown in table 5.

Table 5: test results of the standards

The results show that: the kit provided by the invention can effectively detect a sample with the final concentration of DNA of 1 ng/muL, can effectively distinguish a standard substance with the final concentration as low as 1 copy/muL, and has the advantages of remarkably improved hole entry rate, 97.2% hole entry rate, high accuracy and no false positive reaction.

Example 7: kit for detecting hepatitis B virus

The kit is only different from the kit described in the embodiment 2 in that the concentrations of the HBV-DNA fluorescent probe and the human GAPDH gene fluorescent probe in the primer probe premix are both 1 mu M; the concentration of hot start Taq enzyme in the reaction premix is 2U; the heat-stable pyrophosphatase in the monomolecular amplification enhancer was 0.1U. The kit was used to detect the standards in example 4 by the method described in example 3, and the results are shown in table 6.

Table 6: test results of the standards

The results show that: the kit provided by the invention can effectively detect a sample with the final concentration of DNA of 1 ng/muL, can effectively distinguish a standard substance with the final concentration as low as 1 copy/muL, and has the advantages of remarkably improved hole entry rate, 97.7% hole entry rate, high accuracy and no false positive reaction.

Example 8: kit for detecting hepatitis B virus

The kit is only different from the kit described in the embodiment 2 in that the concentrations of the HBV-DNA fluorescent probe and the human GAPDH gene fluorescent probe in the primer probe premix are both 0.5 mu M; the concentration of hot start Taq enzyme in the reaction premix is 0.5U; the heat-stable pyrophosphatase in the monomolecular amplification enhancer was 0.3U. The kit was used to detect the standards in example 4 by the method described in example 3, and the results are shown in table 7.

Table 7: test results of the standards

The results show that: the kit provided by the invention can effectively detect a sample with the final concentration of DNA of 1 ng/muL, can effectively distinguish a standard substance with the final concentration as low as 1 copy/muL, and has the advantages of obviously improved hole entry rate, 96.6% hole entry rate, high accuracy and no false positive reaction.

Example 9: sample detection of clinical hepatitis B patients

Blood of 200 clinically confirmed hepatitis B patients was collected in an amount of 5mL, and blood samples were tested using the kit of example 2 and the method of example 3. Meanwhile, 5mL of blood is taken, the existing detection kit on the market is used as a contrast reagent for comparative detection, and a gold standard sequencing method is adopted for rechecking.

The method for extracting the DNA in the sample comprises the following steps: 1mL of blood sample was aspirated from the blood collection tube, and DNA in the sample was extracted and purified using QIAamp DNA BloodMini Kit (purchased from QIAGEN, Cat number/ID: 51104). After the quantit is quantified, the total amount is confirmed to be not less than 20ng, and 20 samples are selected from 200 samples as a display, wherein 2 samples with divergence are included. The results are shown in Table 8.

Table 8: test results of clinical specimens

The detection result shows that: the kit provided by the invention has the advantages that the detection result of the kit has 200 positive cases, and the false positive rate is 0; the detection result of the existing detection kit on the market has 198 positive cases, the false positive rate is 0, and the false negative rate is 1%; after rechecking, the accuracy of the kit provided by the invention is 100%, and the accuracy of the existing detection kit on the market is 99%.

The kit provided by the invention not only can meet the technical requirement of 100IU/mL clinically, but also can improve the sensitivity to 10 IU/mL. Can meet the technical requirements of clinical ultra-early screening, and can be widely applied to the dynamic monitoring of HBV activity and the evaluation of the curative effect of antiviral drugs in clinic. Compared with the existing detection kit, the detection kit has the advantages of more convenient operation, stable and reliable data result and greatly reduced detection cost.

The kit provided by the invention can effectively detect HBV-DNA samples with extremely low concentration, can effectively distinguish standard substances with final concentration as low as 1 copy/mu L, has a pore-entering rate of over 96 percent, high accuracy, good stability and no reaction false positive.

Comparative example 1 kit for detecting hepatitis B virus

The kit is different from the kit described in example 2 only in that the concentrations of the HBV-DNA fluorescent probe and the human GAPDH gene fluorescent probe in the primer probe premix are both 0.3. mu.M. The kit was used to detect the standards in example 4 by the method described in example 3, and the results are shown in table 9.

Table 9: the result of the detection

Note: "-" indicates no detection.

The detection result shows that: the kit can effectively distinguish the standard substance with the final concentration as low as 1000 copies/mu L, the hole entering rate is 96.3 percent, the false negative rate of the reaction is high, and the accuracy is low.

