CN115852047A - Primer probe combination for detecting drug-resistant gene of viral hepatitis B, kit and application - Google Patents

Abstract

The application discloses a primer and probe combination, a kit and application for detecting a drug resistance gene of viral hepatitis B, wherein the primer is shown as SEQ ID NO. 1-SEQ ID NO.8, the probe is shown as SEQ ID NO. 11-SEQ ID NO.34, the primer is divided into an outer primer and an inner primer based on nested PCR, and the probes are respectively matched with the inner primer. The kit comprises the primers, the probes, reaction liquid, a closed isolation material, lysis solution, washing solution A, washing solution B and a meltable material based on POCT requirements. The primer probe can detect multiple mutation sites of multiple drug-resistant genes while having high sensitivity and high specificity by combining a real-time fluorescence PCR technology on the basis of nested PCR, and the kit meets the requirement of the automatic reagent of the microfluidic card box and has the advantages of timeliness, specificity, sensitivity, convenience and the like.

Description

Primer probe combination for detecting drug-resistant gene of viral hepatitis B, kit and application

Technical Field

The application belongs to the field of molecular biology detection, and relates to a primer probe combination, a kit and application for detecting a drug-resistant gene of viral hepatitis B.

Background

Hepatitis b is a disease caused by infection with Hepatitis B Virus (HBV) and is distributed globally, with over 20 billion people worldwide infected with HBV, of which 3.5 billion are chronic HBV infected people. The use of nucleoside analogs for treating hepatitis B has become the mainstream of clinical hepatitis B worldwide, and there are 5 common nucleoside hepatitis B-treating drugs: lamivudine, adefovir dipivoxil, entecavir, telbivudine, and tenofovir disoproxil. When the nucleoside is applied to a patient for initial treatment of nucleoside, the incidence rate of drug resistance of some nucleoside drugs is relatively high, such as 70 percent and 29 percent of five-year drug resistance of lamivudine and adefovir respectively.

However, in the process of treating hepatitis B, the meaning of antiviral treatment cannot be correctly understood and the standard antiviral treatment is adopted, so that the hepatitis B virus variation and drug resistance gradually become the largest stumbling stone on the antiviral treatment road, and how to use correct drugs in time is of great importance to the antiviral treatment. Research shows that different gene mutations can cause different drug resistance of viruses, so that drug resistance gene detection gradually becomes one of important means for treating and diagnosing hepatitis B virus.

The current methods for detecting genotype drug resistance mainly include direct sequencing method, real-time fluorescent polymerase chain reaction (real PCR), restriction fragment mass spectrum polymorphism technology and the like. Specifically, the method comprises the following steps: (1) direct sequencing method: is a method for sequencing and analyzing HBV nucleic acid. Sequencing method can detect known and possible unknown drug-resistant mutation sites, is one of the most commonly used genotype drug-resistant detection methods, and is generally used as a gold standard for genotype drug-resistant detection. The disadvantage of this method is that the single sample detection is expensive and time consuming. (2) Real-time fluorescent PCR: the method is simple and convenient to operate, has strong sensitivity, and can detect the drug resistance variation with the variation occurrence rate lower than 10%. But only can detect the mutation of known single site, and the method is a simple, convenient, rapid and cheap method for monitoring a few drug-resistant mutation sites. However, this method is not adequate for simultaneous detection of multi-site variations. (3) Restriction fragment mass spectrometry polymorphism technique: the technology combines a PCR-RFLP technology with a matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology, has high sensitivity, can find variant strains with the quantity less than 1% of that of the HBV quasispecies pool, is expensive, and is difficult to popularize and apply clinically.

In summary, the existing detection for the drug-resistant gene of hepatitis b is complex in operation, high in detection cost, and long in time consumption, and cannot detect multiple mutation sites simultaneously, for example, when a single site mutation detection method is adopted, a large amount of plasma samples need to be collected, so that the detection cost is high, the detection time is long, discomfort of a patient can be caused, and even the health of the patient can be damaged by serious patients.

Disclosure of Invention

Aiming at the defects in the prior art, the application aims to provide a primer and probe combination for detecting the drug-resistant genes of the viral hepatitis B, wherein the primer combination is combined with a real-time fluorescent PCR technology on the basis of nested PCR, and can detect a plurality of mutation sites of a plurality of drug-resistant genes at the same time with high sensitivity and high specificity.

On the other hand, the application provides a kit for detecting the drug-resistant gene of the hepatitis B virus based on the point-of-care testing (POCT) and the primer and probe system of the application, and the kit can detect a plurality of drug-resistant genes of the hepatitis B virus at one time based on the POCT technology and has guiding significance for clinical medication.

