CN112795696A - Hepatitis B virus pgRNA chip digital PCR absolute quantitative detection kit and method - Google Patents
Hepatitis B virus pgRNA chip digital PCR absolute quantitative detection kit and method Download PDFInfo
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- 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
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Abstract
The invention discloses a hepatitis B virus pgRNA chip digital PCR absolute quantitative detection kit and a method. The invention provides application of a primer probe composition in preparing a reagent or a kit for detecting hepatitis B virus pgRNA by using digital PCR; the primer probe composition consists of a primer HBV-F, a primer HBV-R and a probe HBV-P; the primer HBV-F is a single-stranded DNA molecule shown in a sequence 1 in a sequence table; the primer HBV-R is a single-stranded DNA molecule shown in a sequence 2 in a sequence table; one terminal of the probe HBV-P is provided with a fluorescence reporter group, the other terminal is provided with a fluorescence quenching group, and the nucleotide sequence of the probe HBV-P is shown as a sequence 3 in a sequence table. The invention can detect the hepatitis B virus pgRNA and the load capacity with high sensitivity and high accuracy.
Description
Technical Field
The invention belongs to the field of virus detection, and particularly relates to a hepatitis B virus pgRNA chip digital PCR absolute quantitative detection kit and a method.
Background
Hepatitis b is a disease caused by infection with Hepatitis B Virus (HBV) and is transmitted mainly through blood and blood products, mother and infant, and sexual contact. The clinical manifestations of hepatitis B virus infected persons are diversified, and the most common clinical manifestations include anorexia, nausea, vomiting, abdominal distension, hypodynamia and other symptoms. Patients with chronic hepatitis B can progress to cirrhosis or liver cancer, and the treatment is very difficult.
HBV DNA exists in the form of relaxed circular DNA (rcDNA) in the whole virus, after HBV viral particles enter liver cells, the outer capsid is removed, the rcDNA enters the nucleus to form covalently closed circular DNA (cccDNA), the cccDNA is taken as a template and is transcribed to form pregenomic RNA (pgRNA) and other mRNA, the pgRNA forms the rcDNA under the action of reverse transcriptase, and the rcDNA is assembled with related proteins to form the HBV viral particles. The HBV genome of hepatitis B virus is about 3.2kb in length and is a partial double-stranded circular DNA. It can encode four mRNAs of different lengths, 3.5kb, 2.4kb, 2.1kb and 0.7kb, wherein the mRNA of 3.5kb is the pregenomic RNA of hepatitis B virus. The pgRNA can be used as mRNA to transcribe protein necessary for synthesizing HBV on one hand, and can be used as a template for reverse transcription to synthesize HBV DNA on the other hand, and the action mechanism of the nucleoside analogue is to inhibit the process from the pgRNA to the HBV DNA.
The results of some clinical trials have shown that hepatitis B patients with negative HBV DNA detection continue to have the disease condition, and the fundamental reason for this is that the core components of the viral particles are not cleared and stopped. Among them, HBV cccDNA is one of the currently clinically accepted "core components". However, the existing NA antiviral drugs such as Lamivudine (LAM) can not completely remove HBV cccDNA, and after a long period of antiviral treatment, the stably infected cells of hepatitis B patients still maintain the intracellular cccDNA at a certain level, and each hepatocyte has about 5-50 copies. However, HBV cccDNA is mainly present in liver cells, and the material drawing and detection are very difficult. Vaccination with hepatitis b vaccine is the most effective method for preventing HBV infection, but is not effective for patients already infected with HBV. For chronic hepatitis b patients, the most basic treatment modality now recognized is antiviral therapy, including Interferon (IFN) and nucleoside (acid) analogs (NA). According to statistics, the incidence rate of drug resistance reaches 14% in 1 year of treatment, and reaches 70% in 5 years. Research shows that under the condition that various indexes of chronic hepatitis B patients receiving antiviral treatment reach the drug withdrawal standard recommended by the guideline, the recurrence rate of hepatitis B of patients who cannot be detected by pgRNA after drug withdrawal is far lower than that of patients with high pgRNA load, so that the detection of pgRNA load has important clinical significance. Different from the conventional HBV DNA detection indexes, HBV pgRNA is expected to become a brand-new potential biological marker for predicting NA withdrawal.
Disclosure of Invention
The technical problem to be solved by the invention is how to detect pgRNA of hepatitis B virus.
In order to solve the above technical problems, the present invention first provides a primer probe composition consisting of a primer HBV-F, a primer HBV-R and a probe HBV-P.
