CN111118222B - HBV detection kit and using method and application thereof - Google Patents

HBV detection kit and using method and application thereof Download PDF

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Publication number
CN111118222B
CN111118222B CN202010122229.8A CN202010122229A CN111118222B CN 111118222 B CN111118222 B CN 111118222B CN 202010122229 A CN202010122229 A CN 202010122229A CN 111118222 B CN111118222 B CN 111118222B
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hbv
detection kit
nucleic acid
kit according
sample
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CN111118222A (en
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吴燕
邓京
欧格
勾宏娜
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Zhuhai Livzon Diagnostics Inc
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Zhuhai Livzon Diagnostics Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Abstract

The invention provides an HBV detection kit and a using method and application thereof, relating to the technical field of molecular biology. The HBV detection kit provided by the invention comprises a nucleic acid amplification reagent and a sample treatment solution, and PCR amplification detection can be carried out only by directly adding a sample to be detected and the sample treatment solution into the nucleic acid amplification reagent during detection, so that the whole process of operation of the kit is greatly simplified. And avoids the loss of nucleic acid caused by multi-step operation or technical problems in the process of extracting and purifying nucleic acid to the maximum extent, and the operation steps are simple and convenient, and large-scale precise nucleic acid extracting and purifying equipment is not needed. In addition, the nucleic acid amplification reagent provided by the invention comprises enzyme, dNTPs, metal cations, a protein protective agent, an HBV specific primer, an HBV specific probe and a buffer system, and the components are matched for use, so that the nucleic acid amplification reagent provided by the invention can be used for specifically and efficiently amplifying a sample to be detected in a PCR reaction.

Description

HBV detection kit and using method and application thereof
Technical Field
The invention relates to the technical field of molecular biology, in particular to an HBV detection kit and a using method and application thereof.
Background
With the development of molecular biological detection technology, fluorescent quantitative PCR technology is increasingly applied to the detection of clinical pathogens. At present, various Hepatitis B Virus (HBV) nucleic acid quantitative detection kits based on fluorescent quantitative PCR technology are applied to clinical detection in China. HBV nucleic acid detection usually comprises two parts of nucleic acid extraction and purification and nucleic acid amplification detection.
Currently, the methods for extracting and purifying HBV nucleic acid on the market are mainly classified into the following 4 types: (1) boiling method: adding the sample (or the concentrated sample) into the lysate, heating to boil, centrifuging at high speed, and taking part of the supernatant for amplification detection. The method has the advantages of small effective sample amount, low detection sensitivity, and easy pollution and false positive due to high-temperature boiling and high-speed centrifugation. (2) centrifugal column extraction method: after the sample is cracked, the nucleic acid is adsorbed on a silicon dioxide membrane, the lysate is removed, washing is carried out for 2-3 times, and then the nucleic acid is eluted and used as a template for amplification detection. The nucleic acid extracted by the column extraction method has high purity, but the steps are complicated, the automation is not easy, the efficiency is lower, and the multi-step operation is easy to cross-pollute. (3) magnetic bead method: after the sample is cracked, the nucleic acid is adsorbed to the surface of the magnetic bead, the cracking solution is removed through magnetic separation, washing is carried out for 1-3 times, and then the nucleic acid is eluted and used as a template for amplification detection. The magnetic bead method can realize automation, but the requirement on automation hardware is high, multi-step operation is still needed, the time consumption is still long, and meanwhile, the risk of cross contamination also exists. (4) room temperature cracking method: and (3) uniformly mixing the sample to be detected with a lysis solution (a nucleic acid releasing agent), treating at room temperature for 5-10 minutes, and adding part or all of the lysis mixture into a PCR reaction reagent for amplification detection. Although the method omits the complicated steps of enrichment and purification and saves labor, the method still needs several minutes of cracking treatment, and the treatment time of different samples can be different when a plurality of samples are treated simultaneously, thereby influencing the stability of the result.
On the other hand, the HBV nucleic acid amplification detection reagents on the market at present are multi-component liquid reagents, are poor in stability and high in storage requirement, and most of the reagents need to be transported and stored at the temperature of-20 ℃. Meanwhile, before use, a plurality of reagents need to be prepared together and uniformly mixed and then subpackaged in a PCR reaction tube, so that the operation is complicated, preparation errors exist among different people, and the risk of pollution exists simultaneously. In order to solve the above problems, a single-tube packaged, ready-to-use, HBV amplification detection kit which does not require any sample treatment, performs both the release of nucleic acid cleavage and the detection of PCR amplification, and has good stability is required.
Application numbers 201811247122.5 and 201811247216.2 disclose a kit for direct extraction and amplification reaction detection. The kit adds a sample to be detected into lysate to realize the cracking and purification of nucleic acid, and the cracked product is directly added into a ready-to-use freeze-dried powder reagent to realize the amplification detection. The method solves the problems of complicated liquid preparation, poor stability, pollution risk and the like of the multi-component liquid reagent. However, the kit still needs to mix the sample and the lysis solution uniformly in advance and invert the mixture for 3 to 5 times, then liquid phase is taken for PCR amplification detection, time is consumed when the sample amount is large, and automation is not easy to realize. In addition, the lysis solution of the kit contains organic solvents such as polyethylene glycol octyl phenyl ether, phenol and the like, which are harmful to human bodies and pollute the environment. On the other hand, the ratio of the lysis solution to the sample in the method is 5, only the liquid phase is taken to be added into the PCR reaction reagent for reaction after lysis, and a certain volume of water is also added, so that the effective sample addition amount is far less than one sixth, and the improvement of the detection sensitivity is greatly limited. Application No. CN201910541268.9 discloses a PCR reaction system and reagents, which can directly add the unlysed sample (or resuspend the unlysed and/or unleaded sample) into a PCR reaction system for reaction, wherein the PCR reaction system contains one or more surfactants, one or more reducing agents, one or more saccharides, one or more alcohols, and also contains an anti-inhibitor, a ribonuclease inhibitor and components required for conventional PCR reaction, and the detectable sample type includes serum, plasma, whole blood, swab, cell or body fluid. The method solves the problem that the sample needs to be pretreated, the sample can be directly added into the PCR reaction reagent, and the proportion of the plasma sample can reach 25 percent of the reaction volume. However, the method only solves the problem that the sample does not need to be processed, and does not solve the problems that the multi-component distribution liquid of the PCR reaction reagent is complex and has high storage and transportation requirements.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
It is a first object of the present invention to provide an HBV detection kit which alleviates at least one of the technical problems of the prior art.
The second purpose of the invention is to provide a using method of the HBV detection kit.
The third purpose of the invention is to provide the application of the HBV detection kit.
The invention provides an HBV detection kit, which comprises a nucleic acid amplification reagent and a sample treatment solution;
the HBV nucleic acid amplification reagent comprises: enzyme, dNTPs, metal cations, a protein protective agent, an HBV specific primer, an HBV specific probe and a buffer system.
Further, the nucleic acid amplification reagent is a dry powder reagent, preferably a freeze-dried powder reagent;
preferably, the enzymes include DNA polymerase and UNG enzyme; the DNA polymerase is preferably a hot start Taq enzyme;
preferably, the dNTPs comprise dATP, dCTP, dGTP and dUTP;
preferably, the metal cations include magnesium ions and potassium ions;
preferably, the protein protectant comprises mannitol and/or sucrose;
preferably, the buffer system comprises a buffer system with a pH of 7.8-8.6, preferably a Tris-HCl buffer system;
preferably, the concentration of the hot start Taq enzyme is 3-10U/reaction, preferably 5-8U/reaction;
preferably, the concentration of UNG enzyme is 0.3-1U/reaction, preferably 0.5-0.8U/reaction;
preferably, the concentrations of dATP, dCTP and dGTP are 200-300 μ M independently, and the concentration of dUTP is 350-450 μ M;
preferably, the concentration of the magnesium ions is 1-10mM, preferably 3-6mM;
preferably, the concentration of potassium ions is 45-55mM, preferably 50mM;
preferably, the mannitol concentration is 0.2% -2% w/V, preferably 0.5% -1.8% w/V, more preferably 0.7% -1.5% w/V;
preferably, the sucrose concentration is 5% -20% w/V, preferably 8% -18% w/V, more preferably 10.5% -16% w/V;
preferably, the concentration of the Tris-HCl buffer system is 10-30mM, preferably 20mM.
Further, the HBV specific primers comprise a first primer pair and/or a second primer pair;
the first primer pair comprises primers of nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO. 2;
the second primer pair comprises primers of nucleotide sequences shown as SEQ ID NO.3 and SEQ ID NO. 4;
preferably, the HBV-specific probe comprises a first probe and/or a second probe;
the first probe has a nucleotide sequence shown as SEQ ID NO. 5;
the second probe has a nucleotide sequence shown as SEQ ID NO. 6;
preferably, two ends of the HBV specific probe are respectively marked with a fluorescent group and a quenching group;
preferably, the nucleic acid amplification reagents further comprise an internal standard-specific probe;
preferably, the internal standard specific probe has a nucleotide sequence shown as SEQ ID NO. 7;
preferably, two ends of the internal standard specific probe are respectively marked with a fluorescent group and a quenching group, and the fluorescent group in the internal standard specific probe is different from the fluorescent group in the HBV specific probe;
preferably, the concentration of each primer sequence of the HBV-specific primers is independently 0.5-1. Mu.M, preferably 0.6. Mu.M;
preferably, the concentration of each probe sequence of the HBV-specific probes is independently 0.1-0.2. Mu.M, preferably 0.1. Mu.M.