Comparative example 2 kit for detecting hepatitis B virus

This kit differs from the kit described in example 2 only in that the concentration of hot start Taq enzyme in the reaction premix was 0.3U. The kit was used to detect the standards in example 4 by the method described in example 3, and the results are shown in table 10.

Table 10: the result of the detection

Note: "-" indicates no detection.

The detection result shows that: the kit can effectively distinguish the standard substance with the final concentration of 100 copies/mu L, the hole entering rate is 96.5%, the false negative rate of the reaction is high, and the accuracy is low.

Comparative example 3 kit for detecting hepatitis B virus

This kit differs from the kit described in example 2 only in that the heat stable pyrophosphatase enzyme in the single molecule amplification enhancer was 0.5U. The kit was used to detect the standards in example 4 by the method described in example 3, and the results are shown in table 11.

Table 11: the result of the detection

Note: "-" indicates no detection.

The detection result shows that: the kit can effectively distinguish the standard substance with the final concentration of 10 copies/mu L, the hole entering rate is 96.8 percent, the false negative rate of the reaction is high, and the accuracy is low.

Comparative example 4 kit for detecting hepatitis B virus

This kit differs from the kit described in example 2 only in that the fluorescent probe used is not modified by a locked nucleic acid. The kit was used to detect the standard described in example 4, and the detection method was the method described in example 3. The results are shown in Table 12.

Table 12: the result of the detection

Note: "-" indicates no detection.

The detection result shows that: the kit provided by the invention can detect a sample with the final concentration of DNA of only 1 ng/mu L, can effectively distinguish a standard substance with the final concentration of 100 copies/mu L, has a pore-entering rate of 96.5%, a high false negative rate of reaction, low accuracy and poor stability.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

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Claims

1. A fluorescent probe for detecting hepatitis B virus, which comprises a fluorescent probe for detecting HBV-DNA and a fluorescent probe for human GAPDH gene;

2. The fluorescent probe of claim 1, wherein the 5 th and 10 th bases of the 5' end of the fluorescent probe of HBV-DNA are locked nucleic acid modified bases;

the 3 rd and 8 th base positions of the 5' end of the fluorescent probe of the human GAPDH gene are locked with nucleic acid modified bases.

3. The fluorescent probe according to claim 1, wherein the fluorescent probe comprises a fluorescent reporter group FAM and/or CY3 at the 5 'end, and a fluorescent quencher group BHQ-1 and/or BHQ-2 at the 3' end.

4. The kit for detecting the hepatitis B virus is characterized by comprising primer probe premix;

the primer probe premix comprises a primer pair for detecting HBV-DNA, a primer pair of human GAPDH gene, the fluorescent probe for HBV-DNA and the fluorescent probe for human GAPDH gene according to claim 1;

5. The kit according to claim 4, wherein the fluorescent probe of HBV-DNA and/or human GAPDH gene comprises a fluorescent reporter group FAM and/or CY3 at its 5 'end and a fluorescent quencher group BHQ-1 and/or BHQ-2 at its 3' end.

6. The kit according to claim 4, wherein the concentration ratio of the primer pair for detecting HBV-DNA, the primer pair for human GAPDH gene, the fluorescent probe for HBV-DNA and the fluorescent probe for human GAPDH gene in the primer probe premix is 2:2:0.5-2: 0.5-2.

7. The kit of claim 4, further comprising a reaction premix;

the reaction premix comprises: dATP, dCTP, dGTP, dUTP, BSA, hot start Taq enzyme, uracil-DNA-glycosylase, ROX and a single molecule amplification enhancer;

the monomolecular amplification enhancer comprises 0.2 percent of TritonX-100 and 0.1-0.3U of thermostable pyrophosphatase by volume ratio.

8. The kit according to claim 7, wherein the reaction premix contains dATP, dCTP, dGTP and dUTP in a concentration of 0.4mM, BSA in a concentration of 10. mu.g/. mu.L, ROX in a concentration of 100nM, UDG in a concentration of 1U, and Hot Start Taq in a concentration of 0.5-2U.

9. The kit of claim 4, wherein the kit further comprises a positive quality control material and a negative quality control material;

the positive quality control product contains 10000 copies/mu L HBV-DNA;

the negative quality control product is 10 ng/. mu.L human normal liver cell line DNA.

10. Use of the kit according to any one of claims 1 to 9 for the detection of HBV-DNA on a chip-based digital PCR platform.