As one embodiment of the present application, there is provided a primer set for detecting a drug-resistant gene of viral hepatitis b, the primer set comprising an outer primer and an inner primer:

the nucleotide sequence of the outer primer is shown as an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO. 2;

the inner primers are divided into an inner primer A, an inner primer B and an inner primer C; the nucleotide sequence of the inner primer A is shown as an upstream primer shown by SEQ ID NO.3 and a downstream primer shown by SEQ ID NO.4, the nucleotide sequence of the inner primer B is shown as an upstream primer shown by SEQ ID NO.5 and a downstream primer shown by SEQ ID NO.6, and the nucleotide sequence of the inner primer C is shown as an upstream primer shown by SEQ ID NO.7 and a downstream primer shown by SEQ ID NO. 8.

The primer combination is based on the principle of nested PCR, firstly, the outer primer is used for amplification, then, a product obtained by amplification of the outer primer is used as a substrate, and the inner primer is used for further amplification to obtain a target gene fragment.

Furthermore, the application also provides a specific probe for the mutation site aiming at the amplified fragment of the inner primer, and the nucleotide sequence of the specific probe is shown as SEQ ID NO. 11-SEQ ID NO. 27.

Wherein, the binding positions of the specific probes with the nucleotide sequences shown as SEQ ID NO. 11-SEQ ID NO.12 are respectively positioned between the upstream primer (SEQ ID NO. 3) and the downstream primer (SEQ ID NO. 4) of the inner primer A;

the binding positions of the specific probes with the nucleotide sequences shown as SEQ ID NO. 13-SEQ ID NO.24 are respectively positioned between the upstream primer (SEQ ID NO. 5) and the downstream primer (SEQ ID NO. 6) of the inner primer B;

the binding positions of the specific probes with the nucleotide sequences shown as SEQ ID NO. 25-SEQ ID NO.27 are respectively positioned between the upstream primer (SEQ ID NO. 7) and the downstream primer (SEQ ID NO. 8) of the inner primer C.

Furthermore, the 5 'end of the specific probe is labeled with a fluorescent group, the 3' end of the specific probe is labeled with a quenching group, the fluorescent group is selected from FAM, ROX or CY5, and the quenching group is MGB.

In order to improve the accuracy of the detection result and reduce the false positive result, the inner primers further comprise internal standard primers, wherein the internal standard primers are an upstream primer with a nucleotide sequence shown as SEQ ID NO.9 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 10.

Further, an internal standard probe matched with the internal standard primer is provided, and the nucleotide sequence of the internal standard probe is shown in SEQ ID NO. 35.

As a second embodiment of the present application, there is provided a kit for detecting a hepatitis B virus drug resistance gene, the kit comprising the primer combination, the specific probe, and a PCR reaction solution. Preferably, the PCR reaction solution is selected from a hot start real-time fluorescent PCR reaction premix (hotspot real time PCR (probe) MasterMix).

In this embodiment, the pre-mix for hot-start real-time fluorescence PCR reaction mainly contains HotStartTaq enzyme that is expressed in Mg ²⁺ Under the activation condition, the kit has high-efficiency amplification activity, can resist the high temperature (20-95 ℃) required by melting of meltable materials in the kit when in use, can be stored for a long time at the temperature of 2-8 ℃, can be compatible with multiplex PCR amplification and can be compatible with fluorescence PCR with different TM values.

Preferably, the pre-mixed solution for the hot start real-time fluorescent PCR reaction comprises 10-100mM Tris-HCl or Tricine-KOH and 20-100mM K ⁺ ，1-5mM Mg ²⁺ 2-5wt% glycerol, 25-250. Mu.M dNTPs,0.5-2U HotStartTaq enzyme.

Wherein said Mg is contained ²⁺ The reagent is prepared from magnesium chloride hexahydrate, trehalose and water, and is specifically obtained by mixing 2-4 ul of 50mM magnesium chloride hexahydrate aqueous solution with 1-2 ul of 40wt% trehalose aqueous solution, and supplementing 5ul of the mixture with sterilized ultrapure water.

Further in this embodiment, the reaction system of the first nested PCR is: 10-100mM Tris-HCl or Tricine-KOH,20-100mM K ⁺ ，1-5mM Mg ²⁺ 2-5wt% glycerol, 25-250. Mu.M dNTPs,0.5-2U HotStartTaq enzyme, 0.1-10 μ M of each primer.

Further in this embodiment, the reaction system of the second nested PCR is: 10-100mM Tris-HCl or Tricine-KOH,20-100mM K ⁺ ，1-5mM Mg ²⁺ 2-5wt% glycerol, 25-250 μ M dNTPs,0.5-2U HotStartTaq enzyme, 0.1-10 μ M primers, 0.1-10 μ M probes.

Further, the kit also comprises a sealing isolation material, a lysis solution, a washing solution A, a washing solution B and a meltable material.

The density of the sealing and isolating material is 0.1-1.5g/ml, the viscosity is 1000-200000CSTG/ml, the arrangement can ensure that the hydrophobic liquid sealing material can isolate liquid, magnetic beads loaded with samples can pass through the hydrophobic liquid sealing material, and the hydrophobic liquid sealing material has a dispersing effect on the magnetic beads, so that nonpolar impurities on the magnetic beads can be clear.

Preferably, the closed isolation material is silicone oil, or similar silicone oil, a silicone base or other high molecular substances.

