CN112725535A - Fluorescent quantitative PCR kit for simultaneously detecting full-length and truncated HBV pgRNA and application thereof - Google Patents
Fluorescent quantitative PCR kit for simultaneously detecting full-length and truncated HBV pgRNA and application thereof Download PDFInfo
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Abstract
The invention discloses a fluorescent quantitative PCR kit for simultaneously detecting full-length and truncated HBV pgRNA and application thereof, wherein the kit comprises a PCR reaction solution I, and the PCR reaction solution I comprises specific primers shown by SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6, and probes shown by SEQ ID NO 7, SEQ ID NO 8 and SEQ ID NO 9. The invention simultaneously detects the full length and the content of truncated HBV pgRNA in blood, and can provide more reference data for clinicians to evaluate Nucleotide (NAs) drugs in CHB antiviral treatment effect and predict recurrence risk after drug withdrawal. The invention designs primer probes for 8 genotypes (A-H) PreC/C and X conservative areas of HBV respectively, and adds degenerate primers in the system, so that the specificity is high, and the off-target detection probability can be effectively reduced. The internal standard and the PreC/C region share one pair of amplification primers, but the internal standard and the target sequence have respective probes to indicate the amplification condition of each PCR reaction tube, so that the occurrence of false negative or partial inhibition results is avoided.
Description
Technical Field
The invention belongs to the technical field of analytical biology, and relates to a hepatitis B postmedication treatment effect and recurrence risk evaluation method, in particular to a fluorescence quantitative PCR kit for simultaneously detecting full-length and truncated HBV pgRNA and application thereof.
Background
Hepatitis B Virus (HBV) infection is prevalent worldwide, but the prevalence of HBV infection varies widely in different regions. There are about 3.5 billion chronic HBV infected people worldwide reported by WHO, and the Africa and Western Pacific areas account for 68%. The prevalence of hepatitis b surface antigen (HBsAg) in the general population of the southeast asia and western pacific regions is 2% (3900 ten thousand cases) and 6.2% (1.15 hundred million cases), respectively. The local prevalence of Asian HBV varies, with most Asian regions being the moderate to high prevalence areas and a few being the low prevalence areas. It is estimated that the prevalence rate of HBsAg of general population in China is 6-8%, about 9000 more than ten thousand cases of chronic HBV infected persons, and about 2000-3000 ten thousand cases of Chronic Hepatitis B (CHB) patients.
HBV belongs to the hepadnavirus family, the genome DNA of the HBV is double-stranded relaxed circular DNA (rcDNA), the total length is about 3.2Kb, the HBV invades human liver cells, nucleocapsids are removed in cytoplasm, and the rcDNA enters the liver cell nucleus to release terminal protein connected with the 5 'end of a negative strand and RNA residual segment at the 5' end of a positive strand; host cell-dependent DNA polymerase and DNA ligase fill gaps on both strands, and further fold and twist covalently closed circular DNA (cccDNA) forming a supercoiled structure, and transcribe to mrnas of different sizes (3.5Kb, 2.4Kb, 2.1Kb, 0.8Kb) using cccDNA as a template, thereby translating various viral proteins. Wherein pregenomic RNA (pgRNA) of 3.5Kb in length contains all the genetic information on HBV DNA, full-length genomic negative strand DNA is synthesized using viral reverse transcriptase with pgRNA as a reverse transcription template, and positive strand DNA is synthesized by viral DNA polymerase with negative strand DNA as a template, and constitutes new HBV rcDNA together with the negative strand DNA. On one hand, newly synthesized HBV rcDNA enters into cell nucleus and is converted into new cccDNA to supplement the cccDNA library in the cell; on the other hand, the HBV is assembled with viral protein to form new complete HBV, and the new complete HBV is released to the outside of the cell to infect healthy liver cells.
