CN116590469A - Kit and digital PCR detection method for YVDD drug-resistant mutation of hepatitis B virus - Google Patents
Kit and digital PCR detection method for YVDD drug-resistant mutation of hepatitis B virus Download PDFInfo
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Abstract
The application particularly provides a kit and a digital PCR detection method for hepatitis B virus YVDD drug-resistant mutation. The kit is used for detecting the YVDD drug-resistant mutation of the hepatitis B virus; the kit comprises a digital PCR detection reagent, wherein the digital PCR detection reagent comprises an upstream primer of a nucleotide sequence shown as SEQ ID NO.1, a downstream primer of a nucleotide sequence shown as SEQ ID NO.2, a mutation probe of a nucleotide sequence shown as SEQ ID NO.3 and a wild type probe of a nucleotide sequence shown as SEQ ID NO. 4; the 5' end of the mutant probe and the 5' end of the wild probe are both marked with fluorescent groups, and the 3' end of the mutant probe and the wild probe are both marked with quenching groups. When the kit is used for carrying out digital PCR amplification on hepatitis B virus, the amplification of DNA low load can be realized, and the detection limit on YVDD drug-resistant mutation reaches 0.01%.
Description
Technical Field
The application belongs to the technical field of hepatitis B virus drug-resistant mutation detection, and particularly relates to a kit and a digital PCR detection method for hepatitis B virus YVDD drug-resistant mutation.
Background
Hepatitis B Virus (HBV) belongs to hepadnaviridae, is the smallest DNA virus, and mainly infects liver. Hepadnaviruses are classified into hepadnavirus genus and avian hepadnavirus genus, and can infect mammals and birds, respectively, and the infected person is hepadnavirus genus. HBV can cause acute and chronic infections, and the virus itself does not directly cause cytopathy, liver damage is usually caused by the host's immune response to the virus. Chronic viral hepatitis b (Hepatitis B virus; abbreviated as CHB) may cause end-stage liver disease such as cirrhosis, liver failure, hepatocellular carcinoma, etc., if not controlled effectively.
Current methods of anti-viral treatment of CHB may benefit patients. There are two main types of antiviral drugs in clinic: polyethylene glycol interferon (pegylated interferon; abbreviated as PegIFNα) and NAs. The limited course of pegifnα treatment was 1 year to control viral replication by activating cytotoxic T cells to target HBV-infected hepatocyte lysis and cytokine release, with pegifnα treatment achieving viral suppression in about 25% of patients. NAs include pyrimidine nucleoside drugs: lamivudine (LAM/3 TC) and telbivudine tradenames of bivudine (LDT), the purine nucleoside drug entecavir tradename of boriding (ETV), the purine nucleotide drug adefovir dipivoxil tradename He Weili (ADV) and tenofovir dipivoxil tradename Wei Ruide (TDF). NAs medicine acts on target spot is HBV polymerase reverse transcription process, affects HBV DNA synthesis, and has no direct effect on RNA, HBeAg, HBsAg and HBcAg of HBV. LAM, ADV and TDF have significant activity against human immunodeficiency virus (Human immunodeficiency virus; abbreviated HIV). LDT and TDF are recommended for pregnant women with high viral loads when maternal and infant blockage occurs. NAs drugs are not directly targeted to cccDNA, so the continued presence of cccDNA in infected hepatocytes is a major cause of virologic rebound and disease recurrence after drug withdrawal. All CHB guidelines recommend long-term use of NAs antiviral therapy, even for life-long medications. Long-term use of NAs drugs may induce drug-resistant mutations such as YIDD caused by the G.fwdarw.T mutation at base 741 of HBV, YVDD caused by the A.fwdarw.G mutation at base 739 of HBV, etc., in addition to the examination of compliance with patients. Timely detection of drug-resistant mutant strains is helpful for clinicians to judge drug resistance and adjust treatment schemes as early as possible, and consolidates treatment results.