Wherein the lysis solution comprises 1-1000mM acetic acid-sodium acetate, 0.1-10wt% polyethylene glycol, 0.1-10M guanidinium isothiocyanate, 0.1-10wt% TritonX-100, 1-5wt% sodium lauryl sulfate.

Wherein the lotion A comprises 1-500mM Tris-HCl of PH8.0, 10-500mM KCl and 0.1-10wt% polyethylene glycol.

Wherein the washing liquid B comprises: 1-500mM Tris-HCl of PH8.0, 10-500mM KCl, 0.1-10wt% polyethylene glycol and 0.75wt% non-protein blocking agent. The non-protein blocking agent is from Blockmaster TMPA1080 of JSR company.

The melting point range of the meltable material is 20-95 ℃, the magnesium ion solution in the reaction liquid is embedded by using the meltable material at a low temperature, and the magnesium ion solution in the reaction liquid can be mixed with the reaction liquid after being melted at a high temperature. When in use, the PCR product is melted to form oily substances, so that the PCR product of the aerosol can be prevented from polluting a laboratory. .

Preferably, the meltable material is selected from any one or more of paraffin substances, dodecane, tetradecane, hexadecane and other alkane substances.

In addition, the application of the primer probe combination or the kit in the detection of hepatitis B virus drug resistance genes, and

the application of the kit in detecting the drug resistance gene of the hepatitis B virus based on POCT is also in the protection scope of the application.

The beneficial effect of this application does:

the application provides a primer probe combination for detecting a drug-resistant gene of viral hepatitis B, wherein the primer combination is divided into an outer primer and an inner primer based on a nested PCR principle, a small amount of samples are amplified through the outer primer, a limited target sequence can be increased, more target sequences are provided for secondary PCR, the requirement on the sample sampling amount can be reduced, the harm to patients is reduced, and the sampling consumable is reduced. Meanwhile, the secondary PCR of the application adopts the real-time fluorescence principle, and multiple detection of multiple hepatitis B drug-resistant genes can be realized by designing secondary primers and probes, and the detection can be completed only by a small amount of samples on the basis of ensuring the specificity and the sensitivity.

In addition, the application provides a kit based on POCT technology, the kit meets the reagent requirements of automation and multiple anti-pollution of the microfluidic card box, integrates the functions of nucleic acid extraction, amplification, detection and the like, can realize the amplification detection of a plurality of hepatitis B virus drug-resistant genes simultaneously by one-time sample loading, ensures high specificity, and is superior to other hepatitis B virus drug-resistant gene molecular diagnostic kits in the aspects of detection timeliness, specificity, sensitivity, convenience, technical parameters and the like.

Detailed Description

The technical solutions of the present application will be described clearly and completely with reference to specific embodiments of the present application, and it should be understood that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments in the present application without making any creative effort belong to the protection scope of the present application.

In a first embodiment of the present application, a multiplex PCR detection primer probe set for a drug resistance gene of hepatitis B virus is provided, wherein a primer amplification system is divided into an outer primer for a first nested PCR and an inner primer for a second nested PCR based on the nested PCR mechanism. Nested PCR is a variant Polymerase Chain Reaction (PCR) that uses two (but not one) pair of PCR primers to amplify the entire fragment. The first pair of PCR primers amplified fragments similar to those of ordinary PCR. The second pair of primers is called nested primers (because they are in the first PCR amplification fragment) combined in the first PCR product, so that the second PCR amplification fragment is shorter than the first amplification. Nested PCR has the advantage that if the first amplification produces the wrong fragment, the second amplification has a very low probability of primer pairing and amplification on the wrong fragment, and thus the amplification by nested PCR is very specific. In addition, because the second PCR amplification takes the first PCR amplification fragment as a template, multiple PCR can be realized during the second PCR amplification by collecting a small amount of samples, and a plurality of different mutation sites can be simultaneously detected, thereby realizing the technical purpose of detecting drug-resistant genes of various hepatitis B viruses.

In order to simultaneously detect a plurality of drug-resistant genes of hepatitis B virus, the present example provides a primer set for detecting a drug-resistant gene of hepatitis B virus, which includes an outer primer for the first PCR and an inner primer for the second PCR, the inner primer being located between the upstream and downstream primers of the outer primer at the position of the target gene.

The outer primers are specifically as follows:

HBV upstream primer: 5 'AGAACATCGCATCGAGGACTCC-3' (SEQ ID NO: 1);

HBV downstream primer: 5 'GCCTGTTTACAGGAAGTTCC-3' (SEQ ID NO: 2).

In order to analyze whether the DNA of a sample to be detected is normally amplified so as to eliminate abnormal or failed PCR amplification caused by missed reagent or sample, PCR inhibitor contained in the sample, low DNA recovery rate and the like, internal standard primers for amplifying internal reference genes are also added during first nested PCR amplification, and the internal standard primers are specifically as follows:

an internal standard upstream primer: 5 'AGATTTGGACCTGCGAGCG-doped 3' (SEQ ID NO: 9);

internal standard downstream primer: 5 'GAGCGGCTGTCTCCACAAGTT-containing 3' (SEQ ID NO: 10).