The current antiviral drugs for treating hepatitis B mainly fall into two categories: one is a cytokine, represented by interferon; another class are nucleoside (acid) analogs such as adefovir, lamivudine, and entecavir. The interferon is a broad-spectrum antiviral drug, the mechanism is that the interferon is combined with a cell membrane specific receptor, a cell Jak-STAT signal transduction system is induced to activate a target gene, antiviral protein is encoded and synthesized to establish the antiviral state of the cell, and meanwhile, the interferon can also enhance the immune function of an organism. The long-term use of interferon can inhibit the replication of HBV DNA and the formation process of cccDNA, but cannot completely eliminate the cccDNA, and has more adverse reactions, such as the inhibition of hematopoietic cells and the induction of autoimmune diseases. Adefovir, lamivudine and entecavir are safe and effective drugs for treating chronic hepatitis B. The mechanism of action of the three drugs is inhibition of viral DNA polymerase, but inhibition of HBV is reversible, the possibility of relapse within a short period after drug withdrawal is very high, and cccDNA cannot be cleared, even though HBV DNA integrated with cellular genomic DNA is not cleared.
The level of cccDNA has great relevance to hepatitis B relapse, the quantification of cccDNA is an important index for clinically evaluating the treatment effect and predicting the drug withdrawal end point, the detection of cccDNA needs to puncture and sample the liver of a patient, and invasive inspection can cause certain damage to the patient. Serum HBV RNA, as a transcription product of cccDNA, is an ideal serological surrogate marker reflecting cccDNA activity. In recent years, researchers have further revealed that HBV RNA in serum is essentially pgRNA present in the nucleocapsid without complete or partially complete reverse transcription. Subsequent studies found that HBV RNA generated by the action of nucleotide-based drugs (NAs) is not all full-length pgRNA, but that several truncated pgRNA fragments exist, which are confirmed to be splice variants of pgRNA and 3' -terminal truncations of different lengths. As explained in the mechanism, the 3 '-end truncation is generated because the pgRNA, which performs the function of transcription template when the P protein is subjected to reverse transcription, is degraded from the 3' -end by the RNase H activity of the P protein. China 'Chronic hepatitis B prevention and treatment guidelines' (2019 edition) indicates that the amount of HBV RNA in blood is related to the cccDNA transcriptional activity in liver cells, and is worthy of intensive research in the aspects of evaluating the treatment effect of nucleotide (acid) drugs (NAs) and predicting the relapse risk after drug withdrawal. In 2019, in "chronic hepatitis b virus infection drug development guidance (draft) published by FDA in the united states, serum HBV RNA was used as one of the end-point indicators for evaluating the efficacy of a new drug.
Disclosure of Invention
The technical problem to be solved is as follows: in order to overcome the defects of the prior art, the full-length or truncated pgRNA can be detected simultaneously, the comprehensiveness of the detection range of the action products of the nucleotide (acid) drugs is realized, and accurate guidance is provided for clinical evaluation of treatment effect and prediction of drug withdrawal endpoints; in view of this, the invention provides a fluorescent quantitative PCR kit for simultaneously detecting full-length and truncated HBV pgRNA and application thereof.
The technical scheme is as follows: the fluorescent quantitative PCR kit for simultaneously detecting full-length HBV pgRNA and truncated HBV comprises PCR reaction liquid I, wherein the PCR reaction liquid I comprises specific primers shown as SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6, and probes shown as SEQ ID NO 7, SEQ ID NO 8 and SEQ ID NO 9; wherein, SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 7 are PreC/C region primers or probes, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6 and SEQ ID NO 8 are X region primers or probes, and SEQ ID NO 9 is an internal standard probe sharing a pair of amplification primers with the PreC/C region.
Preferably, the final concentration of the specific primers and probes in the amplification system is: 1 is 0.3. mu.M; 2 is 0.1. mu.M; 3 is 0.3. mu.M; SEQ ID NO 4 is 0.36. mu.M; SEQ ID NO 5 is 0.36. mu.M; 6 is 0.12. mu.M of SEQ ID NO; SEQ ID NO 7 is 0.15. mu.M; SEQ ID NO 8 is 0.2. mu.M; SEQ ID NO 9 was 0.1. mu.M. The primer and probe sequences are shown in table 1:
TABLE 1 primer Probe information
Preferably, the probe is labeled with a fluorescent group at the 5' end, the fluorescent group is one of FAM, VIC, CY5 and TAMRA, and the fluorescent label adopted by each group is different; the 3' end of the compound is marked with a quenching group, and the quenching group is MGB or BHQ.