At present, a polymerase chain reaction (Polymerase Chain Reaction; abbreviated as PCR) product direct sequencing method, a pyrosequencing method, a gene chip method, a linear probe reverse hybridization method and the like are generally adopted to monitor drug-resistant mutation, but the monitoring methods have difficulty in amplification when HBV DNA loading is low and are easy to miss detection when the early drug-resistant mutant strain ratio of drug resistance is low, and particularly the PCR product direct sequencing method and the fluorescent PCR method can detect when the mutation is required to be more than 20%.
Disclosure of Invention
The first aim of the embodiment of the application is to provide a kit for solving the problems that the existing hepatitis B virus DNA with low loading capacity is difficult to amplify and YVDD drug-resistant mutation is difficult to detect in early stage.
The technical scheme adopted by the embodiment of the application is as follows:
a kit for detecting a drug-resistant mutation of hepatitis B virus YVDD;
the kit comprises:
the digital PCR detection reagent comprises an upstream primer of a nucleotide sequence shown as SEQ ID NO.1, a downstream primer of a nucleotide sequence shown as SEQ ID NO.2, a mutation probe of a nucleotide sequence shown as SEQ ID NO.3 and a wild type probe of a nucleotide sequence shown as SEQ ID NO. 4; the 5' end of the mutant probe and the 5' end of the wild probe are both marked with fluorescent groups, and the 3' end of the mutant probe and the wild probe are both marked with quenching groups.
Compared with the prior art, the kit provided by the embodiment of the application comprises an upstream primer of a nucleotide sequence shown in SEQ ID NO.1, a downstream primer of a nucleotide sequence shown in SEQ ID NO.2, a mutation probe of a nucleotide sequence shown in SEQ ID NO.3 and a wild type probe of a nucleotide sequence shown in SEQ ID NO.4, can realize amplification of low-load DNA of the hepatitis B virus when being used for carrying out microdroplet digital PCR amplification on the hepatitis B virus, can realize early screening of YVDD drug-resistant mutation of the hepatitis B virus, and can realize detection limit of YVDD drug-resistant mutation of up to 0.01%.
The second object of the embodiment of the application is to provide a digital PCR detection method for drug-resistant mutation of hepatitis B virus YVDD, which adopts the following specific technical scheme:
a digital PCR detection method for the YVDD drug-resistant mutation of hepatitis B virus, which is based on the above kit, comprises the following steps:
extracting DNA of a sample to be detected;
mixing the digital PCR detection reagent with the DNA to obtain a digital PCR reaction system;
adding the digital PCR reaction system and the droplet generation oil into a droplet generation card, adding buffer solution for filling, and placing the mixture in a droplet preparation device to obtain droplets;
transferring the microdroplet into a microplate for PCR amplification reaction;
placing the amplified microplate in a droplet reader for reading;
and automatically calculating and obtaining the copy number of wild type DNA and the copy number of mutant DNA in the hepatitis B virus in the digital PCR reaction system according to the poisson distribution principle.
Preferably, the conditions of the PCR amplification reaction are: enzyme activation at 95℃for 10min; denaturation at 94℃for 30s, annealing at 55.7-56.9℃for 1min, and cycling 40 times; then inactivated at 98℃for 10min and stored at 4 ℃.
Compared with the prior art, the digital PCR detection method for the YVDD drug-resistant mutation of the hepatitis B virus provided by the embodiment of the application can achieve the detection limit of single-digit copy per microliter for wild type and mutant type of hepatitis B virus plants on the premise of being based on kit components, can calculate the ratio of the wild type to the mutant type, and can achieve the lowest detection limit of 0.01% for the YVDD drug-resistant mutation, thereby being capable of timely finding the drug-resistant mutation of the YVDD of a patient in early stage.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are required to be used in the embodiments will be briefly described below. It is evident that the drawings in the following description are only some embodiments of the present application and that other drawings may be obtained from these drawings without inventive effort for a person of ordinary skill in the art.