Further, the nucleic acid amplification reagent comprises: 5U/reaction Hot Start Taq enzyme, 0.5U/reaction UNG enzyme, dATP, dCTP and dGTP concentrations each independently of 250. Mu.M, 400. Mu.M dUTP, 5mM MgCl 2 50mM KCl, 1.0% w/V mannitol, 12.5% w/V sucrose, primers represented by SEQ ID nos. 1 to 4 at concentrations each independently of 0.6. Mu.M, probes represented by SEQ ID nos. 5 to 6 at concentrations each independently of 0.1. Mu.M, and a 20mM Tris-HCl buffer system, the pH of the nucleic acid amplification reagent being 8.2.
Further, the sample processing liquid includes: surfactants and reducing agents.
Further, the surfactant comprises an ionic surfactant and a non-ionic surfactant;
preferably, the ionic surfactant comprises an anionic surfactant, preferably comprising sodium lauryl sulfate;
preferably, the non-ionic surfactant comprises Triton X-100 and Tween 80;
preferably, the reducing agent comprises a reducing sugar, preferably lactose;
preferably, the concentration of sodium lauryl sulfate is 0.01% -0.09% w/V;
preferably, the concentration of said Triton X-100 is 0.02% -0.08% V/V;
preferably, the concentration of Tween80 is 0.01% -0.08%/V;
preferably, the lactose concentration is 2% -12% w/V, preferably 4% -10% w/V, more preferably 5% -9% w/V;
preferably, the sample processing fluid further comprises dimethyl sulfoxide;
preferably, the concentration of dimethyl sulfoxide is 0.01% -0.1% w/V, preferably 0.03% -0.08% w/V, more preferably 0.04% -0.07% w/V;
preferably, the sample processing liquid includes: 0.03% w/V sodium dodecyl sulfate, 0.06% V/V Triton X-100, 0.02% V/V Tween80, 0.05% w/V dimethyl sulfoxide and 6% w/V lactose.
Further, the kit also comprises an internal standard solution, wherein the internal standard solution comprises an internal standard sequence, and two ends of the internal standard sequence comprise sequences identical to HBV specific primer sequences; the middle of the internal standard sequence comprises a complementary sequence which is the same as or opposite to the internal standard specific probe sequence;
preferably, the HBV detection kit further comprises a quality control product;
preferably, the quality control material comprises at least one of a negative quality control material, a critical positive quality control material and a strong positive quality control material.
The invention also provides a using method of the HBV detection kit, wherein a sample to be detected and the sample treatment solution are added into the nucleic acid amplification reagent, and after uniform mixing, the obtained reaction solution is placed in a PCR instrument for amplification detection.
Further, adding a sample to be detected, a sample treatment solution and an internal standard solution into the nucleic acid amplification reagent, uniformly mixing, and placing the obtained reaction solution into a PCR instrument for amplification detection;
preferably, the volume of the sample to be detected is 5% -40% of the volume of the reaction solution, and is preferably 15% -30%;
preferably, the volume of the sample processing solution is 5% -60%, preferably 10% -50% of the volume of the reaction solution;
preferably, the sample to be tested is a plasma sample or a serum sample.
In addition, the invention also provides the application of the HBV detection kit in preparing products for detecting HBV;
preferably, the detection is a quantitative detection.
The HBV detection kit provided by the invention comprises a nucleic acid amplification reagent and a sample treatment solution, and PCR amplification detection can be carried out only by directly adding a sample to be detected and the sample treatment solution into the nucleic acid amplification reagent during detection, so that not only is a complicated nucleic acid extraction and purification or sample cracking step omitted, but also a preparation step of a PCR reaction solution is omitted, the effect of instant use is effectively achieved, and the whole process of kit operation is greatly simplified. In addition, the sample to be detected is directly added into the nucleic acid amplification reagent, so that the nucleic acid loss caused by multi-step operation or technical problems in the nucleic acid extraction and purification process is avoided to the maximum extent, the operation steps are simple and convenient, large-scale and precise nucleic acid extraction and purification equipment is not needed, the operation can be completed only by a conventional pipette, and the requirements on the equipment are greatly reduced. Meanwhile, the extraction steps are simplified, and the operation time and the consumed manpower are greatly shortened. In addition, the nucleic acid amplification reagent provided by the invention comprises enzyme, dNTPs, metal cations, a protein protective agent, an HBV specific primer, an HBV specific probe and a buffer system, and the components are matched for use, so that the nucleic acid amplification reagent provided by the invention can be used for specifically and efficiently amplifying a sample to be detected in a PCR reaction. Meanwhile, the reasonable use of each component also enables the enzyme component in the composition to effectively keep high activity, prolongs the storage time and effectively reduces the storage cost of the reagent.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1A is a diagram illustrating the result of the fluorescent quantitative PCR experiment of formulation 1 provided in example 1 of the present invention;
FIG. 1B is a diagram showing the result of a fluorescence quantitative PCR experiment of formulation 2 according to example 1 of the present invention;
FIG. 1C is a diagram showing the result of a fluorescent quantitative PCR experiment of formulation 3 provided in example 1 of the present invention;
FIG. 1D is a diagram showing the result of a fluorescent quantitative PCR experiment of formulation 4 provided in example 1 of the present invention;
FIG. 1E is a diagram showing the result of a fluorescent quantitative PCR experiment of formulation 5 provided in example 1 of the present invention;
FIG. 1F is a graph showing the result of a fluorescent quantitative PCR experiment of formulation 6 according to example 1 of the present invention;
FIG. 2A is a graph showing the results of a fluorescent quantitative PCR experiment of formulation 7 according to example 2 of the present invention;
FIG. 2B is a graph showing the result of a fluorescent quantitative PCR experiment of formulation 8 according to example 2 of the present invention;
FIG. 2C is a graph showing the result of a fluorescent quantitative PCR experiment of formulation 9 provided in example 2 of the present invention;
FIG. 2D is a graph showing the result of the fluorescent quantitative PCR experiment of the formulation 10 provided in example 2 of the present invention;
FIG. 2E is a diagram showing the result of the fluorescent quantitative PCR experiment of formulation 11 provided in example 2 of the present invention;
FIG. 2F is a graph showing the result of the fluorescent quantitative PCR experiment of formulation 12 provided in example 2 of the present invention;
FIG. 2G is a graph showing the result of a fluorescent quantitative PCR experiment of formulation 13 provided in example 2 of the present invention;
FIG. 2H is a graph showing the result of a fluorescent quantitative PCR assay for formulation 14 provided in example 2 of the present invention;
FIG. 2I is a graph showing the result of a fluorescent quantitative PCR experiment of the formulation 15 provided in example 2 of the present invention;
FIG. 3A is a graph showing the result of a quantitative PCR assay in the control group according to example 3 of the present invention;
FIG. 3B is a graph showing the result of the fluorescent quantitative PCR experiment of the formulation 16 provided in example 3 of the present invention;
FIG. 3C is a graph showing the results of a fluorescent quantitative PCR experiment of formulation 17 provided in example 3 of the present invention;
FIG. 3D is a graph showing the result of the fluorescent quantitative PCR assay of formulation 18 according to example 3 of the present invention;
FIG. 4A is a graph showing the results of a fluorescence quantitative PCR assay for formulation 19 provided in example 4 of the present invention;
FIG. 4B is a graph showing the results of a fluorescence quantitative PCR experiment of the formulation 20 provided in example 4 of the present invention;
FIG. 4C is a graph showing the results of a fluorescent quantitative PCR experiment of formulation 21 provided in example 4 of the present invention;
FIG. 4D is a graph showing the result of the fluorescent quantitative PCR experiment of the formulation 22 provided in example 4 of the present invention;
FIG. 4E is a graph showing the results of a fluorescent quantitative PCR experiment of formulation 23 provided in example 4 of the present invention;
fig. 5A is an external view of a lyophilized powder formulation of formulation 24 provided in example 5 of the present invention;
FIG. 5B is an external view of a lyophilized powder preparation of formulation 25 provided in example 5 of the present invention;
FIG. 5C is an external view of a lyophilized powder preparation of formulation 26 provided in example 5 of the present invention;
fig. 5D is an external view of a lyophilized powder formulation of formulation 27 provided in example 5 of the present invention;
FIG. 5E is an external view of a lyophilized powder formulation of formulation 28 provided in example 5 of the present invention;
FIG. 5F is an external view of a lyophilized powder formulation of formulation 29 provided in example 5 of the present invention;
fig. 5G is an appearance diagram of a lyophilized powder formulation of formulation 30 provided in example 5 of the present invention;
FIG. 6A is a graph showing the results of a fluorescence quantitative PCR experiment of formulation 25 provided in example 6 of the present invention;
FIG. 6B is a graph showing the result of the fluorescent quantitative PCR assay of the formulation 26 provided in example 6 of the present invention;
FIG. 6C is a graph showing the results of a fluorescence quantitative PCR assay for formulation 27 provided in example 6 of the present invention;
FIG. 6D is a graph showing the result of a fluorescent quantitative PCR experiment of the formulation 28 provided in example 6 of the present invention;
FIG. 6E is a graph showing the results of a fluorescence quantitative PCR assay for formulation 29 provided in example 6 of the present invention;
FIG. 7A is a graph showing the results of a fluorescence quantitative PCR experiment of the formulation 30 provided in example 7 of the present invention;
FIG. 7B is a graph showing the results of a fluorescence quantitative PCR assay for formulation 31 provided in example 7 of the present invention;
FIG. 7C is a graph showing the results of a fluorescent quantitative PCR experiment of formulation 32 provided in example 7 of the present invention;
FIG. 7D is a graph showing the results of a fluorescent quantitative PCR experiment of the formulation 33 provided in example 7 of the present invention;
FIG. 7E is a graph showing the results of a fluorescence quantitative PCR assay for formulation 34 provided in example 7 of the present invention;
FIG. 7F is a graph showing the results of a fluorescent quantitative PCR experiment of formulation 35 provided in example 7 of the present invention;
FIG. 7G is a graph showing the result of the fluorescent quantitative PCR experiment of the formulation 36 provided in example 7 of the present invention;
FIG. 8A is a diagram showing the result of a fluorescence quantitative PCR assay on a quantitative standard substance of a commercial kit according to example 11 of the present invention;
FIG. 8B is a standard curve of a commercial kit according to example 11 of the present invention;
FIG. 8C is a diagram showing the result of a fluorescence quantitative PCR assay on a national standard of a commercial kit provided in example 11 of the present invention;
FIG. 8D is a graph showing the result of a fluorescent quantitative PCR assay with a commercial kit of 60IU/mL according to example 11 of the present invention;
FIG. 8E is a graph showing the result of a fluorescent quantitative PCR assay with 30IU/mL in a commercial kit according to example 11 of the present invention;
FIG. 8F is a diagram showing the result of a fluorescent quantitative PCR assay performed on a national standard substance of the kit of the present invention provided in example 11 of the present invention;
FIG. 8G is a graph showing the result of a fluorescent quantitative PCR experiment of 60IU/mL using the kit of the present invention provided in example 11 of the present invention;
FIG. 8H is a graph showing the result of a fluorescent quantitative PCR experiment using 30IU/mL of the kit of the present invention provided in example 11 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
According to an aspect of the present invention, there is provided an HBV detection kit comprising a nucleic acid amplification reagent and a sample-treating solution;
the HBV nucleic acid amplification reagent comprises: enzyme, dNTPs, metal cations, protein protective agents, HBV specific primers, HBV specific probes and a buffer system.