Preferably, the PCR reaction solution I comprises 200mM Tris-HCl with pH 8.5, 0.5M KCl and 200mM MgCl20.5 XROX, dATP, dGTP, dUTP and dCTP.
Preferably, the kit comprises a PCR reaction solution II, and the PCR reaction solution II comprises A-Taq enzyme, UNG enzyme, MMLV reverse transcriptase and RNase inhibitor.
Preferably, the kit comprises a competitive internal standard, the internal standard and the PreC/C region share one pair of amplification primers, and the internal standard is pseudovirus.
Preferably, the kit comprises a positive serum control, a negative serum control and a quantitative calibrator, wherein the positive serum control is an HBV positive serum sample, the negative serum control is an HBV negative serum sample, and the quantitative calibrator is a constant-value HBV positive serum sample.
Preferably, the kit comprises a pgRNA extraction reagent, wherein the pgRNA extraction reagent comprises lysis solution, proteinase K, washing solution I, washing solution II, eluent and DNA digestion reaction solution.
Preferably, the lysis solution is guanidine hydrochloride, guanidine isothiocyanate and dithiothreitol, the washing solution I is composed of guanidine thiocyanate and absolute ethyl alcohol, the washing solution II is composed of sodium chloride and absolute ethyl alcohol, the eluent is RNase-free water, and the DNA digestion reaction solution is DNase I.
The application of any one of the above kits in evaluating the antiviral effect of nucleoside drugs on hepatitis B.
The application of any one of the kits in predicting hepatitis B recurrence risk after nucleoside drug withdrawal.
Has the advantages that: (1) the invention simultaneously detects the full length and the content of truncated HBV pgRNA in blood, and can provide more reference data for clinicians to evaluate Nucleotide (NAs) drugs in CHB antiviral treatment effect and predict recurrence risk after drug withdrawal. (2) According to the invention, primer probes are respectively designed for 8 genotypes (A-H) PreC/C and X conserved regions of HBV, and degenerate primers are added into the system, so that the specificity is high, and the detection off-target probability can be effectively reduced; (3) the invention introduces the artificially modified internal control DNA into a reaction system, the internal standard and the PreC/C region share one pair of amplification primers, but the internal standard and the target sequence have respective probes to indicate the amplification condition of each PCR reaction tube, thereby avoiding the occurrence of false negative or partial inhibition results; (4) the internal reference fluorescence ROX is added into the PCR detection system and used for correcting sample adding errors and tube-to-tube differences, and the quantitative result is more accurate.
Drawings
FIG. 1 shows the conserved sequences of PreC/C and X of HBV A-H genotypes analyzed by DNAMAN software, wherein A is the conserved sequence analyzed in the PreC/C region and B is the conserved sequence analyzed in the X region;
FIG. 2A is the melting curve of the primer designed for PreC/C region during amplification, and FIG. 2B is the melting curve of the primer designed for X region during amplification;
FIG. 3 shows the detection result of the sensitivity of the PreC/C region of the fluorescent quantitative PCR detection method according to the embodiment of the present invention, wherein A is the Ct value of the hepatitis B virus RNA detection reagent with different concentrations (lot No. 340008-;
FIG. 4 shows the result of detecting the sensitivity of the X region in the fluorescence quantitative PCR detection method according to the embodiment of the present invention, wherein A is the Ct value of the hepatitis B virus RNA detection reagent with different concentrations (lot No. 340008-.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and substance of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1
A fluorescence quantitative PCR detection kit for simultaneously and respectively detecting full-length and truncated hepatitis B virus pregenomic RNA (pgRNA) in blood comprises components shown in a table 2, wherein sequences of a primer and a probe are shown as SEQ ID NO 1-SEQ ID NO 9.