FIG. 1 is an annealing temperature probe map of FAM-MGB probe to wild plasmid in the example of the present application;
FIG. 2 is an annealing temperature probe pattern of FAM-MGB probe to mutant plasmid in the example of the present application;
FIG. 3 is an annealing temperature probe map of HEX3-MGB probe to wild type plasmid in the example of the application;
FIG. 4 is an annealing temperature probe map of HEX3-MGB probe to mutant plasmid in the examples of the application;
FIG. 5 is a bar graph of theoretical and actual detection values of a wild-type plasmid for precision detection by microdroplet digital PCR in an example of the present application;
FIG. 6 is a bar graph of theoretical and actual values of precision detection of mutant plasmids by digital droplet PCR in an embodiment of the application;
FIG. 7 is a bar graph of actual values of microdroplet digital PCR for precision detection of plasmids containing both wild type and mutant types in an example of the present application;
FIG. 8 is a comparison of microdroplet digital PCR and direct PCR amplification performed by patient G103 in an embodiment of the present application;
FIG. 9 is a comparison of microdroplet digital PCR and direct PCR amplification performed by patient G113 in an embodiment of the application;
in each figure: ch1 Amplitude represents the Amplitude of channel 1; ch2 Amplitude represents the Amplitude of channel 2; event Number represents an Event Number; pos 3431 indicates that the number of positive droplets is 3431; neg 127546 indicates that the number of negative droplets is 127546; gradient dilution gradient dilution; plasmid represents a plasmid; copies/. Mu.L indicates the copy number per microliter.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the application is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application.
The application at least relates to two aspects of the application, wherein the first aspect of the application provides a kit for detecting the drug-resistant mutation of the hepatitis B virus YVDD.
Specifically, the kit comprises: the digital PCR detection reagent comprises an upstream primer of a nucleotide sequence shown as SEQ ID NO.1, a downstream primer of a nucleotide sequence shown as SEQ ID NO.2, a mutation probe of a nucleotide sequence shown as SEQ ID NO.3 and a wild type probe of a nucleotide sequence shown as SEQ ID NO. 4; the 5' end of the mutant probe and the 5' end of the wild probe are both marked with fluorescent groups, and the 3' end of the mutant probe and the wild probe are both marked with quenching groups.
Specifically, the nucleotide sequence shown in SEQ ID NO.1 is 5'-ttgttcagtggttcgtagg-3'; the nucleotide sequence shown in SEQ ID NO.2 is 5'-aaagggactcaagatgttgt-3'; the nucleotide sequence shown in SEQ ID NO.3 is 5'-ctttcagttatgtg-3'; the nucleotide sequence shown in SEQ ID NO.4 is 5'-ctttcagttatatg-3'.
In some embodiments, the fluorescent group at the 5 'end of the mutant probe is selected from 6-FAM and the quenching group at the 3' end is selected from MGB. In some embodiments, the fluorescent moiety at the 5 'end of the wild-type probe is selected from HEX and the quenching moiety at the 3' end is selected from MGB.
In some embodiments, the digital PCR detection reagent further comprises a premix containing a DNA polymerase, dNTPs, magnesium ions, a buffer, and water.
In some embodiments, the kit further comprises a quality control, a calibrator, a microplate, and a sealing membrane. In some embodiments, the quality control comprises enzyme-free water. In some embodiments, the microwell plate is a 96 well plate, and the 96 well plate is used as a component of the kit, so that the detection flux can be effectively improved, and the automatic detection is facilitated.
In some embodiments, the digital PCR detection reagent may be obtained according to the following method:
(1) The upstream primer and the downstream primer are respectively added into first stock solution and second stock solution which are diluted to 100 mu M by water, and the mutant probe and the wild probe are added into third stock solution and fourth stock solution which are diluted to 100 mu M by water.
(2) The first stock solution, the second stock solution, the third stock solution, and the fourth stock solution were mixed with water to prepare 100. Mu.L of working solution, the volumes of the respective components in the working solution are shown in Table 1.
TABLE 1
Component (A) | Volume/. Mu.L |
Upstream primer | 10 |
Downstream primer | 10 |
Probe with a probe tip | 5(1:1) |
Water and its preparation method | 75 |
Total volume of | 100 |
Note that: in Table 1, the two probes were mixed in a ratio of 1:1
(3) Mixing the working solution and the premix, and adding water when necessary to obtain the digital PCR detection reagent.
The kit is suitable for digital PCR (digital PCR; abbreviated as dPCR) amplification reaction to detect the drug-resistant mutation of the hepatitis B virus YVDD. dPCR includes any of micro-chamber digital PCR (mdPCR), microfluidic chip digital PCR (mcdPCR), microdroplet digital PCR (ddPCR).