After the first nested PCR amplification is carried out, the expression quantity of the internal standard primer amplification fragment can be measured or agarose gel electrophoresis is carried out to judge whether the internal standard gene is expressed or not, when the internal standard gene is expressed normally, the sample to be detected is complete, and then the second nested PCR is carried out.

Specifically, the first nested PCR primer mixture is:

the premix solution for the first nested PCR reaction is as follows: 10-100mM Tris-HCl or Tricine-KOH,20-100mM K ⁺ ，1-5mM Mg ²⁺ 2-5% glycerol, 25-250. Mu.M dNTPs,0.5-2U HotStart Taq enzyme.

Preferably, the first nested PCR reaction premix is: 10mM Tris-HCl or Tricine-KOH,70mM K ⁺ ，1-5mM Mg ²⁺ 2-5% glycerol, 200. Mu.M dNTPs,2U HotStart Taq enzyme.

Mixing the reaction premix and the primer mixture according to the volume ratio of 3-4 to 1-3, and carrying out nested primary PCR, wherein the amplification procedure is as follows:

the inner primers are specifically as follows:

HBV upstream primer 1:5 'GAACACCCGTGTGTCTTGCC-3' (SEQ ID NO: 3);

HBV downstream primer 1:5 'TGCATAGGAAGATATAAAC-3' (SEQ ID NO: 4);

HBV upstream primer 2:5 'AACTGCACCTGTATTCCCATC-3' (SEQ ID NO: 5);

HBV downstream primer 2:5 'AAGGGACTCAAGATGTTGTAC-3' (SEQ ID NO: 6);

HBV upstream primer 3:5 'ATTTGTTCAGTGGTTCGTAGG-3' (SEQ ID NO: 7);

HBV downstream primer 3:5 'TTTCAACACAATATGTTCCTG-doped 3' (SEQ I D NO: 8).

In order to detect mutation sites in the amplified fragments of the primer pair, a real-time fluorescence PCR technology is adopted, and a specific probe aiming at the mutation sites is designed, wherein the specific nucleotide sequences of the specific probe are sequentially as follows:

HBV probe 1:5 'ATTTGTCCTGGGTA-3' (SEQ ID NO: 11);

HBV probe 2:5 'ATTTGTCCTGGATA-doped 3' (SEQ ID NO: 12);

HBV probe 3:5 'CACTCCCATAGGTGT-3' (SEQ ID NO: 13);

HBV probe 4:5 'and TTCGTAGGACTTTC-3' (SEQ ID NO: 14);

HBV probe 5:5 'TACTATGGGATTGG-3' (SEQ ID NO: 15);

HBV probe 6:5 'CCGTTTCTCATGGCTT-3' (SEQ ID NO: 16);

HBV probe 7:5 '-Bisdotttcttggttcagt-3' (SEQ ID NO: 17);

HBV probe 8:5 '-Bisdevttcttgaactcagt-3' (SEQ ID NO: 18);

HBV probe 9:5 'CTCAGTTTGGTAGC-3' (SEQ ID NO: 19);

HBV probe 10:5 'CTCAGTTTTCTAGC-3' (SEQ ID NO: 20);

HBV probe 11:5 '-CTGGCTTTCATTAT-3' (SEQ ID NO: 21);

HBV probe 12:5'-CTGGCTTTCGGTTAT-3' (SEQ ID NO: 22);

HBV probe 13:5 'TTCAGTTATATCGAT-3' (SEQ ID NO: 23);

HBV probe 14:5 'and TTCAGTTATGTGGAT-3' (SEQ ID NO: 24);

HBV probe 15:5 'CAAGTCTGTATGACA-3' (SEQ ID NO: 25);

HBV probe 16:5 'TATACATTTTAACCC-3' (SEQ ID NO: 26);

HBV probe 17: 5-.

Wherein, the 5 'end of each specific probe is marked with a fluorescent group, the 3' end of each specific probe is marked with a quenching group, the fluorescent group is selected from FAM, ROX or CY5, and the quenching group is MGB. It should be noted that the fluorescent groups of the probes in the same reaction system are different from each other in order to distinguish and receive the fluorescent signals of the probes during the reaction. Preferably, the 5 'end labels of the probes with the nucleotide sequences shown as SEQ ID NO. 11-SEQ ID NO.12, SEQ ID NO. 13-SEQ ID NO.14, SEQ ID NO. 15-SEQ ID NO.16, SEQ ID NO. 17-SEQ ID NO.18, SEQ ID NO. 19-SEQ ID NO.20, SEQ ID NO. 21-SEQ ID NO.22, and SEQ ID NO. 23-SEQ ID NO.24 are respectively and sequentially labeled with fluorescent groups ROX and CY5, and the 5' end labels of the probes with the nucleotide sequences shown as SEQ ID NO. 25-SEQ ID NO.27 are respectively and sequentially labeled with fluorescent groups FAM, ROX and CY5.