TABLE 2 kit composition information
Wherein dATP, dUTP, dGTP and dCTP in the PCR reaction solution I are 0.1-1mM and Mg2+50-200mM, 0.1-0.5. mu.M primer and 0.1-0.4. mu.M probe.
The gene sequence of the cloned plasmid in the internal standard is (SEQ ID NO: 10): TGCCTACAGACCACCAAATCTTTAATCCCTTAGAAGTTCCCCTCCTCAACACTTCCGTACTATGCTCTGGCGCTCGCCTCGCAGACGAAGG are provided.
The quantitative calibrator A, the quantitative calibrator B, the quantitative calibrator C and the quantitative calibrator D are respectively 3 multiplied by 104IU/mL、3×105IU/mL、3×106IU/mL、3×107IU/mL of positive serum samples.
The positive quality control product is 3 multiplied by 104 to 3 multiplied by 105IU/mL of positive serum samples.
Example 2
The kit of example 1 is used for detecting HBV pgRNA in blood, and the specific steps are as follows:
1. viral nucleic acid extraction
1) N centrifuge tubes of 1.5mL were counted (N ═ HBV serum number + positive quality control × 1+ negative quality control × 1+ quantitative calibrator × 4), 20 μ L of proteinase K, 200 μ L of sample, and 240 μ L of lysate + competitive internal control pseudovirus (lysate: internal standard pseudovirus 240:1, ready for use), shaking for 20-30s, and mixing thoroughly; performing instantaneous centrifugation, placing into a water bath kettle preheated to 65 deg.C, and standing for 15 min;
2) adding 100 μ l isopropanol and 20 μ l magnetic bead, shaking for 20-30s, mixing well, standing at room temperature for 5 min;
3) placing the centrifuge tube on a magnetic frame, standing for 3min to make the magnetic beads be completely adsorbed, and discarding the supernatant;
4) adding 200 mul of washing solution I into a 1.5mL centrifuge tube, and shaking for 5-10s to fully mix the solution;
5) placing the centrifuge tube on a magnetic frame, standing for 2-3min to make the magnetic beads be completely adsorbed, and discarding the supernatant;
6) adding 200 mul of washing solution II into a 1.5mL centrifuge tube, and shaking for 5-10s to fully mix the solution;
7) placing the centrifuge tube on a magnetic frame, standing for 2-3min to make the magnetic beads be completely adsorbed, and discarding the supernatant;
8) standing at room temperature to volatilize the residual liquid in the tube;
9) adding 50 μ l of eluent, shaking for 10-20s to completely wash the magnetic beads from the tube wall, mixing with the eluent, and standing at 65 deg.C for 5 min;
10) placing a 1.5mL centrifuge tube on a magnetic frame, standing for 2-3min to enable the magnetic beads to be completely adsorbed, and sucking the supernatant into a new clean centrifuge tube to obtain a mixture of HBV pgRNA and DNA.
2. Purification of HBV pgRNA: purification of the extracted HBV pgRNA Using DNase I
1) The following DNase I treatment system (20. mu.L) was set up
DNase I10 multiplied by reaction buffer 2 mul;
DNaseⅠ1μL；
eluent 17 μ L;
2) incubating at 37 ℃ for 20 min;
3) incubating for 10min at 75 ℃ to inactivate DNase I and obtain a pure product of HBV pgRNA;
3. preparation of PCR reaction system and sample adding
Taking the PCR reaction solution I and the PCR reaction solution II, and preparing a PCR reaction system according to the sample example according to the following system: PCR reaction solution I25. mu.L XN; PCR reaction solution II 5. mu.L XN; after fully mixing, 30 mu L of each tube is subpackaged into PCR tubes, 20 mu L of the pure HBV pgRNA after digestion is added into each tube, the final system is 50 mu L per tube, and the mixture is mixed and centrifuged instantly.
4. PCR amplification detection
The PCR reaction tube was placed in ABI 7500 or QuantStaudio DX fluorescence PCR instrument and the program setup was as shown in Table 3.