Based on the kit, the application scheme of the second aspect of the application provides a digital PCR detection method for the drug-resistant mutation of the hepatitis B virus YVDD.
The detection is based on the kit, and specifically comprises the following steps:
extracting DNA of a sample to be detected;
mixing the digital PCR detection reagent with the DNA to obtain a digital PCR reaction system;
adding the digital PCR reaction system and the droplet generation oil into a droplet generation card, adding buffer solution for filling, and placing the mixture in a droplet preparation device to obtain droplets;
transferring the microdroplet into a microplate for PCR amplification reaction;
placing the amplified microplate in a droplet reader for reading;
and automatically calculating and obtaining the copy number of wild type DNA and the copy number of mutant DNA in the hepatitis B virus in the digital PCR reaction system according to the poisson distribution principle.
In some embodiments, the sample to be tested comprises any one of plasma, serum. The DNA extraction process of the sample to be tested according to the present application will be described below by taking DNA extraction of a serum sample as an example.
Extraction was performed using EasyPure Viral DNA/RNA kit (full gold, cat# ER 201-01) and RNase inhibitors were sprayed periodically during the course of the experiment.
The operation steps are as follows:
before use, 48mL of absolute ethanol was added to the elution Buffer (Wash Buffer; abbreviated as WB 5).
(1) Add 20. Mu.L of protease k (proteinase K) to a 1.5mL sterile centrifuge tube.
(2) 150. Mu.L of RNase-free water was added to the centrifuge tube, 50. Mu.L of serum sample was added, the total volume was 200. Mu.L, and the insufficient sample was filled with water.
(3) 200. Mu.L of the mixture (BB5+Carrier RNA) was added thereto, and the mixture was vortexed and mixed at a rotational speed of 2000 to 3000r for 15 seconds.
(4) Incubate at 56℃for 15min.
(5) 250 μl of absolute ethanol was added, and the mixture was vortexed at a rotational speed of 1500-2000r for 15s, followed by standing at room temperature for 5min.
(6) The solution was added to the column together with the precipitate, centrifuged at 12,000g for 1min and the effluent was discarded.
(7) 500. Mu.L of WB5 was added and centrifuged at 12,000g for 1min, and the effluent was discarded.
(8) Repeating the step (7) once.
(9) Centrifuging at room temperature for 2min at 12,000g, thoroughly removing residual ethanol, standing in an ice box for 5min, and thoroughly airing the centrifugal column.
(10) The column was transferred to a 1.5mL RNase-free centrifuge tube (full gold cartridge) and 20. Mu.L RNase-free Water (RNase-free Water) was added to the center of the column and allowed to stand at room temperature for 5min.
(11) Centrifuging at 12,000g for 1min at room temperature, eluting to obtain DNA/RAN.
(12) The DNA was stored at-20℃and the RNA was stored at-80 ℃.
In some embodiments, the conditions of the PCR amplification reaction are: enzyme activation at 95℃for 10min; denaturation at 94℃for 30s, annealing at 55.7-56.9℃for 1min, and cycling 40 times; then inactivated at 98℃for 10min and stored at 4 ℃. Preferably, the annealing temperature is 56 ℃.
To determine the annealing temperature range of the primers and probes, primers 5 and oligo 6 software were used to design primers and probes for detecting hepatitis B virus YVDD drug resistance mutation, wherein FAM-MGB probe (i.e., SEQ ID NO. 3) was used for detecting mutation, and HEX3-MGB probe (i.e., SEQ ID NO. 4) was used for detecting wild type.
To find out suitable detection conditions and systems, wild-type plasmid pBlueBac4.5 HBV1.2M204 (YMDD; wherein Y represents tyrosine; M represents methionine; D represents aspartic acid) and mutant plasmid pBlueBac4.5 HBV 1.2V 204 (YVDD; wherein V represents valine) were constructed. The application searches the temperature suitable for specificity and effective amplification of PCR reaction by setting the temperature gradient, and the results are shown in figures 1 to 4.