According to the HBV detection kit provided by the invention, during detection, PCR amplification detection can be carried out only by directly adding a sample to be detected and a sample treatment solution into a nucleic acid amplification reagent, so that not only is a complicated nucleic acid extraction and purification or sample cracking step omitted, but also a preparation step of a PCR reaction solution is omitted, the effect of instant use is effectively achieved, and the whole process of the kit operation is greatly simplified. In addition, the sample to be detected is directly added into the nucleic acid amplification reagent, so that the nucleic acid loss caused by multi-step operation or technical problems in the nucleic acid extraction and purification process is avoided to the maximum extent, the operation steps are simple and convenient, large-scale and precise nucleic acid extraction and purification equipment is not needed, the operation can be completed only by a conventional pipette, and the requirements on the equipment are greatly reduced. Meanwhile, the extraction steps are simplified, and the operation time and the consumed manpower are greatly shortened. In addition, the components in the nucleic acid amplification reagent provided by the invention are matched for use, so that the nucleic acid amplification reagent provided by the invention can be used for specifically and efficiently amplifying a sample to be detected in a PCR reaction. Meanwhile, the reasonable use of each component also enables the enzyme component in the composition to effectively keep high activity, prolongs the storage time and effectively reduces the storage cost of the reagent.
Optionally, the nucleic acid amplification reagent can be separately pre-loaded in the PCR reaction tube, and when the HBV detection kit provided by the invention is used for detection, a sample to be detected and the sample treatment solution are directly added into the PCR reaction tube filled with the nucleic acid amplification reagent, so that the reaction reagent does not need to be transferred, and the detection error possibly caused by multi-step operation can be effectively avoided.
The sample treatment solution provided by the present invention is not limited to specific components, and any sample treatment solution that can promote sample lysis and nucleic acid release may be used, and preferably includes a surfactant and a reducing agent.
In some preferred embodiments, the nucleic acid amplification reagent is a dry powder reagent, preferably a lyophilized powder reagent. The nucleic acid amplification reagent in the form of freeze-dried powder can ensure that all components in the HBV detection kit provided by the invention can be stored for at least 24 months at the storage temperature of 2-8 ℃, thereby greatly reducing the cost of reagent transportation and storage.
Lyophilization is a complex phase transition process, and various stresses (including low temperature stress, freezing stress, drying stress, etc.) during lyophilization are often factors that directly or indirectly cause denaturation of proteins in lyophilized products, and thus the use of protein protectants in lyophilized products is of paramount importance. The enzyme in the amplification reaction reagent provided by the invention is protein, and whether the enzyme is denatured or not is directly related to the activity of the reaction reagent. Therefore, in order to maintain a high degree of activity of the enzyme, mannitol and/or sucrose are preferably used as the protein protectant in this embodiment.
Mannitol is a polyhydroxy compound, has stable property, is not easy to be oxidized, forms crystals when being frozen at a slow speed, thereby providing a support structure for nucleic acid amplification reagent components, can be used as an excellent excipient in a freeze-drying process, ensures that a freeze-drying reagent has good easy redissolution, and can be effectively used as a freeze-drying protective agent for protein at a proper concentration. Sucrose is a non-reducing sugar, forms a special protective film in the process of drying and dehydration, prevents secondary structures of molecules such as enzyme in a nucleic acid amplification reagent from being damaged, prevents the extension and aggregation of protein polypeptide chains in the process of freeze-drying treatment and in the storage period, maintains the original activity, has higher glass transition temperature and lower hygroscopicity, and therefore can be used as a very good freeze-drying protective agent.
It should be noted that "and/or" in the present invention means "and" or ", for example, the protein protectant may be mannitol, or sucrose, or a combination of mannitol and sucrose. Mannitol or sucrose is used as a freeze-drying protective agent singly or mannitol and sucrose are used as freeze-drying protective agents jointly, and the freeze-drying protective agent can play a good freeze-drying protective role. When the mannitol and the sucrose are compounded for use, excessive freeze-drying protective agents are not introduced to influence the reaction activity, the freeze-dried powder reagent can have a good solid shape, the high activity of the enzyme is kept, and the freeze-dried powder reagent has good re-solubility and storage stability. Thus, as a preferred embodiment of the present invention, the protein protectant includes mannitol and sucrose.
Preferably, the mannitol concentration is 0.2% -2% W/V, such as, but not limited to, 0.2% W/V, 0.5% W/V, 0.8% W/V, 1% W/V, 1.2% W/V, 1.5% W/V, 1.8% W/V or 2% W/V, preferably 0.5% -1.8% W/V, more preferably 0.7% -1.5% W/V; the sucrose concentration is 5% -20% W/V, which may be, for example, but not limited to, 5% -W/V, 8% -W/V, 10% -W/V, 12% -W/V, 15% -W/V, 18% -W/V or 20% -W/V, preferably 8% -18% -W/V, more preferably 10.5% -16% -W/V. The use concentrations of mannitol and sucrose are further optimized, so that the mannitol and sucrose can play better roles in protection and shaping after being compounded and used, and the re-solubility and the storage stability are further improved on the basis of keeping the enzyme activity.
In some preferred embodiments, the enzymes include DNA polymerase and UNG enzyme, and the use of UNG enzyme can prevent false positive caused by contamination of PCR amplification products, effectively ensuring the accuracy of PCR results. In order to effectively avoid some non-specific amplification and formation of primer dimer and improve the amplification success rate of target gene, hot start Taq enzyme is preferably used as DNA polymerase.
Wherein, the concentration of the hot start Taq enzyme is preferably 3-10U/reaction, for example, but not limited to 3U/reaction, 4U/reaction, 5U/reaction, 6U/reaction, 7U/reaction, 8U/reaction, 9U/reaction or 10U/reaction, more preferably 5-8U/reaction; the concentration of UNG enzyme is preferably 0.3 to 1U/reaction, and may be, for example, but not limited to, 0.3U/reaction, 0.4U/reaction, 0.5U/reaction, 0.6U/reaction, 0.7U/reaction, 0.8U/reaction, 0.9U/reaction or 1U/reaction, and more preferably 0.5 to 0.8U/reaction. By adjusting the reaction concentration of the enzyme, unnecessary waste can be avoided on the basis of ensuring the action function of the enzyme, and the cost is effectively controlled.
Preferably, the dNTPs comprise dATP, dCTP, dGTP and dUTP. Wherein the concentration of dATP, dCTP and dGTP is 200-300. Mu.M independently, and can be, for example, but not limited to, 200. Mu.M, 210. Mu.M, 220. Mu.M, 230. Mu.M, 240. Mu.M, 250. Mu.M, 260. Mu.M, 270. Mu.M, 280. Mu.M, 290. Mu.M or 300. Mu.M; the concentration of dUTP is 350-450. Mu.M, and may be, for example, but not limited to, 350. Mu.M, 360. Mu.M, 370. Mu.M, 380. Mu.M, 390. Mu.M, 400. Mu.M, 410. Mu.M, 420. Mu.M, 430. Mu.M, 440. Mu.M, or 450. Mu.M. By adjusting the concentration of dNTPs, unnecessary waste can be avoided on the basis of ensuring the function of the dNTPs, and the cost is effectively controlled.
Preferably, the metal cations include magnesium ions and potassium ions. The concentration of magnesium ions is preferably 1 to 10mM, and may be, for example, but not limited to, 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, or 10mM, and more preferably 3 to 6mM; the concentration of potassium ions is 45 to 55mM, and may be, for example, but not limited to, 45mM, 46mM, 47mM, 48mM, 49mM, 50mM, 51mM, 52mM, 53mM, 54mM, or 55mM, and more preferably 50mM. The source of the magnesium ion and the potassium ion is not limited, and any soluble salt capable of providing the magnesium ion or the potassium ion may be used, and may be, for example, magnesium chloride or magnesium sulfate, potassium chloride or potassium sulfate.
Preferably, the buffer system comprises a buffer system with a pH of 7.8-8.6, such as, but not limited to, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5 or 8.6, and any buffer system with a pH value within the above range can effectively buffer the nucleic acid amplification reagent provided by the present invention, preferably comprising a Tris-HCl buffer system.