The specific probe is used for detecting a mutation site of a target drug-resistant gene, and a mutual corresponding relation exists between the mutation site and an inner primer for amplifying the target drug-resistant gene. Specifically, the probes with the nucleotide sequences shown as SEQ ID NO. 11-SEQ ID NO.12 are used for detecting the mutation sites of the target drug-resistant genes amplified by the primers with the nucleotide sequences shown as SEQ ID NO. 3-SEQ ID NO.4, the probes with the nucleotide sequences shown as SEQ ID NO. 13-SEQ ID NO.24 are used for detecting the mutation sites of the target drug-resistant genes amplified by the primers with the nucleotide sequences shown as SEQ ID NO. 5-SEQ ID NO.6, and the probes with the nucleotide sequences shown as SEQ ID NO. 25-SEQ ID NO.27 are used for detecting the mutation sites of the target drug-resistant genes amplified by the primers with the nucleotide sequences shown as SEQ ID NO. 7-SEQ ID NO. 8.

In order to analyze whether the DNA of a sample to be detected is normally amplified so as to eliminate abnormal or failed PCR amplification caused by missed reagent or sample, PCR inhibitor contained in the sample, low DNA recovery rate and the like, internal standard primers for amplifying internal reference genes are also added during the second nested PCR amplification, and the internal standard primers are specifically as follows:

an internal standard upstream primer: 5 'AGATTTGGACCTGCGAGCG-doped 3' (SEQ ID NO: 9);

internal standard downstream primer: 5 'GAGCGGCTGTCTCCACAAGTT-doped 3' (SEQ ID NO: 10).

Correspondingly, an internal standard probe for detecting the internal standard gene amplified by the internal standard primer is also added in the probe set, and the internal standard probe specifically comprises the following components:

an internal standard probe: 5't CTGACCTGAAGGCTCTCTTGCGCG-3' (SEQ ID NO: 35).

Wherein, the 5 'end of the internal standard probe is connected with a fluorescence label HEX, and the 3' end of the internal standard probe is connected with a quenching group BHQ1.

Specifically, the second nested PCR primer probe mixture is:

the second nest type PCR reaction premixed solution is the same as the first nest type PCR reaction premixed solution: 10-100mM Tris-HCl or Tricine-KOH,20-100mM MK ⁺ ，1-5mMMg ²⁺ 2-5% glycerol, 25-250. Mu.M dNTPs,0.5-2UHotStartTaq enzyme.

Preferably, the second nested PCR reaction premix is: 10mM Tris-HCl or Tricine-KOH,70mM MK ⁺ ，1-5mMMg ²⁺ 2-5% glycerol, 200. Mu.M dNTPs,2UHotStartTaq enzyme.

Mixing the PCR reaction premix solution and the primer probe mixed solution according to the volume ratio of 3-4 to 1-3, and carrying out the second nested PCR, wherein the amplification procedure is as follows:

in another embodiment of the present application, the kit for detecting hepatitis b virus drug resistance gene can be assembled by subpackaging and labeling the primer mixture, the primer probe mixture and the PCR reaction premix.

Specifically, the kit comprises a first nested PCR primer mixed solution, a second nested PCR primer probe mixed solution, a first nested PCR reaction premixed solution and a second nested PCR reaction premixed solution.

When the Kit is used, since the Kit does not contain an Extraction reagent, a commercially available nucleic acid Extraction Kit such as QIAamp MinElute Virus Spin Kit (cat # 57704) from QIAGEN, viral genomic DNA/RNA Extraction Kit (cat # DP 315) from Tiangen Biochemical technology Ltd or TaKaRaMiniBEST Viral RNA/DNA Extraction Kit Ver.5.0 (cat # 9766) can be used for the blood sample, and the specific Extraction steps refer to the corresponding Extraction Kit instructions.

And adding the extracted sample DNA into a reaction system containing a first nested PCR primer mixed solution and a first PCR reaction premixed solution, covering a tube cover, centrifuging for several seconds, moving to an amplification detection area, and putting the PCR reaction tube into a fluorescent PCR amplification instrument for amplification detection.

The result judging method comprises the following steps:

when the fluorescent group of the drug-resistant gene corresponding to the specific probe shows that the amplification curve is not S-shaped, and the fluorescent group HEX of the internal standard probe shows that the amplification curve is S-shaped and Ct value is less than or equal to 35, the drug-resistant gene without mutation is shown;

when the fluorescent group of the drug-resistant gene corresponding to the specific probe shows that the amplification curve is S-shaped and the Ct value is less than or equal to 35, the existence of the mutant drug-resistant gene of the specific probe is indicated.

In another embodiment of the present application, the kit further comprises a blocking material, a lysis solution, a washing solution a, a washing solution B and a meltable material, in addition to the above kit.