TABLE 3 qRT-PCR thermal cycling parameters
Wherein the fluorescent signal is collected in stage 3: the fluorescence signal collected is the probe-labeled fluorescence (FAM/VIC/TAMRA) at 60 ℃ for 30sec, and the Reference fluorescence (Passive Reference) is set at ROX.
5. Analysis of results
The software quantitated automatically by adjusting the Start and Stop values and thresholds (flattening the curve of the negative control or below the threshold line) of the baseline appropriately from the images automatically analyzed by the instrument.
6. Quality control
1) Negative control: the FAM channel and the VIC channel have no Ct value, and the Ct value of the TAMRA channel is less than or equal to 35;
2) positive control: the detected concentration value is 3 × 104-3×105IU/mL；
3) Four quantitative calibrators: all detected as positive, and the correlation R of the standard curve2≥0.99；
4) The above requirements need to be met in the same experiment, otherwise, the experiment is invalid;
example 3
The kit of example 1 was subjected to detection of sensitivity.
And (3) diluting the national standard substance of the HBV hepatitis B virus RNA detection reagent to 3E +7, 3E +6, 3E +5, 3E +4, 3E +3, 3E +2 and 3E +1IU/mL by using negative serum, and quantitatively detecting the doubly diluted standard substance by using the determined detection system and the circulating parameters. The result shows that the fluorescence quantitative detection method has higher sensitivity, the linear range of the PreC/C and the X region can be as low as 3.00E +01IU/mL, wherein the linear regression equation of the standard curve of the PreC/C region is as follows: y is-3.36 x +42.234, and the square of the correlation coefficient R2 is 0.997 (fig. 3A, B, C); the linear regression equation of the standard curve in the X region is as follows: y is-3.43 x +45.945 and the square of the correlation coefficient R2 is 0.999 (fig. 4A, B, C).
Example 4
The kit of example 1 was used to perform quantitative comparative detection of full-length and truncated HBV pgRNA in 4 positive blood samples treated with nucleotide-based drugs (NAs) for 3 months. Meanwhile, the HBV DNA level of blood is detected by a commercial HBV DNA quantitative detection kit (Yapei), and the result shows that 4 cases of HBV DNA detection of blood samples treated by nucleotide (acid) drugs (NAs) for 3 months are all positive, and the replication level of HBV cccDNA in liver tissues needs to be reflected by detecting the amount of HBV pgRNA in serum, so that more reference data are provided for a clinician to evaluate the antiviral treatment effect of the nucleotide (acid) drugs (NAs) on CHB.
The fluorescence quantitative PCR method of the invention is used for detecting 4 cases of HBV pgRNA in blood of patients with chronic hepatitis B treated by Nucleotide (NAs) medicines for 3 months. The results showed that the amount of HBV pgRNA in the PreC/C region was significantly higher than that in the X region, and that the amount of PreC/C region pgRNA in samples Nos. 1-4 was 7.04, 2.78, 7.04, 13.96 times that in the X region, respectively (Table 4). It is demonstrated that the reverse transcription and DNA synthesis process of the virus is significantly inhibited by the treatment of Nucleotide (NAs) drugs for 3 months, the synthesis of negative-strand HBV DNA is terminated early, the produced HBV RNA is not all full-length pgRNA, and some truncated pgRNA fragments exist. The primers designed from the X conserved region of the present invention can amplify the full-length pgRNA (FIG. 1B), the primers designed from the PreC/C conserved region can amplify both full-length pgRNA and pgRNA truncation (FIG. 1A), and some truncated pgRNA fragments are detected by the PreC/C region primer probe system.
