Long fragment amplification analysis method and amplification kit for HBV RNA spliceosome
Through HBV-specific primers and optimized PCR amplification conditions, combined with the library sequencing analysis process, the problem of low levels of HBV RNA and splices in HBeAg-negative patients was solved, efficient amplification and accurate analysis of HBV RNA splices were achieved, and the characteristics of HBV RNA splices were accurately identified in HBV infected patients.
Patent Information
- Application Number
- CN202510166535.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-14
- Publication Date
- 2025-05-16
AI Technical Summary
The levels of HBV RNA and splices are low in HBeAg-negative patients. It is difficult to obtain high-quality sequencing fragments directly by direct sequencing of single-molecule long fragments of conventional RNA. In addition, there are chimeric sequences from integrated sources and HBx-encoded RNA in the serum, which affects the amplification and analysis of HBV RNA splices.
Reverse transcription PCR was performed using HBV-specific primers, and amplification primers that could recognize the full-length region of the HBV genome were selected for PCR amplification. Combined with the optimized library sequencing analysis process, chimeric sequences from the integration source and HBx-encoded RNA were removed to achieve long fragment amplification and accurate analysis of HBV RNA splices.
Efficient amplification and accurate analysis of HBV RNA splices in the serum of HBeAg-negative patients were achieved, which can accurately identify the characteristics of HBV RNA splices, including types and proportions of HBV infected patients.
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Figure CN120006033A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical technology, and in particular to a long-segment amplification and analysis method for HBV RNA spliceosomes and an amplification kit. Background Art
[0003] The levels of HBV RNA and spliceosomes are low in HBeAg-negative patients infected with hepatitis B virus. Secondly, mutations in the pre-C region and core promoter region are common in HBeAg-negative patients. These mutations may lead to changes in the transcription level of HBV RNA. It is difficult to obtain higher sequencing fragments for analysis using conventional RNA single-molecule long fragment direct sequencing. Secondly, HBV RNA in serum also contains host-virus RNA with chimeric sequences from integration sources and HBx-encoded RNA. The conventional PolyT-tailed amplification and library construction will inevitably contain these two RNA sequences, thereby affecting the amplification and analysis of HBV RNA spliceosomes. Summary of the invention
[0004] The purpose of the present invention is to provide a long-fragment amplification analysis method and an amplification kit for HBV RNA spliceosomes, so as to solve the problem in the prior art that it is difficult to obtain a higher sequencing fragment by conventional single-molecule long-fragment sequencing for HBeAg-negative patients.
[0005] To achieve the above object, the present invention provides a method for amplifying and analyzing long fragments of HBV RNA spliceosomes, comprising the following steps:
[0006] S1: Extract viral nucleic acid and remove DNA from viral nucleic acid;
[0007] S2: cDNA was obtained by reverse transcription using HBV-specific primers;
[0008] S3: Select amplification primers that can recognize the full-length region of the HBV genome in the conserved region of the HBV genome for further PCR amplification;
[0009] S4: The obtained PCR amplification products are subjected to library construction, sequencing and analysis to obtain the types and proportions of HBV RNA spliceosomes.
[0010] Furthermore, the HBV-specific primer is HBV RT1799, and its sequence is shown in SEQ ID NO.1.
[0011] Furthermore, the amplification primers include a first amplification primer and a second amplification primer, wherein the first amplification primer is HBV 1827FP, whose sequence is shown in SEQ ID NO.2, and the second amplification primer is HBV 1602RP, whose sequence is shown in SEQ ID NO.3.