The kit system can be applied to the detection of the drug resistance gene of the hepatitis B virus by a point-of-care testing (POCT) technology. The point-of-care (POCT) is a new integrated gene analysis technology integrating nucleic acid extraction, real-time fluorescent gene detection and microfluidic card boxes, only manual sample adding is needed, and then the nucleic acid extraction, detection and analysis processes are completed by machines, so that the totally-closed automatic output from a sample to a gene detection result is realized, and the POCT has the characteristics of simplicity in operation, high specificity, low cost, high speed, accurate result and the like. The integrated nucleic acid rapid detection kit based on the POCT technology can integrally extract the nucleic acid and perform PCR amplification on the hepatitis B virus under the condition of relatively low cost, further can directly perform data collection and analysis required by optical detection, and does not need a nucleic acid detection laboratory (comprising a reagent preparation room, a nucleic acid extraction room and a PCR amplification room) which needs secondary biosafety qualification and professional technicians to prepare a manual PCR amplification system, sample adding of extracted nucleic acid, detection of amplified genes, analysis of fluorescence data and judgment of experimental results like the conventional nucleic acid detection technology. The method can be used for really realizing automation from sample to result output and helping to realize rapid nucleic acid detection in areas with limited conditions.

The patent refers to publication No. CN114015567A, entitled pneumatic integrated PCR detection kit based on rotary plunger and detection method thereof, which describes an integrated PCR detection microfluidic cartridge, which is provided with at least one mixing chamber for sample cleaning and elution, a plurality of liquid storage chambers for storing different reagents and a plurality of reaction chambers; the box body is also provided with a plunger cavity, the plunger cavity is internally provided with a rotary plunger which can rotate to different angles along the axial direction, the rotary plunger is provided with a plurality of different liquid flow passages, and each liquid flow passage is respectively communicated with the mixing bin and the liquid storage bin when the rotary plunger rotates to different angles.

The closed isolation material is a hydrophobic liquid material, can isolate a plurality of functional cavities, prevents liquid among the cavities from overflowing, can perform cracking, cleaning and purification and amplification reactions on biological samples in the cavities respectively, and can bring nucleic acid into the cavities in sequence by using magnetic beads in an attachment mode, wherein the hydrophobic liquid closed material can have a dispersion effect on the magnetic beads, can allow the magnetic beads to pass through the magnetic beads and the nucleic acid on the magnetic beads, and can block other impurities.

Specifically, the density of the sealing and isolating material is 0.1-1.5g/ml, the viscosity is 1000-200000CSTG/ml, the arrangement can ensure that the hydrophobic liquid sealing material can isolate liquid, magnetic beads loaded with samples can pass through the sealing and isolating material, and the sealing and isolating material has a dispersing effect on the magnetic beads, so that nonpolar impurities on the magnetic beads are clear. Preferably, the closed isolation material is silicone oil.

The lysis solution contains 1-1000mM acetic acid-sodium acetate, 0.1-10wt% polyethylene glycol, 0.1-10M guanidinium isothiocyanate, 0.1-10wt% TritonX-100, 1-5wt% sodium lauryl sulfate.

The lotion A contains 1-500m PH8.0 of MTris-HCl, 10-500mM KCl and 0.1-10wt% of polyethylene glycol.

The washing solution B contains 1-500mM Tris-HCl of PH8.0, 10-500mM KCl, 0.1-10wt% PEG and 0.75wt% non-protein blocking agent. Wherein the non-protein blocking agent is from Blockmaster TMPA1080 of JSR company.

The meltable material is used for embedding part of reaction components in the first nested PCR primer mixed solution and the second nested PCR primer probe mixed solution, and specifically, magnesium ion liquid is embedded in the meltable material, so that the reaction liquid of a reaction liquid system is separated from enzymatic metal ions, the metal ions are prevented from stimulating enzyme protein, and the effective period of the card box is prolonged. Specifically, the meltable material is any one or combination of more of paraffin substances such as paraffin, dodecane, tetradecane, hexadecane and the like, the melting point range is 20-95 ℃, and paraffin is preferred. The paraffin oil melted into liquid state when in use can prevent PCR products forming aerosol from polluting a laboratory; the paraffin oil has weak thermal conductivity, and the temperature condition of PCR is effectively ensured; the low-temperature solidification characteristic of the paraffin can fix the position of the magnetic beads after the temperature of the magnetic bead back dragging is reduced; the paraffin is meltable so that the inclusion allows the reaction solution to be combined from a separate state.