TABLE 4 blood HBV pgRNA (region X and PreC/C) levels in patients with chronic hepatitis B for 3 months treated with nucleoside (acid) -based drugs (NAs)
In conclusion, the kit provided by the invention can be used for simultaneously detecting the content of full-length and truncated HBV pgRNA in blood, and can provide more reference data for clinicians to evaluate the treatment effect of nucleotide (acid) drugs (NAs) on CHB antiviral treatment and predict the recurrence risk after drug withdrawal. The competitive PCR method internal standard reference and the template to be detected are synchronously amplified by using the same reaction condition, the same reaction tube and the same pair of primers, so that the reaction system can be effectively controlled and the amplification efficiency can be reflected, the inhibition of a large amount of complex unknown components on the PCR reaction can be more effectively controlled, and the quantification can be more accurately carried out. The HBV pgRNA detection kit provided by the invention has the advantages of high sensitivity, strong specificity, simple operation, accurate detection and the like, and is suitable for quantitative detection of HBV pgRNA in clinic and scientific research.
Sequence listing
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Claims (10)
1. The fluorescent quantitative PCR kit for simultaneously detecting full-length HBV pgRNA and truncated HBV is characterized by comprising a PCR reaction solution I, wherein the PCR reaction solution I comprises specific primers shown as SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6 and probes shown as SEQ ID NO 7, SEQ ID NO 8 and SEQ ID NO 9; wherein, SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 7 are PreC/C region primers or probes, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6 and SEQ ID NO 8 are X region primers or probes, and SEQ ID NO 9 is an internal standard probe sharing a pair of amplification primers with the PreC/C region.
2. The fluorescence quantitative PCR kit for simultaneous detection of full-length and truncated HBV pgRNA according to claim 1, wherein the final concentration of the specific primers and probes in the amplification system is: 1 is 0.3. mu.M; 2 is 0.1. mu.M; 3 is 0.3. mu.M; SEQ ID NO 4 is 0.36. mu.M; SEQ ID NO 5 is 0.36. mu.M; 6 is 0.12. mu.M of SEQ ID NO; SEQ ID NO 7 is 0.15. mu.M; SEQ ID NO 8 is 0.2. mu.M; SEQ ID NO 9 was 0.1. mu.M.
3. The fluorescence quantitative PCR kit for simultaneous detection of full-length and truncated HBV pgRNA according to claim 1, wherein the PCR reaction solution I comprises 200mM Tris-HCl pH 8.5, 0.5M KCl, 200mM MgCl20.5 XROX, dATP, dGTP, dUTP and dCTP.
4. The fluorescence quantitative PCR kit for simultaneous detection of full-length and truncated HBV pgRNA according to claim 1, wherein the kit comprises PCR reaction solution II, and the PCR reaction solution II comprises A-Taq enzyme, UNG enzyme, MMLV reverse transcriptase and RNase inhibitor.
5. The fluorescence quantitative PCR kit for simultaneous detection of full-length and truncated HBV pgRNA according to claim 1, wherein the kit comprises a competitive internal standard, the internal standard and the PreC/C region share a pair of amplification primers, and the internal standard is pseudovirus.
6. The fluorescence quantitative PCR kit for simultaneous detection of full-length and truncated HBV pgRNA according to claim 1, wherein the kit comprises a positive serum control, a negative serum control and a quantitative calibrator, wherein the positive serum control is an HBV positive serum sample, the negative serum control is an HBV negative serum sample, and the quantitative calibrator is a constant HBV positive serum sample.
7. The fluorescence quantitative PCR kit for simultaneously detecting full-length and truncated HBV pgRNA according to claim 1, wherein the kit comprises a pgRNA extraction reagent, and the pgRNA extraction reagent comprises lysis solution, proteinase K, washing solution I, washing solution II, eluent and DNA digestion reaction solution.
8. The fluorescence quantitative PCR kit for simultaneous detection of full-length and truncated HBV pgRNA according to claim 7, wherein the lysis solution is guanidine hydrochloride, guanidine isothiocyanate and dithiothreitol, the washing solution I is composed of guanidine thiocyanate and absolute ethanol, the washing solution II is composed of sodium chloride and absolute ethanol, the eluent is RNase-free water, and the DNA digestion reaction solution is DNase I.
9. Use of the kit of any one of claims 1 to 8 for evaluating the antiviral effect of nucleoside drugs on hepatitis b.
10. Use of the kit of any one of claims 1 to 8 for predicting the risk of hepatitis b recurrence following nucleoside drug withdrawal.
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