[0012] See also Figure 2 Furthermore, the library construction and sequencing analysis process described in step S4 specifically includes the following steps:
[0013] S11: Use the kit to construct a library with the PCR amplification product, and ensure that the total amount of the library is greater than the value calculated by Smrtlink by testing the concentration and size;
[0014] S12: Combine the library before loading onto the machine, so that the library is combined with sequencing primers and polymerase;
[0015] S13: purify the final reaction product and sequence it;
[0016] S14: Use minimap2 to align the circular consensus sequence reads of each sample to the corresponding pregenomic RNA reference sequence;
[0017] S15: Use samtools to build a BAM file index, and generate a BED format file based on the BAM file using the bam2Bed12 program of flair;
[0018] S16: Use the correct module of flair to correct the misaligned or ambiguously located splice sites in the long read data;
[0019] S17: Use the script to perform secondary correction on the remaining abnormal sites, and use the correct module of flair to define high-confidence splicing variants from the corrected reads;
[0020] S18: Convert BED format output files into genotype-specific GTF annotation files using bedToGenePred and genePredToGtf tools;
[0021] S19: Classify and count the known HBV RNA spliceosomes based on the annotation files and quantitative file results to obtain the types and proportions of spliceosomes.
[0022] Furthermore, the kit is SMRTbell Express Template Prep Kit 2.0.
[0023] Furthermore, the identification of splice sites was completed by 2passtools, and the parameters were set to filter sites with jump alignment depth less than 4 and support counts less than 10.
[0024] Furthermore, the purification is AMpure PB Beads purification.
[0025] The present invention also discloses an amplification kit, which is applied to the long-fragment amplification analysis method as described above, and includes a first-chain cDNA synthesis reagent, a DNA digestion reaction solution, an HBV-specific reverse transcription primer, a buffer, an inhibitor, a dNTP mixture, distilled water and a reverse transcriptase, wherein the buffer has a 5-fold concentration, the inhibitor is RiboLock RNase Inhibitor, the dosage is 20U / μL, the dosage of the dNTP mixture is 10mM, and the reverse transcriptase is RevertAidM-MuLVRT, the dosage is 200U / μL.
[0026] The present invention discloses a long-segment amplification and analysis method for HBV RNA spliceosomes. Aiming at the problem of low HBV RNA and spliceosome levels in HBeAg-negative patients infected with hepatitis B virus, and removing chimeric host-virus RNA and HBx-encoded RNA from integration sources, the PCR amplification conditions and primer design are optimized, and the established optimized library construction sequencing analysis process is adopted to realize the identification of the characteristics of HBV RNA spliceosomes in the serum of HBV infected persons, such as the types and proportions, to realize accurate analysis, and accordingly an amplification kit is proposed, and specific primers are designed to improve the amplification efficiency of HBV RNA spliceosomes in the serum of HBeAg-negative chronic hepatitis B patients. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0028] Figure 1 This is a HBV RNA amplification diagram of a long-segment amplification analysis method for HBV RNA spliceosomes of the present invention.
[0029] Figure 2 It is a schematic diagram of the library construction, sequencing and analysis process of a long-fragment amplification and analysis method of HBV RNA spliceosomes of the present invention.
[0030] Figure 3 It is the types and relative proportions of different HBV RNA spliceosomes in the serum of chronic hepatitis B patients obtained after long-fragment amplification analysis in a specific embodiment of a long-fragment amplification analysis method of HBV RNA spliceosomes of the present invention. DETAILED DESCRIPTION
[0031] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.
[0032] See also Figure 1 The present invention provides a long-segment amplification analysis method and an amplification kit for HBV RNA spliceosomes:
[0033] The specific embodiments are as follows:
[0034] Serum samples from 4 HBeAg-positive and 4 HBeAg-negative chronic hepatitis B patients were collected to extract viral nucleic acid, and RNA in HBV-infected HepG2-NTCP cells was used as a control. Figure 1 The experimental process shown is as follows: first, HBV-specific primers were used for reverse transcription PCR (RT-PCR) reaction to enrich HBV RNA spliceosomes, and then primers designed for the full-length HBV genome were used for PCR amplification to enrich HBV RNA and spliceosomes in serum. The amplified products were then used to construct libraries using SMRTbell Express Template Prep Kit 2.0 (PacBio, USA), and the concentration (Qubit) and size (Agilent2100) were tested to ensure that the total amount of the library was greater than the Smrtlink calculated value. The PacbioBinding kit was used to bind the library to be loaded onto the machine, so that the library was bound to the sequencing primers and Polymerase. The final reaction product was purified by AMpure PB Beads and then placed on the SequelⅡ sequencer for sequencing. The obtained sequencing data were statistically analyzed: minimap2 was used to align the CCS reads (circular consensus sequence read length) of each sample to the corresponding pregenomic RNA (pgRNA) reference sequence.