The primer HBV-F is (a1) or (a 2):
(a1) a single-stranded DNA molecule shown in sequence 1 of the sequence table;
(a2) and (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 1 and has the same function as the sequence 1.
The primer HBV-R is (a3) or (a 4):
(a3) a single-stranded DNA molecule shown in a sequence 2 of a sequence table;
(a4) and (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 2 and has the same function as the sequence 2.
The probe HBV-P has a fluorescence reporter group at one end and a fluorescence quenching group at the other end, and the nucleotide sequence is as follows (a5) or (a 6):
(a5) as shown in sequence 3 of the sequence table;
(a6) as shown by the substitution and/or deletion and/or addition of one or more nucleotides to the sequence 3.
Specifically, the probe HBV-P has a fluorescent group FAM at the 5 'end and a fluorescence quencher group BHQ-1 at the 3' end.
In order to solve the technical problems, the invention also provides application of the primer probe composition in preparing a reagent or a kit for detecting hepatitis B virus pgRNA by using digital PCR.
The invention also protects the application of the primer probe composition, which is (b1), (b2), (b3), (b4), (b5) or (b6) or (b7) or (b 8):
(b1) identifying the hepatitis B virus;
(b2) preparing a kit for identifying hepatitis B virus;
(b3) detecting whether the sample to be detected contains hepatitis B virus pgRNA;
(b4) preparing a kit for detecting whether a sample to be detected contains hepatitis B virus pgRNA or not;
(b5) detecting the load capacity of the hepatitis B virus pgRNA in a sample to be detected;
(b6) preparing a kit for detecting the pgRNA load of the hepatitis B virus in a sample to be detected;
(7b) predicting the recurrence rate of hepatitis B;
(8b) preparing a product for predicting the recurrence rate of hepatitis B.
The invention also provides a kit, which comprises the primer probe composition; the kit has the functions of any one of the following (c1) to (c 4):
(c1) identifying the hepatitis B virus;
(c2) detecting whether the sample to be detected contains hepatitis B virus pgRNA;
(c3) detecting the load capacity of the hepatitis B virus pgRNA in a sample to be detected;
(c4) predicting the recurrence rate of hepatitis B.
In order to solve the technical problem, the invention also provides a method for detecting whether a sample to be detected contains hepatitis B virus pgRNA, which comprises the following steps: performing digital PCR by using cDNA of a sample to be detected as a template and the primer probe composition; if the detection result is positive, the sample to be detected contains hepatitis B virus pgRNA; and if the detection result is negative, the sample to be detected does not contain hepatitis B virus pgRNA.
In order to solve the technical problems, the invention also provides a method for detecting the load of hepatitis B virus pgRNA in a sample to be detected, which comprises the following steps: performing digital PCR by using cDNA of a sample to be detected as a template and the primer probe composition; and obtaining the load capacity of the hepatitis B virus pgRNA in the sample to be detected according to the hepatitis B virus pgRNA positive fluorescent spot.
In the method, the reaction system of the digital PCR can be composed of a primer HBV-F, a probe HBV-P and a Quant StudioTM3D Digital PCR Master Mix and template. In the digital PCR reaction, the concentration of the primer HBV-F was 0.33. mu.M, the concentration of the primer HBV-R was 0.33. mu.M, and the concentration of the probe HBV-P was 0.67. mu.M.
In the above method, the reaction procedure of the digital PCR may be: pre-denaturation at 96 ℃ for 10 min; denaturation at 98 ℃ for 30s, annealing at 60 ℃ for 2min, and 40 cycles; infinity at 10 ℃.
The invention also provides a premixed solution for detecting the hepatitis B virus pgRNA through digital PCR, which contains the primer HBV-F, the probe HBV-P and the Quant Studio in the primer probe compositionTM3D Digital PCR Master Mix; in the premix, the concentration of the primer HBV-F is 0.53. mu.M, the concentration of the primer HBV-R is 0.53. mu.M, and the concentration of the probe HBV-P is 1.05. mu.M. The application of the premix is as follows: the following (c1), (c2) or (c3) or (c 4): (c1) identifying the hepatitis B virus; (c2) detecting whether the sample to be detected contains hepatitis B virus pgRNA; (c3) detecting the content of hepatitis B virus pgRNA in a sample to be detected; (c4) predicting the recurrence rate of hepatitis B.
The invention also provides a kit for detecting hepatitis B virus pgRNA by digital RT-PCR, which comprises the premix, a negative control and a positive control. Wherein the negative control is human hepatitis B virus negative plasma; the positive control is plasma containing hepatitis B virus pgRNA.