Specifically, in this example, the applicant also designed probes shown in SEQ ID NO.28 for wild-type hepatitis B virus primers shown in SEQ ID NO. 3-SEQ ID NO.4 and probes shown in SEQ ID NO. 29-34 for wild-type hepatitis B virus primers shown in SEQ ID NO. 5-SEQ ID NO.6, respectively. And the 5' end of the probe of which the probe is shown as SEQ ID NO. 28-34 is marked with a fluorescent group FAM. The method comprises the following specific steps:

HBV probe 18:5'-FAM-ATTTGTCCTGGTTA-MGB-3' (SEQ ID NO: 28);

HBV probe 19:5 '-FAM-CACTCCATAGGTAT-MGB-3' (SEQ ID NO: 29);

HBV probe 20:5 '-FAM-TACTATGGGAGTGG-MGB-3' (SEQ ID NO: 30);

HBV probe 21:5'-FAM-TTCTCTTGGCTCAGT-MGB-3' (SEQ ID NO: 31);

HBV probe 22:5'-FAM-CTCAGTTTACTAGC-MGB-3' (SEQ ID NO: 32);

HBV probe 23:5'-FAM-CTGGCTTTCAGTTAT-MGB-3' (SEQ ID NO: 33);

HBV probe 24:5'-FAM-TTCAGTTATATGGAT-MGB-3' (SEQ ID NO: 34).

The second time nest type PCR primer probe mixed liquid is:

wherein, reaction storehouse 1 constitutes:

wherein, reaction bin 2 constitutes:

wherein, reaction bin 3 constitutes:

wherein, reaction chamber 4 constitutes:

wherein, reaction bin 5 constitutes:

wherein, reaction chamber 6 constitutes:

wherein, reaction bin 7 constitutes:

wherein, reaction chamber 8 constitutes:

the corresponding genes of the reaction bins are as follows:

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the effect of the technical scheme of the application is described below by taking an integrated nucleic acid rapid detection kit based on POCT technology as an example.

Examples of the experiments

In order to verify the accuracy of the detection of the drug resistance of the hepatitis B virus by the kit, 30 HBV patients are detected.

1) Preparation for detection

(1) Preparing an instrument: please operate the machine according to the instrument manual. And turning on a power supply of the full-automatic medical PCR analyzer (nested). And (5) after the self-checking of the instrument is finished, the instrument is ready for use.

(2) And (3) program confirmation: if the kit is used for the first time, please click on an instrument operation interface [ New Experiment ], click on a [ Scan two-dimensional code ] Scan QR code after interface conversion, scan the two-dimensional code on the side surface of the outer package of the kit, and quit the interface after the program is successfully established (if the program is stored in the instrument, the step is executed without interference). Part of the instrument already has the program built in, and no new program is needed, i.e. no step is needed.

(3) Preparation of reagents: and taking out the detection card box and the magnetic beads, and rewarming at room temperature. The beads were thoroughly shaken and mixed well before use.

2) Sample detection:

(1) the lid of the cartridge was opened and the sealing film was opened and 200. Mu.l of the sample was added. Then, 10. Mu.l of magnetic beads (thoroughly suspended by shaking before use) was added thereto, and the reagent was pipetted and mixed well, followed by closing the cap.

(2) And (4) selecting [ fast operation ]/Rapid Run on a main interface. Click the scan icon of [ detection Name ]/[ Detect Name ] column. The bar code on the test card is aligned with the scanner of the instrument and the bar code is scanned to load the test program. Then click on the sample information column and enter the sample information.

(3) And after the software prompt appears, selecting one channel, inserting the detection card box into the detection channel to be operated, and pressing tightly.

(4) And clicking the instrument to start detection, and displaying an operation interface by the software.

3) And (3) data analysis:

after the detection is finished, after the data comes out, the Analysis data/Analysis is selected on a screen, and the instrument directly analyzes the result and automatically judges the qualitative detection result of the sample to be detected.

4) And (4) analyzing results:

(1) after the operation of the detection program is finished, the detection result is automatically reported by an instrument.

(2) The results show the detection results of the target gene (FAM/ROX/CY 5) and the reference gene (HEX):

when the HEX channel amplification curve is S-shaped and the Ct value is less than or equal to 35, the FAM, ROX and CY5 channel amplification curves are not S-shaped, and the condition that the mutated drug resistance gene does not exist is indicated;

when the HEX channel amplification curve is S-shaped and the Ct value is less than or equal to 35, the FAM, ROX or CY5 channel amplification curve is S-shaped and the Ct value is less than or equal to 35, the existence of the drug-resistant gene corresponding to the corresponding channel probe is indicated. The results are shown in Table 1.

TABLE 1 results of blood sample test of 30 HBV patients

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And (4) conclusion: in the detection results of 30 samples, 30 samples are effective data, the coincidence rate of positive samples of the effective data of the samples is 100%, and except for the samples 15 in which no mutation site is detected, the other samples have gene mutation.

In order to further verify the accuracy of the detection result, the PCR amplification product is subjected to sequencing analysis. And performing Sanger sequencing on the products of the nested secondary PCR, comparing the sequencing map with the sequence of the HBV reference strain by using sequence analysis software Sequencher after the sequencing is finished, and further determining the mutation condition of the site to be analyzed to determine the drug-resistant mutation site of the HBV nucleoside analogue. The results are shown in Table 2.

TABLE 2 mutation sites of drug-resistant genes in blood samples of 30 HBV patients

And (4) conclusion: as can be seen from the comparison of the results in tables 1 and 2, the drug resistance gene of sample 15 was not detected by the method of the present application, and the other samples were consistent with the Sanger sequencing results.