As can be seen from FIGS. 1 and 2, the FAM-MGB probe has a higher signal intensity for mutant plasmids and wild type plasmids as the temperature decreases, and a higher signal intensity for mutant plasmids detected at 56.9deg.C down to 55.7deg.C, and a highest signal intensity for mutant plasmids detected at 56 deg.C, allowing efficient separation of wild and mutant signals.
As can be seen from FIGS. 3 and 4, the signal intensity of HEX3-MGB probe was continuously enhanced for both wild type and mutant plasmids, and the wild type plasmid was detected to have higher signal intensity when the temperature was lowered to 55.7℃at 56.9℃and the wild type plasmid was detected to have the highest signal intensity when the temperature was lowered to 56 ℃.
From the results of FIGS. 1 to 4, it was confirmed that the annealing temperature of the primer and probe of the present application was suitably 55.7℃to 56.9℃and the optimum reaction annealing temperature condition was 56 ℃.
In order to further evaluate the detection efficacy and lower limit of the established digital PCR detection method, the present application uses plasmids of known copy number as standards, and sets a series of samples of different concentration gradients by continuously subjecting the plasmids to an equal ratio dilution, thereby defining the detection accuracy and linear range of the digital PCR, and the results are shown in fig. 5 to 7.
As can be seen from FIG. 5, when the pure wild type plasmid is detected by digital PCR, the theoretical value and the actual detection value after dilution are listed in the figure, and the consistency of the theoretical value and the detection value is good in the range of 2.95-2950 copies/. Mu.L, and the theoretical value and the detection value belong to the stable linear range of results; and after the concentration is less than 2.95 copies/. Mu.L, larger deviation appears between the theoretical value and the detection value, and the detection value is unstable. FIG. 6 shows the results of detecting mutant plasmids by digital PCR, and it can be seen that the theoretical value and the detection value are not greatly different in the range of 6.43-6430 copies/. Mu.L, but are greatly deviated after less than 6.43 copies/. Mu.L.
Meanwhile, in order to evaluate the condition that the wild type and the mutant exist simultaneously, the wild type sample and the mutant sample with known copy numbers are mixed so as to simulate the condition that the wild type and the mutant coexist in a patient. By diluting and mixing mutant samples, a series of mixed samples with different mutant duty ratios were constructed, and fig. 7 shows the result of detecting the mixed samples using digital PCR. As can be seen from the figure, even if the mutant plasmid accounts for only 0.01%, the digital PCR can still detect the mutant plasmid, and the advantages not possessed by the fluorescent quantitative PCR are exhibited.
In summary, the detection range of the digital PCR on the wild type is 2.95-2950 copies/. Mu.L, the detection range of the digital PCR on the mutant type is 6.43-6430 copies/. Mu.L, the lowest detection range of the digital PCR on the wild type and the mutant type can reach single digit copies per microliter, and samples smaller than the single digit copies per microliter can also be detected. For the detection of the wild type and mutant samples, the detection of the mutant in the wild type is carried out at a ratio, and the lowest detection limit can reach 0.01%.
Detection of drug resistance variation by digital PCR and direct sequencing technology
The application uses two samples with different drug resistance variation ratios as research objects, and uses a droplet digital PCR and a direct sequencing technology to test the samples, wherein the specific results are shown in fig. 8 and 9, fig. 8 is the detection result of a patient G103, fig. 9 is the detection result of a patient G113, wherein fig. 8-A and 9-A are the results of droplet digital PCR detection, and fig. 8-B and 9-B are the results of direct sequencing. From FIGS. 8-A and 9-A, it can be seen that the wild type (lower right hand corner) and the variant (upper left hand corner) can be distinguished significantly in the microdroplet digital PCR, while the variant duty cycle can also be calculated. In fig. 8-B and 9-B, the M204V bimodal sample is not only indistinguishable, but also cannot be used to calculate the duty cycle of the variant.