The invention also protects the application of the premix or the kit, which is (c1), (c2) or (c3) or (c 4): (c1) identifying the hepatitis B virus; (c2) detecting whether the sample to be detected contains hepatitis B virus pgRNA; (c3) detecting the content of hepatitis B virus pgRNA in a sample to be detected; (c4) predicting the recurrence rate of hepatitis B.
The invention provides a digital PCR kit which has high sensitivity, high specificity, high accuracy and high precision and can realize accurate quantification, and the kit can be used for detecting the hepatitis B virus pgRNA. The kit and the method provided by the invention have the following advantages:
1) the invention can carry out detection by using a blood sample, has no wound, can be applied on a large scale conventionally, and solves the problem that HBV cccDNA depends on liver puncture.
2) The invention surpasses the conventional pathogen RT-PCR technology and the fluorescence quantitative qPCR technology, and can detect the pgRNA with ultra-low load in blood.
3) The invention solves the problem of relapse after drug withdrawal after clinical dependence on HBV DNA indexes and judgment of antiviral treatment withdrawal, and provides a scheme for solving the problem of searching clinical standard and biological marker for predicting NA withdrawal of hepatitis B, which is one of ten problems to be solved in clinical guidelines of hepatitis B.
4) The application of the invention can deeply research the hepatitis B pgRNA form in China and provide basic research data for developing pgRNA medicaments.
Drawings
FIG. 1 shows the results of digital PCR detection of pgRNA of hepatitis B virus; wherein, the ordinate and the abscissa are logarithms with a base 10 respectively.
FIG. 2 shows the results of the digital PCR detection of the sensitivity of hepatitis B virus pgRNA; wherein A is a detection result of a negative control, B is a detection result of a positive control, C is a detection result of a sample to be detected with a concentration of 50copies/mL, D is a detection result of a sample to be detected with a concentration of 25copies/mL, E is a detection result of a sample to be detected with a concentration of 10copies/mL, and F is a detection result of a sample to be detected with a concentration of 5 copies/mL.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The digital PCR in the examples all adopt Quant StudioTM3D Digital PCR System (ThermoFisher Scientific) System (including Quantstrudio)TM3D Digital PCR Instrument,QuantStudioTM3D Digital PCR Chip Loader and ProFlexTM2x Flat Block Thermal Cycler)。
Example 1 preparation of a kit for detecting hepatitis B Virus pgRNA
Design of primers and probes
The kit for detecting the hepatitis B virus pgRNA consists of a primer pair HBV-F/R and a probe HBV-P. The primer pair HBV-F/R consists of a primer HBV-F: 5'-GAGGCTGTAGGCATAAATTGGT-3' (sequence 1 in the sequence table) and a primer HBV-R: 5'-CTAGACTGGAATCGCGATTCAT-3' (sequence 2 in the sequence table). The probe HBV-P is: 5'-CACCAGCACCATGCAACTTTTTCACC-3' (SEQ ID NO: 3 in the sequence Listing). The 5 'end of the probe HBV-P is marked with a fluorescent group FAM, and the 3' end is marked with a fluorescence quenching group BHQ-1.
Preparation of respective reagents
The solution A is a premixed solution of a dPCR probe method. The composition of solution A per 9.5. mu.L was as follows: 7.5 μ L QuantStudioTM3D Digital PCR Master Mix, 0.5. mu.L of HBV-F solution (the concentration of HBV-F in the HBV-F solution is 10. mu.M), 0.5. mu.L of HBV-R solution (the concentration of HBV-R in the HBV-R solution is 10. mu.M), 1. mu.L of HBV-P solution (the concentration of probe in the HBV-P solution is 10. mu.M).
Solution B was a positive control. The preparation method of the solution B comprises the following steps: recombinant virus containing pgRNA coding sequences was diluted to E3copies/mL in human hepatitis B virus negative plasma.
Solution C was a negative control. Solution C was human hepatitis b virus negative plasma.
Assembly of three, digital PCR absolute quantitative detection kit
The kit comprises the following components: and respectively and independently packaging the solution A, the solution B and the solution C.
Example 2 establishment of a method for detecting hepatitis B Virus pgRNA by digital PCR
1. Extraction of total RNA from test sample
A recombinant vector obtained by replacing the fragment between XhoI and XbaI recognition sites on the vector pLVX-Puro with a DNA fragment (encoding hepatitis B virus pgRNA) shown in sequence 4 and keeping other nucleotides of pLVX-Puro unchanged; the recombinant vector was transfected into HEK293T cells to obtain recombinant viruses containing pgRNA coding sequences.