In some preferred embodiments, the HBV-specific primers comprise a first primer pair and/or a second primer pair. The first primer pair or the second primer pair is used alone or the first primer pair and the second primer pair are used in combination to realize the specific detection of HBV. In order to further improve the accuracy of detection, it is preferable to use the first primer pair and the second primer pair in combination as HBV-specific primers. The concentration of each primer sequence of the HBV-specific primers is preferably 0.5-1. Mu.M, and may be, for example, but not limited to, 0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM, or 1mM, more preferably 0.6. Mu.M.
Wherein the first primer pair comprises primers of nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO. 2; the second primer pair comprises primers of nucleotide sequences shown as SEQ ID NO.3 and SEQ ID NO. 4. The primer pairs have the characteristics of strong specificity and high sensitivity, and can specifically and efficiently detect HBV.
In some preferred embodiments, the HBV-specific probe comprises a first probe and/or a second probe. The first probe or the second probe can be used alone or in combination with the first probe and the second probe, and can be matched with HBV specific primers to realize specific detection of HBV. In order to further improve the accuracy of detection, it is preferable to use the first probe and the second probe in combination as HBV-specific probes. The concentration of each probe sequence of the HBV-specific probes is preferably 0.1-0.2. Mu.M each, and may be, for example, but not limited to, 0.1mM, 0.12mM, 0.15mM, 0.18mM, or 0.2mM, more preferably 0.1. Mu.M.
Wherein the first probe has a nucleotide sequence shown as SEQ ID NO. 5; the second probe has a nucleotide sequence shown as SEQ ID NO. 6; the probes have the characteristics of strong specificity and high sensitivity, and can be matched with HBV specific primers to specifically and efficiently detect HBV.
Preferably, both ends of the HBV-specific probe are labeled with a fluorescent group and a quenching group, respectively, for example, the 5 'end is labeled with a fluorescent group, and the 3' end is labeled with a quenching group; or 5 'end is marked with quenching group, 3' end is marked with fluorescent group.
In some preferred embodiments, the nucleic acid amplification reagent further comprises an internal standard specific probe, and the internal standard specific probe can be used as a positive control and can be used for monitoring whether a PCR inhibitor exists or not so as to avoid false negatives. Preferably, the internal standard specific probe has a nucleotide sequence shown in SEQ ID No.7, and both ends of the internal standard specific probe are respectively labeled with a fluorescent group and a quenching group, for example, a 5 'end is labeled with a fluorescent group, and a 3' end is labeled with a quenching group; or the 5 'end is marked with a quenching group, the 3' end is marked with a fluorescent group, and the fluorescent group in the internal standard specific probe is different from the fluorescent group in the HBV specific probe.
In some preferred embodiments, the surfactant in the sample processing solution includes an ionic surfactant and a non-ionic surfactant. Among them, the ionic surfactant preferably includes an anionic surfactant, and more preferably includes sodium lauryl sulfate. The nonionic surfactant preferably includes Triton X-100 and Tween 80. Sodium dodecyl sulfate is a common anionic surfactant, triton X-100 and Tween80 are nonionic surfactants, and the combination of the surfactants can effectively crack a sample and release nucleic acid substances in the sample.
Wherein the concentration of sodium dodecyl sulfate is preferably 0.01% -0.09% W/V, and for example, but not limited to, 0.01% -W/V, 0.02% -W/V, 0.03% -W/V, 0.04% -W/V, 0.05% -W/V, 0.06% -W/V, 0.07% -W/V, 0.08% -W/V or 0.09% -W/V; a concentration of Triton X-100 of 0.02% -0.08% V/V, such as, but not limited to, 0.02% V/V, 0.03% V/V, 0.04% V/V, 0.05% V/V, 0.06% V/V, 0.07% V/V or 0.08% V/V; the concentration of Tween80 is 0.01% -0.08% V/V, and for example, but not limited to, 0.01% V/V, 0.02% V/V, 0.03% V/V, 0.04% V/V, 0.05% V/V, 0.06% V/V, 0.07% V/V or 0.08% V/V. Through further optimization and limitation of the use amounts of different surfactants, the waste of raw materials can be avoided on the premise of ensuring the cracking function, and unnecessary adverse effects on the reaction can also be avoided.
Due to the lack of protein denaturants, nucleic acids and proteins remain bound together after the outer viral membrane is cleaved, affecting the effectiveness of PCR amplification. Lactose is reducing disaccharide, the reducibility of lactose can enable nucleic acid and protein to be better separated, and simultaneously, the lactose can be effectively combined with protein substances, anticoagulants, lipids and other substances in a sample under the combined action of mannitol and sucrose in a nucleic acid amplification reagent, and the lactose is denatured and precipitated at high temperature, so that the inhibition effect on PCR reaction is reduced. Therefore, lactose is preferably used as the reducing agent. The concentration of lactose is preferably 2% -12% W/V, and can be, for example, but not limited to, 2% -W/V, 3% -W/V, 4% -W/V, 5% -W/V, 6% -W/V, 7% -W/V, 8% -W/V, 9% -W/V, 10% -W/V, 11% -W/V or 12% -W/V, preferably 4% -10% -W/V, more preferably 5% -9% -W/V. The dosage of the lactose is further limited, the matching efficiency of the lactose and the mannitol and the sucrose is higher, the matching effect is better, and the inhibition effect of protein substances, anticoagulant, lipid and other substances in a sample on PCR reaction is further reduced.
Preferably, the sample processing solution further comprises dimethyl sulfoxide, wherein the dimethyl sulfoxide is a sulfur-containing organic compound and has strong permeability, so that the outer membrane of the virus can be cracked more easily, and the combined use of three surfactants, namely sodium dodecyl sulfate, triton X-100 and Tween80, in the sample processing solution and the dimethyl sulfoxide with strong permeability can fully crack the outer membrane of the virus without the action of strong modifiers such as guanidine salt and the like, so that nucleic acid, protein, lipid and the like in the outer membrane can be released, and the inhibition of the use of guanidine salt on PCR amplification can be avoided. Meanwhile, after the dimethyl sulfoxide is added into the PCR reaction, the formation of primer dimer can be inhibited, and the specificity is enhanced. The concentration of dimethyl sulfoxide is preferably 0.01% -0.1% W/V, and may be, for example, but not limited to, 0.01% W/V, 0.02% W/V, 0.03% W/V, 0.04% W/V, 0.05% W/V, 0.06% W/V, 0.07% W/V, 0.08% W/V, 0.09% W/V or 0.1% W/V, preferably 0.03% -0.08% W/V, more preferably 0.04% -0.07% W/V.
In some preferred embodiments, the HBV detection kit provided by the present invention further comprises an internal standard solution comprising an internal standard sequence, both ends of the internal standard sequence comprising a sequence identical to HBV-specific primer sequence; the middle of the internal standard sequence comprises a complementary sequence which is identical with or opposite to the internal standard specific probe sequence. For example, one end of the internal standard sequence is the nucleotide sequence shown in SEQ ID NO.1, and the other end is the nucleotide sequence shown in SEQ ID NO.2, or one end of the internal standard sequence is the nucleotide sequence shown in SEQ ID NO.3, and the other end is the nucleotide sequence shown in SEQ ID NO. 4. Optionally, the internal standard sequence has a nucleotide sequence shown as SEQ ID No. 8.
The method adopts a unique internal standard method for quantification, the HBV concentration in the sample is quantified through the internal standard with known concentration, the internal standard and the target gene are synchronously amplified during reaction, the concentration of the corresponding sample can be calculated through the known internal standard concentration, an additional quantitative calibrator is not required to be utilized to construct a calibration curve, the operation is simplified, the utilization rate of the reagent is improved, invalid results and repeated detection caused by failed construction of the calibration curve are avoided, and meanwhile, the quantification error caused by the existence of an inhibitor in the sample can be effectively corrected.
Preferably, the HBV detection kit further comprises a quality control product, wherein the quality control product can be a negative quality control product, or a critical positive quality control product, or a strong positive quality control product, or a combination of any two of the negative quality control product, the critical positive quality control product, and the strong positive quality control product, or simultaneously comprises the negative quality control product, the critical positive quality control product, and the strong positive quality control product. Through setting up quality control article, can further ensure the accuracy and the validity of testing result.
In some preferred embodiments, the HBV detection kit provided by the present invention, the nucleic acid amplification reagent comprises 5U/reaction of hot start Taq enzyme, 0.5U/reaction of UNG enzyme, dATP, dCTP and dGTP at a concentration of 250. Mu.M each independently, 400. Mu.M of dUTP, 5mM MgCl 2 50mM KCl, 1.0% w/V mannitol, 12.5% w/V sucrose, primers represented by SEQ ID nos. 1 to 4 at concentrations each independently of 0.6. Mu.M, probes represented by SEQ ID nos. 5 to 6 at concentrations each independently of 0.1. Mu.M, and a 20mM Tris-HCl buffer system, the pH of the nucleic acid amplification reagent being 8.2. Uniformly mixing all components of the nucleic acid amplification reagent according to an optimized formula, pre-distributing the mixture into a PCR reaction tube, and freeze-drying to obtain the pre-distributed ready-to-use HBV nucleic acid quantitative detection reagent.
In the HBV detection kit provided by the present invention, the sample treatment solution contained 0.03% of W/V sodium dodecyl sulfate, 0.06% of V/V Triton X-100, 0.02% of V/V Tween80, 0.05% of W/V dimethyl sulfoxide and 6% of W/V lactose.
The invention also provides a using method of the HBV detection kit, wherein a sample to be detected and a sample treatment solution are added into a nucleic acid amplification reagent, and after uniform mixing, the obtained reaction solution is placed in a PCR instrument for amplification detection.