[0035] The BAM file index was constructed using samtools, and the BED format file was generated based on the BAM file using the bam2Bed12 program of flair (v2.0.0). The identification of splice sites was completed using 2passtools, and the parameters were set to filter sites with a jump alignment depth (JAD) less than 4 and support counts less than 10. The correct module of flair was further used to correct the splice sites with incorrect alignment or ambiguity in the long read data. After secondary correction of the remaining abnormal sites using a custom script, the correct module of flair was used to define high-confidence splice variants from the corrected reads to ensure data accuracy. Finally, the BED format output file was converted into a genotype-specific GTF annotation file using the bedToGenePred and genePredToGtf tools.
[0036] Finally, the known HBV RNA splice variants were classified and counted according to the annotation files and quantitative file results to obtain the types and proportions of splice variants.
[0037] See also Figure 3 There are multiple HBV RNA spliceosomes in the serum of chronic HBV infected patients, and the reported proportion of HBV RNA spliceosomes is (10.1%-37.2%), including SP1, SP2, SP3, SP4, SP5, SP6 and other reported RNA spliceosomes. Among them, SP1 can be detected in every sample and is the spliceosome with the highest proportion (35%-62%). In addition, we also identified 41 new spliceosomes, and the proportion of new HBV RNA spliceosomes was (0%-2.3%), of which 6 new spliceosomes were repeatedly detected in ≥2 independent samples.
[0038] Serum samples
[0039] All serum samples were obtained from the First Affiliated Hospital of Chongqing Medical University and stored at -80°C.
[0040] Reagents
[0041] Viral RNA Mini Kit was purchased from QIAGEN (Cat. No. 52904); DNase I was purchased from Thermoscienfitic (Cat. No. EN0529); High-sensitivity One-Step RT-qPCR premix was purchased from Apbai, and sequencing primers and polymerase were Sequel II Sequencing 2.0 Bundle purchased from Pac-Bio.
[0042] The specific example of HBV RNA long fragment amplification is as follows:
[0043] Use viral nucleic acid extraction kit Viral RNA Mini Kit (QIAGEN, Germany) was used to extract and purify viral nucleic acid in serum;
[0044] Incubate with DNase I (Thermo Fisher Scientific, USA) at 37°C for 30 minutes to remove HBV DNA from viral nucleic acid; add EDTA (50 mM) and incubate at 60°C for 10 minutes to terminate the digestion;
[0045] RevertAid first-strand cDNA synthesis reagent was used for reverse transcription PCR reaction, the reaction system included DNA digestion reaction solution, HBV-specific reverse transcription primer HBV RT1799 (10 μΜ), 5×Reaction Buffer, RiboLock RNaseInhibitor (20 U / μL), 10 mM dNTP Mix, RevertAidM-MuLVRT (200 U / μL), and the reaction program was 42°C, 60 min→70°C, 5 min; LATaq was used to amplify the cDNA obtained by reverse transcription by PCR, and the reaction was carried out on a PCR instrument, and the program was as follows:
[0046]
[0047] HBV RT1799, the sequence of which is: 5'-GCATGGTGCTGGTGAACAGAC-3'
[0048] HBV 1827FP: Its sequence is 5'-TCACCTCTGCCTARTCATCTCAT-3'
[0049] HBV 1602RP: Its sequence is 5'-GTGCAGAGGTGAAGCGAAGT-3'.