The constructed recombinant virus containing pgRNA coding sequence was diluted 10-fold to 10E5copies/mL, 10E4copies/mL, 10E3copies/mL and 10E2copies/mL, and the total Nucleic Acid of the sample was extracted with Mag Pure Viral Acid Maxi LQ Kit (Meiji Biotech, Guangzhou).
2. Obtaining cDNA of sample to be tested
The total nucleic acid of the sample to be tested was extracted, and pgRNA was Reverse transcribed into cDNA using as a template solution according to the procedure of the Reverse Transcription Kit (High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor, Thermo Fisher Scientific, No. 4374967).
3. Digital PCR amplification reaction
(1) Digital PCR reaction System: add 9.5. mu.L of solution A to 5.5. mu.L of template solution. An equal volume of solution C was used as a negative control treatment instead of the template solution. An equal volume of solution B was used instead of template solution as a positive control treatment.
(2) Taking the PCR reaction system in the step (1), and loading the PCR reaction system on a chip according to a Quant Studio deviceTM3D Digital PCR Chip Loader, Thermo Fisher Scientific) descriptionLoading and packaging a chip by the book; chip in plate PCR instrument (Pro Flex)TM2x Flat Block Thermal Cycler, Thermo Fisher Scientific), PCR was performed by setting PCR Thermal cycling conditions according to the instructions of the corresponding PCR apparatus. PCR amplification procedure: (1) pre-denaturation at 96 ℃ for 10 min; (2) denaturation at 98 ℃ for 30s, annealing at 60 ℃ for 2min, and 40 cycles; (3) infinity at 10 ℃.
(3) After the reaction is finished, the chip is placed into a digital PCR chip reader for chip scanning, and the test result is analyzed by using the software matched with the digital PCR chip reader. Then, the following judgment is carried out: if the sample to be detected generates a positive signal, the sample to be detected contains hepatitis B virus pgRNA; if the sample to be tested does not generate a positive signal, the sample to be tested does not contain hepatitis B virus pgRNA.
The results show (as in fig. 1): positive signals are detected in template solutions with the dilution concentrations of 10E5copies/mL, 10E4copies/mL, 10E3copies/mL and 10E2copies/mL respectively; the linear range of the established pgRNA detection method is 10E2copies/mL to 10E5 copies/mL; the linear regression equation is that y is 1.115 x-0.49; the square of the correlation coefficient R2 is 0.9999.
Example 3 detection of sensitivity
1. Extraction of total RNA from test sample
The 10E2 sample of example 2 was diluted in multiple concentrations of 50copies/mL, 25copies/mL, 10copies/mL, and 5copies/mL, and total Nucleic acids were extracted from the sample using the Mag Pure Viral Nucleic Acid Maxi LQ Kit (Meiji Biotech, Guangzhou).
2. Obtaining cDNA of sample to be tested
Total nucleic acids of samples to be detected are respectively taken, pgRNA is reversely transcribed into cDNA according to the steps of the instructions of a Reverse Transcription Kit (High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor, Thermo Fisher Scientific, No.4374967), and the cDNA is used as a template solution.
3. Digital PCR amplification reaction
(1) Digital PCR reaction System: add 9.5. mu.L of solution A to 5.5. mu.L of template solution. An equal volume of solution C was used as a negative control treatment instead of the template solution. An equal volume of solution B was used instead of template solution as a positive control treatment.
(2) Taking the PCR reaction system in the step (1), and loading the PCR reaction system on a chip according to a Quant Studio deviceTM3D Digital PCR Chip Loader, Thermo Fisher Scientific) instructions for Chip loading and packaging; chip in plate PCR instrument (Pro Flex)TM2x Flat Block Thermal Cycler, Thermo Fisher Scientific), PCR was performed by setting PCR Thermal cycling conditions according to the instructions of the corresponding PCR apparatus. PCR amplification procedure: (1) pre-denaturation at 96 ℃ for 10 min; (2) denaturation at 98 ℃ for 30s, annealing at 60 ℃ for 2min, and 40 cycles; (3) infinity at 10 ℃.
(3) After the reaction is finished, the chip is placed into a digital PCR chip reader for chip scanning, and the test result is analyzed by using the software matched with the digital PCR chip reader. Then, the following judgment is carried out: if the sample to be detected generates a positive signal, the sample to be detected contains hepatitis B virus pgRNA; if the sample to be tested does not generate a positive signal, the sample to be tested does not contain hepatitis B virus pgRNA.