The application method of the HBV detection kit provided by the invention is simple to operate, PCR amplification detection can be carried out only by adding a sample to be detected and a sample treatment solution into a nucleic acid amplification reagent and uniformly mixing, so that not only is the complicated step of nucleic acid extraction and purification or sample cracking omitted, but also the step of preparing a PCR reaction solution is omitted.
In some preferred embodiments, the sample to be detected, the sample treatment solution and the internal standard solution are added into the nucleic acid amplification reagent, and after uniform mixing, the obtained reaction solution is placed in a PCR instrument for amplification detection. After the sample to be detected, the internal standard solution and the sample treatment solution are added into the nucleic acid amplification reagent, the freeze-dried reagent is instantly redissolved, and all components are uniformly mixed together. Dimethyl sulfoxide can inhibit the formation of primer dimer and enhance the specificity of reaction. Under the combined action of sodium dodecyl sulfate, triton X-100, tween80 and dimethyl sulfoxide in the sample processing solution, the virus outer membrane in the sample is destroyed, and substances such as nucleic acid, protein and the like are released. The lactose in the sample processing liquid has reducibility, so that nucleic acid substances and proteins can be better separated, and simultaneously, the lactose and mannitol and sucrose in the nucleic acid amplification reagent have combined action, can be effectively combined with protein substances, anticoagulants, lipids and other substances in the sample, and are denatured and precipitated at high temperature, so that the inhibition effect on PCR reaction is reduced. The cleaved nucleic acid is directly used as a template and is continuously copied in a PCR amplification cycle, and a Ct value is generated when the value exceeds a threshold value line. And calculating the original concentration of the HBV virus in the sample according to the Ct value of the internal standard in the same reaction tube and the known internal standard concentration. The use of the internal standard solution for quantification not only eliminates the trouble of constructing a calibration curve by a calibrator, but also corrects the quantitative deviation caused by an inhibitor in a sample.
Preferably, the volume of the sample to be tested is 5% -40% of the volume of the reaction solution, for example, but not limited to, 5%, 10%, 15%, 20%, 25%, 30%, 35% or 40%, preferably 15% -30%;
preferably, the volume of the sample processing solution is 5% to 60% of the volume of the reaction solution, for example, but not limited to, 5%, 10%, 20%, 30%, 40%, 50% or 60%, preferably 10% to 50%;
the PCR reaction can be further optimized by limiting the using amount of each component in a PCR reaction system, and meanwhile, the limited using amount is more suitable for the use mode of the HBV detection kit provided by the invention when the kit is opened, so that the detection is more efficient and the result is more accurate.
Preferably, the sample to be tested is a plasma sample or a serum sample.
Based on the beneficial effects of the HBV detection kit provided by the invention, the invention also provides the application of the HBV detection kit in preparing products for detecting HBV.
When the HBV detection kit provided by the invention contains an internal standard solution, the detection is quantitative detection.
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
Example 1
Sample treatment fluids of different formulations were prepared as shown in the following table:
formulation of SDS Triton X-100 Tween80 Tween20 Dimethyl sulfoxide Lactose
Formulation 1 0.01％ 0.02％ / / / /
Formulation 2 0.01％ / 0.01％ / / /
Formulation 3 0.01％ / / 0.01％ / /
Formulation 4 0.01％ 0.02％ / / 0.01％ /
Formulation 5 0.01％ 0.02％ 0.01％ / 0.01％ /
Formulation 6 0.01％ 0.02％ / / 0.01％ 2％
Preparing a nucleic acid amplification reagent, which comprises the following components: taq enzyme 5U/reaction, UNG enzyme 0.5U/reaction, dATP, dCTP, dGTP 250. Mu.M each, dUTP 400. Mu.M, mgCl 2 5mM, KCl 50mM, tris-HCl 20mM, pH8.2, 0.6. Mu.M HBV amplification primers (SEQ ID NO. 1-4), 0.1. Mu.M HBV-specific probe (SEQ ID NO. 5-6).
HBV positive plasma was diluted to 1.0E +04IU/mL with HBV negative plasma, 5. Mu.L was added directly to the prepared nucleic acid amplification reagent, 20. Mu.L of the sample treatment solution of formulas 1-6 was added, and one set of the samples added with 20. Mu.L of water was set as a control, and the total reaction volume was 50. Mu.L (plasma ratio 10%). And (3) putting the PCR reaction tube into a macro-stone fluorescent quantitative PCR instrument (SLAN-96P) for amplification detection, wherein the amplification procedure comprises the following steps: at 50 ℃ for 2min; at 95 ℃ for 1min;95 ℃,5s,55 ℃ (fluorescence collected), 50s,50cycles;25 ℃ for 10s. The experimental results are shown in fig. 1A to 1F, and it can be seen that Ct values of formula 1, formula 2, formula 4, formula 5 and formula 6 are advanced compared with the control group, fluorescence signal values of formula 1, formula 4, formula 5 and formula 6 are improved, and formula 3 has no obvious difference from the control group, which indicates that the amplification of HBV positive plasma is obviously improved by 5 formulas except formula 3. Wherein, the fluorescence signal value of formula 6 is significantly improved, indicating that the addition of lactose is beneficial to reducing the inhibitory effect of the reactants.
Example 2
Sample treatment fluids of different formulations were prepared as shown in the following table:
formulation(s) SDS Triton X-100 Tween80 Dimethyl sulfoxide Lactose
Formulation 7 0.03％ 0.04％ 0.02％ 0.03％ 2％
Formulation
8 0.03％ 0.06％ 0.02％ 0.03％ 4％
Formulation 9 0.06％ 0.08％ 0.04％ 0.03％ 4％
Formulation
10 0.09％ 0.08％ 0.06％ 0.03％ 4％
Formulation
11 0.03％ 0.04％ 0.08％ 0.03％ 4％
Formulation 12 0.03％ 0.06％ 0.02％ 0.05％ 4％
Formulation 13 0.03％ 0.06％ 0.02％ 0.1％ 4％
Formulation 14 0.03％ 0.06％ 0.02％ 0.05％ 8％
Formulation 15 0.03％ 0.06％ 0.02％ 0.05％ 12％
Preparing a nucleic acid amplification reagent, which comprises the following components: taq enzyme 5U/reaction, UNG enzyme 0.5U/reaction, dATP, dCTP, dGTP each 250. Mu.M, dUTP 400. Mu.M, mgCl 2 5mM, KCl 50mM, tris-HCl 20mM, pH8.2, 0.6. Mu.M HBV amplification primers (SEQ ID NO. 1-4), 0.1. Mu.M HBV-specific probe (SEQ ID NO. 5-6).
HBV positive plasma is diluted to 2.0E +04IU/mL and 2.0E +03IU/mL by HBV negative plasma, 5 mu L of the diluted plasma is directly added into a prepared nucleic acid amplification reagent, and then 20 mu L of sample treatment liquid of formulas 7 to 15 is respectively added, wherein the total reaction volume is 50 mu L (the plasma proportion is 10%). And (3) putting the PCR reaction tube into a garnet fluorescent quantitative PCR instrument (SLAN-96P) for amplification detection, wherein the amplification procedure comprises the following steps: at 50 ℃ for 2min; at 95 ℃ for 1min;95 ℃,5s,55 ℃ (fluorescence collected), 50s,50cycles;25 ℃ for 10s. The results are shown in FIGS. 2A-I, from which it can be seen that formulations 7-15 all favoured the amplification of HBV positive plasma, with formulation 14 being most effective.
Example 3
The basic components of the nucleic acid amplification reagent are as follows: taq enzyme 5U/reaction, UNG enzyme 0.5U/reaction, dATP, dCTP, dGTP 250. Mu.M each, dUTP 400. Mu.M, mgCl 2 5mM，KCl 50mM，Tris-HCl 20mM，pH8.2，0.6. Mu.M HBV amplification primer (SEQ ID NO. 1-4), 0.1. Mu.M HBV-specific probe (SEQ ID NO. 5-6).
Different formulations of protein protecting agents were added to the nucleic acid amplification reagent base as shown in the following table:
formulation of Trehalose Mannitol Sucrose
Formulation
16 6％ 0.2％ /
Formulation 17 / 0.2％ 5％
Formulation 18 6％ 0.2％ 5％
HBV positive plasma was diluted with HBV negative plasma to 2.0E +04IU/mL, 2.0E +03IU/mL and 2.0E +02IU/mL, 5. Mu.L of the diluted HBV positive plasma was added directly to the nucleic acid amplification reagents of the formulated formulations 16-18 while setting a set of basic nucleic acid amplification reagent formulations as controls, and then 20. Mu.L of sample treatment solution (containing SDS 0.03% W/V, triton X-100.06% V/V, tween800.02% V/V, dimethyl sulfoxide 0.05% W/V and lactose 8% W/V) was added, respectively, to the total reaction volume was 50. Mu.L (plasma ratio 10%). And (3) putting the PCR reaction tube into a macro-stone fluorescent quantitative PCR instrument (SLAN-96P) for amplification detection, wherein the amplification procedure comprises the following steps: at 50 ℃ for 2min; at 95 ℃ for 1min;95 ℃,5s,55 ℃ (fluorescence collected), 50s,50cycles;25 ℃ for 10s. The experimental results are shown in fig. 3A-D, from which it can be seen that formula 16 has an inhibitory effect on the amplification detection of HBV positive plasma at low concentration, and formula 17 has a significant improvement effect on the amplification detection of HBV positive plasma.