In order to provide valuable information to clinic, the application also carries out absolute quantitative detection of droplet digital PCR on 93 patients with double peaks of direct sequencing 204, and the detection method is as follows:
microparticle preparation: a droplet generation card is installed, 21 mu L of digital PCR detection reagent is sucked into a sample hole, 70 mu L of oil is added into the oil hole, redundant holes are filled with buffer solution, epithelial strips are sleeved, and the mixture is placed into a droplet preparation device to start droplet preparation. The sample gun was adjusted to 50. Mu.L, and the prepared droplets were all transferred into a 96-well plate with gentle motion, and PCR amplification was performed.
PCR amplification conditions: enzyme activation at 95℃for 10min; denaturation at 94℃for 30s, annealing at 56℃for 1min, and cycling 40 times; inactivating at 98deg.C for 10min; stored at 4℃in 40. Mu.L.
Reading parameters were set and the amplified 96-well plates were transferred to a droplet reader for reading, with the specific results shown in table 3.
TABLE 3 93 direct sequencing 204 bimodal patient droplet digital PCR absolute quantitative results
As can be seen from Table 3, hepatitis B patients can also be detected when the YVDD resistance mutation ratio is only 0.01%.
In conclusion, the detection method of the application can realize the amplification of the low-load hepatitis B virus DNA based on the kit, and the detection limit of the YVDD drug resistance mutation reaches 0.01%, so that the early screening of the hepatitis B virus YVDD drug resistance mutation can be realized.
The foregoing description of the preferred embodiments of the application is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the application.
Claims (10)
1. A kit, which is characterized in that the kit is used for detecting the drug-resistant mutation of the hepatitis b virus YVDD;
the kit comprises:
the digital PCR detection reagent comprises an upstream primer of a nucleotide sequence shown as SEQ ID NO.1, a downstream primer of a nucleotide sequence shown as SEQ ID NO.2, a mutation probe of a nucleotide sequence shown as SEQ ID NO.3 and a wild type probe of a nucleotide sequence shown as SEQ ID NO. 4; the 5' end of the mutant probe and the 5' end of the wild probe are both marked with fluorescent groups, and the 3' end of the mutant probe and the wild probe are both marked with quenching groups.
2. The kit of claim 1, wherein the fluorescent group at the 5 'end of the mutant probe is selected from 6-FAM and the quenching group at the 3' end is selected from MGB;
the fluorescent group at the 5 'end of the wild-type probe is selected from HEX, and the quenching group at the 3' end is selected from MGB.
3. The kit of claim 1, wherein the digital PCR detection reagent further comprises a premix containing DNA polymerase, dNTPs, magnesium ions, buffer, and water.
4. The kit of claim 1, further comprising a quality control, a calibrator, a microplate, and a sealing membrane.
5. The kit of claim 4, wherein the quality control comprises enzyme-free water.
6. A digital PCR detection method for a hepatitis b virus YVDD resistance mutation, characterized in that the detection is based on the kit of any one of claims 1 to 5, comprising the steps of:
extracting DNA of a sample to be detected;
mixing the digital PCR detection reagent with the DNA to obtain a digital PCR reaction system;
adding the digital PCR reaction system and the droplet generation oil into a droplet generation card, adding buffer solution for filling, and placing the mixture in a droplet preparation device to obtain droplets;
transferring the microdroplet into a microplate for PCR amplification reaction;
placing the amplified microplate in a droplet reader for reading;
and automatically calculating and obtaining the copy number of wild type DNA and the copy number of mutant DNA in the hepatitis B virus in the digital PCR reaction system according to the poisson distribution principle.
7. The method of claim 6, wherein the PCR amplification reaction conditions are: enzyme activation at 95℃for 10min; denaturation at 94℃for 30s, annealing at 55.7-56.9℃for 1min, and cycling 40 times; then inactivated at 98℃for 10min and stored at 4 ℃.
8. The method of claim 7, wherein the annealing temperature is 56 ℃.
9. The method of any one of claims 6 to 8, wherein the sample comprises any one of plasma and serum.
10. The detection method according to any one of claims 6 to 8, wherein the digital PCR is selected from any one of micro-droplet digital PCR, micro-chamber digital PCR, and micro-fluidic chip digital PCR.
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CN116949223B (en) * | 2023-09-19 | 2023-12-29 | 广东凯普生物科技股份有限公司 | Hepatitis B virus drug administration guidance system and application thereof |
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