The results show (as shown in fig. 2): the lowest pgRNA load that could be stably detected was 10 copies/mL.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Sequence listing
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Claims (10)
1. The application of a primer probe composition in preparing a reagent or a kit for detecting hepatitis B virus pgRNA by using digital PCR;
the primer probe composition consists of a primer HBV-F, a primer HBV-R and a probe HBV-P;
the primer HBV-F is (a1) or (a 2):
(a1) a single-stranded DNA molecule shown in sequence 1 of the sequence table;
(a2) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 1 and have the same functions as the sequence 1;
the primer HBV-R is (a3) or (a 4):
(a3) a single-stranded DNA molecule shown in a sequence 2 of a sequence table;
(a4) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 2 and have the same functions as the sequence 2;
the probe HBV-P has a fluorescence reporter group at one end and a fluorescence quencher group at the other end, and the nucleotide sequence of the probe HBV-P is as follows (a5) or (a 6):
(a5) as shown in sequence 3 of the sequence table;
(a6) as shown by the substitution and/or deletion and/or addition of one or more nucleotides to the sequence 3.
2. A primer probe composition, which consists of a primer HBV-F, a primer HBV-R and a probe HBV-P;
the primer HBV-F is (a1) or (a 2):
(a1) a single-stranded DNA molecule shown in sequence 1 of the sequence table;
(a2) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 1 and have the same functions as the sequence 1;
the primer HBV-R is (a3) or (a 4):
(a3) a single-stranded DNA molecule shown in a sequence 2 of a sequence table;
(a4) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 2 and have the same functions as the sequence 2;
the probe HBV-P has a fluorescence reporter group at one end and a fluorescence quenching group at the other end, and the nucleotide sequence is as follows (a5) or (a 6):
(a5) as shown in sequence 3 of the sequence table;
(a6) as shown by the substitution and/or deletion and/or addition of one or more nucleotides to the sequence 3.
3. The primer probe composition of claim 2, which is used in the following (b1), (b2), (b3), (b4), (b5) or (b6) or (b7) or (b 8):
(b1) identifying the hepatitis B virus;
(b2) preparing a kit for identifying hepatitis B virus;
(b3) detecting whether the sample to be detected contains hepatitis B virus pgRNA;
(b4) preparing a kit for detecting whether a sample to be detected contains hepatitis B virus pgRNA or not;
(b5) detecting the load capacity of the hepatitis B virus pgRNA in a sample to be detected;
(b6) preparing a kit for detecting the pgRNA load of the hepatitis B virus in a sample to be detected;
(7b) predicting the recurrence rate of hepatitis B;
(8b) preparing a product for predicting the recurrence rate of hepatitis B.
4. A kit comprising the primer probe composition of claim 2; the function of the kit is as follows (c1), (c2), (c3) or (c 4):
(c1) identifying the hepatitis B virus;
(c2) detecting whether the sample to be detected contains hepatitis B virus pgRNA;
(c3) detecting the load capacity of the hepatitis B virus pgRNA in a sample to be detected;
(c4) predicting the recurrence rate of hepatitis B.
5. A method for detecting whether a sample to be detected contains hepatitis B virus pgRNA or not comprises the following steps: performing digital PCR by using cDNA of a sample to be detected as a template and the primer probe composition of claim 2; if the detection result is positive, the sample to be detected contains hepatitis B virus pgRNA; and if the detection result is negative, the sample to be detected does not contain hepatitis B virus pgRNA.
6. A method for detecting the load of hepatitis B virus pgRNA in a sample to be detected comprises the following steps: performing digital PCR by using cDNA of a sample to be detected as a template and the primer probe composition of claim 2; and obtaining the load capacity of the hepatitis B virus pgRNA in the sample to be detected according to the hepatitis B virus pgRNA positive fluorescent spot.
7. A premix for detecting hepatitis B virus pgRNA by digital PCR comprising the primer HBV-F, the primer HBV-R, the probe HBV-P and QuantStudio as set forth in the primer probe composition of claim 2TM3D Digital PCR Master Mix; in the premix, the concentration of the primer HBV-F is 0.53. mu.M, the concentration of the primer HBV-R is 0.53. mu.M, and the concentration of the probe HBV-P is 1.05. mu.M.
8. A kit for detecting hepatitis b virus pgRNA by digital RT-PCR comprising the premix of claim 7, a negative control and a positive control.
9. The kit of claim 8, wherein: the negative control is plasma negative for the human hepatitis B virus; the positive control is plasma containing hepatitis B virus pgRNA.
10. The premix according to claim 7 or the kit according to claim 8 or 9, which is (c1), (c2) or (c3) or (c 4):
(c1) identifying the hepatitis B virus;
(c2) detecting whether the sample to be detected contains hepatitis B virus pgRNA;
(c3) detecting the content of hepatitis B virus pgRNA in a sample to be detected;
(c4) predicting the recurrence rate of hepatitis B.
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