Example 4
The basic components of the nucleic acid amplification reagent are as follows: taq enzyme 5U/reaction, UNG enzyme 0.5U/reaction, dATP, dCTP, dGTP each 250. Mu.M, dUTP 400. Mu.M, mgCl 2 5mM, KCl 50mM, tris-HCl 20mM, pH8.2, 0.6. Mu.M HBV amplification primers (SEQ ID NO. 1-4), 0.1. Mu.M HBV-specific probe (SEQ ID NO. 5-6), and 0.1. Mu.M internal standard-specific probe (SEQ ID NO. 7).
Various concentrations of protein protectant were added to the nucleic acid amplification reagent base as shown in the following table:
formulation of Mannitol Sucrose
Formulation 19 0.2％ 5％
Formulation
20 0.6％ 8％
Formulation
21 1.0％ 12％
Formulation 22 1.5％ 16％
Formulation 23 2.0％ 20％
HBV positive plasma was diluted with HBV negative plasma to 2.0E +04IU/mL, 2.0E +03IU/mL and 2.0E +02IU/mL, 5. Mu.L of the diluted HBV positive plasma was directly added to the nucleic acid amplification reagent of the formulated formulation 19-23, the internal standard solution (final concentration 500 copies/reaction) was added, and 20. Mu.L of the sample treatment solution (sample treatment solution containing SDS 0.03% W/V, triton X-100 0.06% V/V, twenn 800.02% V/V, dimethyl sulfoxide 0.05% W/V and lactose 8% W/V) was added, respectively, and the total reaction volume was 50. Mu.L (plasma ratio 10%). And (3) putting the PCR reaction tube into a garnet fluorescent quantitative PCR instrument (SLAN-96P) for amplification detection, wherein the amplification procedure comprises the following steps: at 50 ℃ for 2min; at 95 ℃ for 1min;95 ℃,5s,55 ℃ (fluorescence collected), 50s,50cycles;25 ℃ for 10s. As shown in FIGS. 4A-E (dotted line is internal standard curve, solid line is HBV curve) and Table 1, it can be seen that the formulations 19-23 all have significant improvement effect on the amplification detection of low concentration HBV positive plasma, wherein the effect of the formulations 20-23 is better than that of the formulation 19, and the effect of the formulation 21 is the best.
Table 1 example 4 results
Figure BDA0002392262470000091
Example 5
The basic components of the nucleic acid amplification reagent are as follows: taq enzyme 5U/reaction, UNG enzyme 0.5U/reactiondATP, dCTP and dGTP each 250. Mu.M, dUTP 400. Mu.M, mgCl 2 5mM, KCl 50mM, tris-HCl 20mM, pH8.2, 0.6. Mu.M HBV amplification primers (SEQ ID NO. 1-4), 0.1. Mu.M HBV-specific probe (SEQ ID NO. 5-6), and 0.1. Mu.M internal standard-specific probe (SEQ ID NO. 7).
Various concentrations of protein protectant were added to the nucleic acid amplification reagent base as shown in the following table:
formulation of Mannitol Sucrose
Formulation 24 0.3％ 5％
Formulation 25 0.5％ 8％
Formulation
26 0.7％ 10.5％
Formulation 27 1.0％ 12.5％
Formulation 28 1.5％ 16％
Formulation 29 1.8％ 18％
Formulation
30 2.0％ 20％
Subpackaging the prepared nucleic acid amplification reagents with different formulas into a PCR reaction tube, and freeze-drying according to the following parameters: (1) a first stage: pre-freezing, slowly cooling to-45 ℃, wherein the cooling time is 1 hour, and keeping the temperature for 3 hours; and a second stage: primary drying, slowly raising the temperature to-30 deg.C, and maintaining for 8 hr, wherein the air pressure is maintained at 0.2mbar; and a third stage: the drying was intensified, the temperature was kept at 25 ℃, the drying time was 3 hours and the air pressure was kept at 0.1mbar. The appearance of the lyophilized reagents is shown in fig. 5A-G, from which it can be seen that the lyophilized reagents obtained from formulation 24 have some collapse and are not well formed, while the lyophilized reagents obtained from formulations 25-30 have uniform appearance and are well formed and have no collapse, while the lyophilized reagents obtained from formulation 30 have less tendency to fall off and some have wall adhesion, and the lyophilized reagents obtained from formulations 25-29 have better quality than those obtained from formulations 25-29.
Example 6
The lyophilized reagents of the above formulations 25-29 were selected for functional testing. HBV positive plasma was diluted to 1.0E +04IU/mL, 1.0E +03IU/mL, 1.0E +02IU/mL and 50IU/mL with HBV negative plasma, 12.5. Mu.L was directly added to the lyophilized nucleic acid amplification reagents of formulas 25-29, 12.5. Mu.L of internal standard solution (final concentration 500 copies/reaction) was added, and 25. Mu.L of sample treatment solution (sample treatment solution containing SDS 0.03% W/V, triton X-100 0.06% V/V, tween800.02% V/V, dimethyl sulfoxide 0.05% W/V and lactose 8% W/V) was added, respectively, and the total reaction volume was 50. Mu.L (plasma ratio 25%). And (3) putting the PCR reaction tube into a garnet fluorescent quantitative PCR instrument (SLAN-96P) for amplification detection, wherein the amplification procedure comprises the following steps: at 50 ℃ for 2min; at 95 ℃ for 1min;95 ℃,5s,55 ℃ (fluorescence collected), 50s,50cycles;25 ℃ for 10s. As shown in FIGS. 6A-E (dotted line is the internal standard curve, solid line is the HBV curve) and Table 2, it can be seen that the formulations 25-29 all gave better functional results, with the results of the formulations 26-28 being slightly better than the results of the formulations 25 and 29.
Table 2 results of example 6
Figure BDA0002392262470000101
Example 7
Adding different concentrations of dimethyl sulfoxide and lactose, using 0.03% W/V SDS, 0.06% V/V Triton X-100 and 0.02% V/V Tween80 as the basic recipe for the sample treatment solutions, resulting in different recipes of sample treatment solutions as shown in the following table:
formulation of Dimethyl sulfoxide Lactose
Formulation 30 0.03％ 4％
Formulation
31 0.04％ 5％
Formulation 32 0.05％ 6％
Formulation 33 0.06％ 7％
Formulation 34 0.07％ 8％
Formulation 35 0.08％ 9％
Formulation
36 0.09％ 10％
HBV positive plasma is diluted to 1.0E +04IU/mL, 1.0E +03IU/mL, 1.0E +02IU/mL and 50IU/mL by using HBV negative plasma, 15 mu L of the diluted plasma is directly added into the freeze-dried nucleic acid amplification reagent of the formula 27, 10 mu L of internal standard solution (the final concentration is 500 copies/reaction) is added, 25 mu L of sample treatment solution of the formula 30-36 is respectively added, and the total reaction volume is 50 mu L (the plasma proportion is 30%). And (3) putting the PCR reaction tube into a garnet fluorescent quantitative PCR instrument (SLAN-96P) for amplification detection, wherein the amplification procedure comprises the following steps: at 50 ℃ for 2min; at 95 ℃ for 1min;95 ℃,5s,55 ℃ (fluorescence collected), 50s,50cycles;25 ℃ for 10s. The results are shown in FIGS. 7A-G (dotted line is the internal standard curve, solid line is the HBV curve) and Table 3, from which it can be seen that the formulations 30-36 all gave better results, with the results of formulations 31-35 being slightly better than those of formulations 30 and 36.
Table 3 example 7 results
Figure BDA0002392262470000111
Example 8 quantitative accuracy study
According to the formula 27 (the components are as follows: taq enzyme 5U/reaction, UNG enzyme 0.5U/reaction, dATP, dCTP, dGTP each 250. Mu.M, dUTP 400. Mu.M, mgCl 2 5mM，KThe nucleic acid amplification reagents were lyophilized from Cl 50mM, tris-HCl 20mM, pH8.2, 0.6. Mu.M HBV amplification primers (SEQ ID nos. 1-4), 0.1. Mu.M HBV specific probe (SEQ ID nos. 5-6) and 0.1. Mu.M internal standard specific probe (SEQ ID No. 7), 1.0% mannitol, 12.5% sucrose) by the lyophilization procedure: the first stage is as follows: pre-freezing, slowly cooling to-45 ℃, keeping the cooling time for 1 hour, and keeping the temperature for 3 hours; and a second stage: primary drying, slowly raising the temperature to-30 deg.C, and maintaining for 8 hr, wherein the air pressure is maintained at 0.2mbar; and a third stage: drying is enhanced, the temperature is kept at 25 ℃, the drying time is 3 hours, and the air pressure is kept at 0.1mbar.
The sample treatment solutions were prepared as described in formulation 32 (composition: 0.03% W/V SDS, 0.06% V/V Triton X-100, 0.02% V/V Tween80, 0.05% W/V dimethyl sulfoxide, 6% W/V sucrose).
The internal standard plasmid is diluted to proper concentration by using the internal standard diluent, and the value of the internal standard plasmid is determined by using the HBV enterprise standard (the quantitative value is 42000 IU/mL) (the HBV enterprise standard is traced to HBV international standard).
HBV national standard is diluted to 1.0E + 08IU/mL-1.0E +02IU/mL by using HBV negative plasma, and the detection is carried out by using the detection method disclosed by the invention, namely: adding 50% of sample processing solution, 25% of internal standard solution and 25% of plasma sample into the freeze-dried nucleic acid amplification reagent, uniformly mixing, putting the PCR reaction tube into a garnet fluorescent quantitative PCR instrument (SLAN-96P) for amplification detection, and quantifying by using the internal standard with known concentration, wherein the amplification procedure is as follows: at 50 ℃ for 2min; at 95 ℃ for 2min;95 ℃,5s,55 ℃,60s,5cycles;95 ℃ C, 5s,55 ℃ C (fluorescence acquisition), 60s,45cycles. The results are shown in Table 4. According to the results, when the quantitative detection method established by the invention is used for detection, the quantitative results of the kit on the HBV national standard all meet the requirement of accuracy (the deviation of the detection logarithm and the theoretical logarithm is within +/-0.5).