[0050] Library construction and sequencing:
[0051] After the PCR product was purified by 0.6×AMPure PB Beads, the library was constructed using SMRTbell Express Template Prep Kit 2.0 (PacBio). After the SMRTbell library was purified by AMPure PB beads, the size of the library fragment was detected using an Agilent 2100 Bioanalyzer (Agilent technologies, USA). Finally, the Sequel II Sequencing Kit 2.0 (Pacbio, USA) and Sequel II Binding Kit 3.2 (Pacbio, USA) were used for sequencing on a PacBio Sequel II instrument. The above sequencing was completed by Xi'an Haorui Gene Technology Co., Ltd.
[0052] What is disclosed above is only a preferred embodiment of the present invention, and it certainly cannot be used to limit the scope of rights of the present invention. Ordinary technicians in this field can understand that all or part of the processes of the above embodiment and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.
Claims
1. A method for amplifying and analyzing long fragments of HBV RNA spliceosomes, characterized in that: The steps include: S1: Extract viral nucleic acid and remove DNA from viral nucleic acid; S2: cDNA was obtained by reverse transcription using HBV-specific primers; S3: Select amplification primers that can recognize the full-length region of the HBV genome in the conserved region of the HBV genome for further PCR amplification; S4: The obtained PCR amplification products are subjected to library construction, sequencing and analysis to obtain the types and proportions of HBV RNA spliceosomes.
2. The long fragment amplification analysis method according to claim 1, characterized in that: The HBV specific primer is HBV RT1799, and its sequence is shown in SEQ ID NO.
1.
3. The long fragment amplification analysis method according to claim 1, characterized in that: The amplification primers include a first amplification primer and a second amplification primer, wherein the first amplification primer is HBV1827FP, whose sequence is shown in SEQ ID NO.2, and the second amplification primer is HBV 1602RP, whose sequence is shown in SEQ ID NO.
3.
4. The long fragment amplification analysis method according to claim 1, characterized in that: The library construction, sequencing and analysis process described in step S4 specifically includes the following steps: S11: Use the kit to construct a library with the PCR amplification product, and ensure that the total amount of the library is greater than the value calculated by Smrtlink by testing the concentration and size; S12: Combine the library before loading onto the machine, so that the library is combined with sequencing primers and polymerase; S13: purify the final reaction product and sequence it; S14: Use minimap2 to align the circular consensus sequence reads of each sample to the corresponding pregenomic RNA reference sequence; S15: Use samtools to build a BAM file index, and generate a BED format file based on the BAM file using the bam2Bed12 program of flair; S16: Use the correct module of flair to correct the misaligned or ambiguously located splice sites in the long read data; S17: Use the script to perform secondary correction on the remaining abnormal sites, and use the correct module of flair to define high-confidence splicing variants from the corrected reads; S18: Convert BED format output files into genotype-specific GTF annotation files using bedToGenePred and genePredToGtf tools; S19: Classify and count the known HBV RNA spliceosomes based on the annotation files and quantitative file results to obtain the types and proportions of spliceosomes.
5. The long fragment amplification analysis method according to claim 4, characterized in that: The kit is SMRTbell Express Template Prep Kit 2.
0.
6. The long fragment amplification analysis method according to claim 4, characterized in that: The identification of splice sites was done using 2passtools, with parameters set to filter sites with jump alignment depth less than 4 and support counts less than 10.
7. The long fragment amplification analysis method according to claim 4, characterized in that: The purification was carried out using AMpure PB Beads.
8. An amplification kit, used in the long fragment amplification analysis method according to claims 1 to 7, characterized in that: The invention comprises a first-strand cDNA synthesis reagent, a DNA digestion reaction solution, a HBV-specific reverse transcription primer, a buffer, an inhibitor, a dNTP mixture, a reverse transcriptase, distilled water and a heat-stable DNA converting enzyme.