Claims (10)

1. A primer probe combination for detecting a drug-resistant gene of viral hepatitis B is characterized by comprising an outer primer, an inner primer and a specific probe:

the nucleotide sequence of the outer primer is shown as an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO. 2;

the inner primers are divided into an inner primer A, an inner primer B and an inner primer C; the nucleotide sequence of the inner primer A is shown as an upstream primer shown by SEQ ID NO.3 and a downstream primer shown by SEQ ID NO.4, the nucleotide sequence of the inner primer B is shown as an upstream primer shown by SEQ ID NO.5 and a downstream primer shown by SEQ ID NO.6, and the nucleotide sequence of the inner primer C is shown as an upstream primer shown by SEQ ID NO.7 and a downstream primer shown by SEQ ID NO. 8;

the nucleotide sequence of the specific probe is shown in SEQ ID NO. 11-SEQ ID NO. 27.

2. The primer probe combination for detecting the drug resistance gene of viral hepatitis B according to claim 1,

the binding positions of the specific probes with the nucleotide sequences shown as SEQ ID NO. 11-SEQ ID NO.12 are respectively positioned between the upstream primer and the downstream primer of the inner primer A;

the binding positions of the specific probes with the nucleotide sequences shown as SEQ ID NO. 13-SEQ ID NO.24 are respectively positioned between the upstream primer and the downstream primer of the inner primer B;

the binding positions of the specific probes with the nucleotide sequences shown as SEQ ID NO. 25-SEQ ID NO.27 are respectively positioned between the upstream primer and the downstream primer of the inner primer C.

3. The primer-probe combination of claim 1, wherein the specific probe is labeled with a fluorescent group at the 5 'end and a quenching group at the 3' end, the fluorescent group is selected from FAM, ROX or CY5, and the quenching group is MGB.

4. The primer probe combination for detecting the drug-resistant gene of the viral hepatitis B according to claim 1, wherein the inner primers further comprise an internal standard primer, and the internal standard primer is an upstream primer with a nucleotide sequence shown as SEQ ID No.9 and a downstream primer with a nucleotide sequence shown as SEQ ID No. 10; the probe also comprises an internal standard probe matched with the internal standard primer, and the nucleotide sequence of the internal standard probe is shown as SEQ ID NO. 35.

5. A kit for detecting a hepatitis B virus drug resistance gene, comprising the primer probe combination of any one of claims 1 to 4 and a pre-mixed solution for hot-start real-time fluorescent PCR reaction, wherein the pre-mixed solution for hot-start real-time fluorescent PCR reaction comprises 10-100mM Tris-HCl or Tricine-KOH and 20-100mM K ⁺ ，1-5mM Mg ²⁺ 2-5wt% glycerol, 25-250. Mu.M dNTPs,0.5-2U HotStartTaq enzyme.

6. The kit for detecting a drug-resistant gene of hepatitis B virus according to claim 5,

the reaction system of the first nested PCR is as follows: 10-100mM Tris-HCl or Tricine-KOH,20-100mM K ⁺ ，1-5mM Mg ²⁺ 2-5wt% of glycerol, 25-250 mu M of dNTPs,0.5-2U of HotStartTaq enzyme and 0.1-10 mu m of each primer;

the reaction system of the second nested PCR is as follows: 10-100mM Tris-HCl or Tricine-KOH,20-100mM K ⁺ ，1-5mM Mg ²⁺ 2-5wt% glycerol, 25-250 μ M dNTPs,0.5-2U HotStartTaq enzyme, 0.1-10 μm primers, and 0.1-10 μm probes.

7. The kit for detecting the drug-resistant gene of hepatitis B virus according to claim 5, wherein the kit further comprises a blocking isolation material, a lysis solution, a washing solution A, a washing solution B and a meltable material;

the sealing isolation material is silicone oil;

said lysis solution comprises 1-1000mM acetic acid-sodium acetate, 0.1-10wt% polyethylene glycol, 0.1-10M guanidinium isothiocyanate, 0.1-10wt% TritonX-100, 1-5wt% sodium lauryl sulfate;

the lotion A comprises 1-500mM Tris-HCl of PH8.0, 10-500mM KCl and 0.1-10wt% polyethylene glycol;

the washing solution B comprises: 1-500mM Tris-HCl of PH8.0, 10-500mM KCl, 0.1-10wt% polyethylene glycol and 0.75wt% non-protein blocking agent;

the meltable material is selected from any one or more of paraffin, dodecane, tetradecane and hexadecane, and the magnesium ion solution in the hot-start real-time fluorescent PCR reaction premixed solution is embedded in the meltable material.

8. The kit for detecting the drug resistance gene of hepatitis B virus according to claim 7, wherein the silicone oil has a density of 0.1 to 1.5g/ml and a viscosity of 1000 to 200000CSTG/ml.

9. Use of the primer probe combination of claim 1 or the kit of claim 5 for detecting hepatitis b virus drug resistance gene.

10. Use of the kit of claim 7 for the detection of a hepatitis b virus resistance gene based on POCT.