TABLE 4 quantitative accuracy study results
Sample(s) HBV Ct Internal standard Ct Detecting the concentration Detection of logarithm of concentration Logarithm of theoretical concentration Deviation of
NC NoCt 22.61 0.00E+00 / / /
1.0E+02 31.22 22.95 1.36E+02 2.13 2 0.13
1.0E+03 27.75 22.64 1.22E+03 3.09 3 0.09
1.0E+04 24.3 22.94 1.63E+04 4.21 4 0.21
1.0E+05 21.03 22.53 1.18E+05 5.07 5 0.07
1.0E+06 17.57 22.27 1.09E+06 6.04 6 0.04
1.0E+07 14.28 21.99 8.80E+06 6.94 7 -0.06
1.0E+08 10.52 21.68 9.62E+07 7.98 8 -0.02
Example 9 precision study
According to the formula 27 (the components are as follows: taq enzyme 5U/reaction, UNG enzyme 0.5U/reaction, dATP, dCTP, dGTP each 250. Mu.M, dUTP 400. Mu.M, mgCl 2 The nucleic acid amplification reagents were lyophilized with 5mM, KCl 50mM, tris-HCl 20mM, pH8.2, 0.6. Mu.M HBV amplification primers (SEQ ID NO. 1-4), 0.1. Mu.M HBV-specific probe (SEQ ID NO. 5-6) and 0.1. Mu.M internal standard-specific probe (SEQ ID NO. 7), 1.0% mannitol, 12.5% sucrose) by the lyophilization procedure: the first stage is as follows: pre-freezing, slowly cooling to-45 ℃, wherein the cooling time is 1 hour, and keeping the temperature for 3 hours; and a second stage: primary drying, slowly raising the temperature to-30 deg.C, and maintaining for 8 hr, wherein the air pressure is maintained at 0.2mbar; and a third stage: drying is enhanced, the temperature is kept at 25 ℃, the drying time is 3 hours, and the air pressure is kept at 0.1mbar.
The sample treatment solutions were prepared as described in formulation 32 (composition: 0.03% W/V SDS, 0.06% V/V Triton X-100, 0.02% V/V Tween80, 0.05% W/V dimethyl sulfoxide, 6% W/V sucrose).
Diluting the internal standard plasmid to a proper concentration by using an internal standard diluent, and carrying out value determination on the internal standard plasmid by using an HBV enterprise standard (the quantitative value is 42000 IU/mL) (the HBV enterprise standard is traced to HBV international standard).
The HBV national precision reference substance is diluted 1000 times by HBV negative plasma and detected by the detection method in the embodiment, namely: adding 50% of sample processing solution, 25% of internal standard solution and 25% of plasma sample into the freeze-dried nucleic acid amplification reagent, uniformly mixing, putting the PCR reaction tube into a macrolith fluorescent quantitative PCR instrument (SLAN-96P) for amplification detection, and quantifying by using the internal standard with known concentration, wherein the amplification procedure is as follows: at 50 ℃ for 2min; at 95 ℃ for 2min;95 ℃,5s,55 ℃,60s,5cycles;95 ℃ C, 5s,55 ℃ C (fluorescence acquisition), 60s,45cycles. The results are shown in Table 5. According to the results, when the method established by the invention is used for carrying out quantitative detection on the HBV sample, the coefficient of variation (CV,%) of the quantitative result is 2 percent and is superior to the industry-recognized standard (5 percent).
TABLE 5 results of the precision study
Repetition of HBV Ct Internal standard Ct Detecting the concentration Logarithm of concentration
1 23.68 23.23 3.06E+04 4.49
2 23.64 23.11 2.91E+04 4.46
3 23.45 23.45 4.19E+04 4.62
4 23.71 23.44 3.48E+04 4.54
5 23.36 23.78 5.64E+04 4.75
6 23.69 23.54 3.77E+04 4.58
7 23.60 23.48 3.87E+04 4.59
8 23.32 22.70 2.74E+04 4.44
9 23.51 23.04 3.03E+04 4.48
10 23.45 22.97 3.00E+04 4.48
Mean value / / / 4.54
SD / / / 0.10
CV / / / 2％
Example 10 serum sample test results
According to the formula 27 (the components are as follows: taq enzyme 5U/reaction, UNG enzyme 0.5U/reaction, dATP, dCTP, dGTP each 250. Mu.M, dUTP 400. Mu.M, mgCl 2 The nucleic acid amplification reagents were lyophilized with 5mM, KCl 50mM, tris-HCl 20mM, pH8.2, 0.6. Mu.M HBV amplification primers (SEQ ID nos. 1-4), 0.1. Mu.M HBV specific probe (SEQ ID nos. 5-6) and 0.1. Mu.M internal standard specific probe (SEQ ID No. 7), 1.0% mannitol, 12.5% sucrose) by the procedures of: the first stage is as follows: pre-freezing, slowly cooling to-45 ℃, keeping the cooling time for 1 hour, and keeping the temperature for 3 hours; and a second stage: primary drying, slowly raising the temperature to-30 deg.C, and maintaining for 8 hr, wherein the air pressure is maintained at 0.2mbar; and a third stage: the drying was intensified, the temperature was kept at 25 ℃, the drying time was 3 hours and the air pressure was kept at 0.1mbar.
Preparing a sample treatment solution according to the recipe 32 (consisting of 0.03%; W/V SDS, 0.06%; V/V Triton X-100, 0.02%; V/V Tween80, 0.05%; W/V dimethyl sulfoxide, 6% W/V sucrose).
Diluting the internal standard plasmid to a proper concentration by using an internal standard diluent, and carrying out value determination (the quantitative value is 45000 IU/mL) on the internal standard plasmid by using an HBV enterprise standard product (the HBV enterprise standard product is traced to HBV international standard product).
HBV national standard is diluted to 1.0E + 08IU/mL-1.0E +02IU/mL by HBV negative serum, and the detection is carried out by the detection method of the invention, namely: adding 50% of sample processing solution, 25% of internal standard solution and 35% of serum sample into the freeze-dried nucleic acid amplification reagent, uniformly mixing, putting the PCR reaction tube into a macrophyllite fluorescent quantitative PCR instrument (SLAN-96P) for amplification detection, and quantifying by using the internal standard with known concentration, wherein the amplification procedure is as follows: at 50 ℃ for 2min; at 95 ℃ for 2min;95 ℃,5s,55 ℃,60s,5cycles;95 ℃ in the presence of 5s,55 ℃ for fluorescence, 60s,45cycles. The results are shown in Table 6. According to the results, when the quantitative detection method established by the invention is used for detection, the quantitative results of the kit on the HBV national standard all meet the requirement of accuracy (the deviation of the detection logarithm and the theoretical logarithm is within +/-0.5).
TABLE 6 serum sample quantification results
Sample(s) HBV Ct Internal standard Ct Detecting the concentration Detection of logarithm of concentration Logarithm of theoretical concentration Deviation of
NC NoCt 22.22 0.00E+00 / / /
1.0E+02 31.20 22.41 1.02E+02 2.01 2 0.01
1.0E+03 28.06 22.37 8.68E+02 2.94 3 -0.06
1.0E+04 24.68 22.07 7.36E+03 3.87 4 -0.13
1.0E+05 21.22 22.55 1.13E+05 5.05 5 0.05
1.0E+06 17.75 22.26 1.03E+06 6.01 6 0.01
1.0E+07 14.16 21.89 9.58E+06 6.98 7 -0.02
1.0E+08 10.02 21.84 1.63E+08 8.21 8 0.21
Example 11 comparison with commercial kits
According to the formula 27 (the components are as follows: 5U Taq enzyme/reaction, 0.5U UNG enzyme/reaction, 250. Mu.M each of dATP, dCTP and dGTP, 400. Mu.M dUTP, mgCl 2 5mM, KCl 50mM, tris-HCl 20mM, pH8.2, 0.6. Mu.M HBV amplification primers (SEQ ID NO. 1-4), 0.1. Mu.M HBV-specific12 aliquots of single aliquot lyophilized nucleic acid amplification reagents prepared with probes (SEQ ID nos. 5-6) and 0.1 μ M internal standard specific probe (SEQ ID No. 7), 1.0% mannitol, 12.5% sucrose) and lyophilized according to the lyophilization procedure, 30 μ L of sample treatment fluid prepared according to formulation 32 (components: 0.03% W/V SDS, 0.06% V/V Triton X-100, 0.02% V/V Tween80, 0.05% W/V dimethyl sulfoxide, 6% W/V sucrose) and 15 μ L of internal standard solution (concentration is 42000 IU/mL), then respectively adding 15 μ L of HBV national standard substance of series gradient obtained by diluting HBV negative plasma, wherein each of 1 well 2.0E + 07IU/mL-2.0E +02IU/mL, 2 wells 60IU/mL, 3 wells 30IU/mL and 1 well of negative plasma, after uniformly mixing, placing the PCR reaction tube into a macrostone fluorescence quantitative PCR instrument (SLAN-96P) for amplification detection, and performing quantification by using the internal standard with known concentration, wherein the amplification procedure is as follows: at 50 ℃ for 2min; at 95 ℃ for 2min;95 ℃,5s,55 ℃,60s,5cycles;95 ℃ in the presence of 5s,55 ℃ for fluorescence, 60s,45cycles.
The commercial HBV quantitative detection kit comprises the following operation steps: (1) Taking out the PCR reaction solution, the enzyme mixed solution and the internal standard solution from a refrigerator at the temperature of-20 ℃, thawing and uniformly mixing, and preparing the PCR reaction solution, the enzyme mixed solution and the internal standard solution into a PCR premixed solution according to the method of the specification and the ratio of 38; (2) Taking out the sample treatment liquid, thawing, uniformly mixing, taking 5 mu L into a PCR reaction tube with 16 holes in total, adding 5 mu L of a plasma sample into each reaction tube, wherein 2.0E + 07IU/mL-2.0E +02IU/mL is 1 hole in each reaction tube, 60IU/mL is 2 holes, 30IU/mL is 3 holes, 1 hole in negative plasma is 1 hole in each reaction tube, quantitative standards A-D are 1 hole in each reaction tube, each reaction hole is blown by a pipettor and uniformly mixed for 3-5 times to uniformly mix the sample with the lysate, and standing for 10 minutes at room temperature; (3) And (2) fully and uniformly mixing the PCR premix prepared in the step (1), performing instantaneous centrifugation, subpackaging 40 mu L of each hole into a cracked sample, covering a tube cover, performing instantaneous centrifugation, and then putting a PCR reaction tube into a macrophyllite fluorescence quantitative PCR instrument (SLAN-96P) for amplification detection, wherein the amplification procedure is as follows: at 50 ℃ for 2min;94 ℃,5min;94 ℃,15s,57 ℃ (fluorescence collected), 30s,45cycles;25 ℃ for 10s. And (4) making a standard curve according to the Ct values and concentration values of the quantitative standard substances A-D, and quantifying the sample with unknown concentration. The results are shown in FIGS. 8A to 8H and tables 7 and 8.
TABLE 7 comparison of the quantitative accuracy of the kit of the invention and the commercial kits
Figure BDA0002392262470000141
TABLE 8 comparison of detection sensitivity of the kit of the present invention and the commercial kit
Figure BDA0002392262470000142
According to the results of this example, compared with the commercial extraction-free quantitative detection kit for HBV, the method of the present invention greatly simplifies the operation steps and can tolerate a larger sample ratio. As for a sample with the concentration of 30IU/mL, a commercial kit cannot be detected completely, and the method disclosed by the invention can be detected completely and stably, the method disclosed by the invention has better sensitivity and higher fluorescence signal value (Rn).
The method adopts an internal standard method for quantification, does not need to make an additional standard curve of a quantitative standard product, improves the utilization rate of the reagent, has the deviation of the detection logarithm and the theoretical logarithm smaller than that of a commercial kit, and has better quantification accuracy.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> Zhuhailizhu reagent GmbH
<120> HBV detection kit, and use method and application thereof
<160> 8
<170> PatentIn version 3.5
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Claims (43)

1. An HBV detection kit, which is characterized by comprising a nucleic acid amplification reagent and a sample treatment solution;
the nucleic acid amplification reagent comprises: enzyme, dNTPs, metal cations, a protein protective agent, an HBV specific primer, an HBV specific probe and a buffer system;
the nucleic acid amplification reagent is a freeze-dried powder reagent; the protein protective agent is mannitol and sucrose, the concentration of the mannitol is 0.7-1.5% W/V, and the concentration of the sucrose is 10.5-16% W/V;
the sample processing solution consists of 0.03% W/V SDS, 0.06% V/V Triton X-100, 0.02% V/V Tween80, 0.04-0.07% W/V dimethyl sulfoxide and 5-9% W/V lactose.
2. The HBV detection kit according to claim 1, wherein said enzymes comprise DNA polymerase and UNG enzyme.
3. The HBV detection kit according to claim 2, wherein the DNA polymerase is hot start Taq enzyme.
4. The HBV detection kit according to claim 3, wherein the concentration of the hot start Taq enzyme is 3-10U/reaction.
5. The HBV detection kit according to claim 4, wherein the concentration of hot start Taq enzyme is 5-8U/reaction.
6. The HBV detection kit according to claim 2, wherein the concentration of UNG enzyme is 0.3-1U/reaction.
7. The HBV detection kit according to claim 6, wherein the concentration of UNG enzyme is 0.5-0.8U/reaction.
8. The HBV detection kit according to claim 1, wherein the dNTPs comprise dATP, dCTP, dGTP and dUTP.
9. The HBV detection kit according to claim 8, wherein the concentrations of dATP, dCTP and dGTP are each independently 200 to 300. Mu.M.
10. The HBV detection kit according to claim 8, wherein the concentration of dUTP is 350-450. Mu.M.
11. The HBV detection kit according to claim 1, wherein the metal cation comprises magnesium ion and potassium ion.
12. The HBV detection kit according to claim 11, wherein the concentration of the magnesium ion is 1-10mM.
13. The HBV detection kit according to claim 12, wherein the concentration of the magnesium ion is 3-6mM.
14. The HBV detection kit according to claim 11, wherein the concentration of the potassium ion is 45-55mM.
15. The HBV detection kit according to claim 14, wherein the concentration of the potassium ion is 50mM.
16. The HBV detection kit according to claim 1, wherein the buffer system comprises a buffer system having a pH of 7.8 to 8.6.
17. The HBV detection kit of claim 16, wherein the buffer system comprises a Tris-HCl buffer system.
18. The HBV detection kit according to claim 17, wherein the concentration of the Tris-HCl buffer system is 10-30mM.
19. The HBV detection kit according to claim 18, wherein the concentration of the Tris-HCl buffer system is 20mM.
20. The HBV detection kit according to claim 1, wherein the HBV specific primers comprise a first primer pair and a second primer pair;
the first primer pair comprises primers of nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO. 2;
the second primer pair comprises primers of nucleotide sequences shown as SEQ ID NO.3 and SEQ ID NO. 4.
21. The HBV detection kit according to claim 1, wherein the HBV-specific probe comprises a first probe and a second probe;
the first probe has a nucleotide sequence shown as SEQ ID NO. 5;
the second probe has a nucleotide sequence shown as SEQ ID NO. 6.
22. The HBV detection kit according to claim 21, wherein both ends of the HBV-specific probe are labeled with a fluorescent group and a quencher group, respectively.
23. The HBV detection kit according to claim 1, wherein the nucleic acid amplification reagent further comprises an internal standard specific probe.
24. The HBV detection kit according to claim 23, wherein the internal standard specific probe has a nucleotide sequence shown in SEQ ID No. 7.
25. The HBV detection kit according to claim 23, wherein both ends of the internal standard specific probe are respectively labeled with a fluorophore and a quencher, and the fluorophore in the internal standard specific probe is different from the fluorophore in the HBV specific probe.
26. The HBV detection kit according to claim 1, wherein the concentration of each primer sequence of the HBV-specific primers is independently 0.5 to 1. Mu.M.
27. The HBV detection kit of claim 26 wherein each primer sequence of the HBV specific primers is independently present at a concentration of 0.6 μ M.
28. The HBV detection kit according to claim 1, wherein the concentration of each probe sequence of the HBV-specific probe is independently 0.1 to 0.2 μ M.
29. The HBV detection kit of claim 28, wherein the concentration of each probe sequence of the HBV-specific probe is independently 0.1 μ Μ.
30. The HBV detection kit according to any one of claims 1 to 29, wherein the nucleic acid amplification reagents comprise: 5U/reaction Hot Start Taq enzyme, 0.5U/reaction UNG enzyme, dATP, dCTP and dGTP concentrations each independently of 250. Mu.M, 400. Mu.M dUTP, 5mM MgCl 2 50mM KCl, 1.0% W/V mannitol, 12.5% W/V sucrose, primers shown in SEQ ID NO.1-4 with concentrations of 0.6 mu M independently, and SEQ I with concentrations of 0.1 mu M independentlyD, 5-6 and a 20mM Tris-HCl buffer system, wherein the pH of the nucleic acid amplification reagent is 8.2.
31. The HBV detection kit according to claim 1, wherein the sample treatment solution consists of 0.03% W/V sodium dodecyl sulfate, 0.06% V/V Triton X-100, 0.02% V/V Tween80, 0.05% W/V dimethyl sulfoxide and 6% W/V lactose.
32. The HBV detection kit according to claim 1 further comprising an internal standard solution comprising an internal standard sequence, both ends of the internal standard sequence comprising a sequence identical to HBV-specific primer sequence; the middle of the internal standard sequence comprises a complementary sequence which is identical or opposite to the internal standard specific probe sequence.
33. The HBV detection kit of claim 32 wherein the HBV detection kit further comprises a quality control.
34. The HBV detection kit of claim 33 wherein the quality control material comprises at least one of a negative quality control material, a critical positive quality control material, and a strong positive quality control material.
35. A method for amplifying HBV nucleic acid using the HBV detection kit according to any one of claims 1 to 34 for non-diagnostic and therapeutic purposes, wherein a sample to be tested and a sample treatment solution are added to a nucleic acid amplification reagent, and after mixing them uniformly, the resulting reaction solution is placed in a PCR instrument for amplification detection.
36. The method according to claim 35, wherein the sample to be detected, the sample treatment solution and the internal standard solution are added to the nucleic acid amplification reagent, and after uniform mixing, the obtained reaction solution is placed in a PCR instrument for amplification detection.
37. The method of claim 36, wherein the volume of the sample to be tested is 5-40% of the volume of the reaction solution.
38. The method of claim 37, wherein the volume of the sample to be tested is 15-30% of the volume of the reaction solution.
39. The method of claim 36, wherein the volume of the sample processing solution is 5% to 60% of the volume of the reaction solution.
40. The method of claim 39, wherein the volume of the sample processing solution is 10% to 50% of the volume of the reaction solution.
41. The method of claim 36, wherein the test sample is a plasma sample or a serum sample.
42. Use of the HBV detection kit of any one of claims 1 to 34 in the manufacture of a product for the detection of HBV.
43. The use of claim 42, wherein said detection is a quantitative detection.
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