NL2029623B1 - PROBE COMPOSITION FOR DETECTING HBV-cccDNA, APPLICATION AND DETECTION METHOD THEREOF - Google Patents
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Abstract
The present invention relates to a probe composition for detecting HBV-cchN A, which comprises a left, a middle, and a right probe; when all probes are ligated together in accordance with the left probe - the middle probes - the right probe in the 5'-3' direction, which is a polynucleotide with a complete hybridization sequence region, and the complete hybridization sequence region covers the sequence in HBV-cchN A negative strand genome region corresponding to the position DR] at the positive strand of HBV-rcDN A or HBV-cchNA positive strand genome region which corresponds to the position DR2 at the negative strand of HBV—rcDN A. The present utilizes sequential arrangement after specific hybridization, recognizable by ligase to be ligated into a chimera chain, which can be amplified using artificial sequence primers without being disturbed by false cchN A produced by HBV—rcDNA in a PCR reaction environment, therefore detecting the presence of cchN A and/or the load thereof in a sample.
Description
PROBE COMPOSITION FOR DETECTING HBV-cccDNA, APPLICATION AND
DETECTION METHOD THEREOF
[0001] The present disclosure relates to the technical field of medical diagnosis, specifically to a probe composition for detecting HBV-cccDNA, an application and a detection method thereof.
[0002] About 350-400 million people in the world are infected with hepatitis B virus, and the number is more than eight times that of AIDS-infected people. In China, there are about 93 million people suffered chronic hepatitis B. of which more than 20 million symptomatic patients with active hepatitis B need treatment. Hepatitis B virus is a DNA virus, presenting in the body as two forms, rcDNA (relaxed circular DNA) and cccDNA (covalently closed circular DNA). The sequences of the two are identical and both have a double-loop structure, while the difference is that there is a breakpoint in the middle of the negative strand (outer circle) of cDNA (the bottom left of FIG. 1), and the positive strand (inner circle) is incomplete and large fragments may be deleted (the dashed part in FIG. 1); and cccDNA has a complete closed loop structure for both positive and negative strands.
[0003] The study of Professor Nassal’s group from the University Hospital of Freiburg found that HBV infection of liver cells starts when the L protein on the surface of virons is recognized and bound by the sodium-taurocholate cotransporting polypeptide (NTCP) receptor on the surface of liver cells, and the virons may be taken into the cells by endocytosis. The nucleocapsid is released in the cytoplasm. Nucleocapsid-mediated nuclear transport and release of P-linked relaxed circular reDNA at the nuclear pore into the nucleus., where rcDNA is coverting into cccDNA eventually. cccDNA is the template for viral RNAs and subsequent generation of progeny virions, it is the key to the persistence of HBV infection. (PMID: 26048673, Michael Nassal, 2015)
[0004] However, all PCR-based cccDNA detection methods, such as over gap PCR with primers designed at both ends of the deletion fragment in the positive strand of rcDNA, or rolling circle amplification using circular DNA as a template, fail to absolutely rule out the interference of rcDNA. Due to the structural features of rc DNA, when it is in a PCR reaction environment, the positive and negative strands serve as the primers of each other, which can naturally restore all nucleic acid sequences, just like the cccDNA. Therefore, when using the traditional PCR detection technique of a pair of primers plus PCR-probe. the interference of rcDNA cannot be eliminated thothoughly, resulting in false positives or inaccurate quantification. CN104388598A describes to a digital PCR quantitative detecting kit for HBV (hepatitis B virus) cccDNA and an application of the digital PCR quantitative detecting kit for
HBV cceDNA. US2020354800A1 describes that a detection of nucleic acids can be achieved using a chemical ligation-based template assisted rapid assay (TARA-L) without the need for enzymes.
[0005] In addition, JBS SCIENCE INC. (Pennsylvania, U.S.) disclosed a cccDNA detection method that can avoid rcDNA interference (PCT/US2017/017999 2017.08.14). By using sodium bisulfite to treat a nucleic acid sample with mixed cccDNA and rcDNA, this method eliminates the complementary characteristics between the positive and negative strands by transversing the nucleotide cytosine into uracil, and prevents rcDNA from forming interfering cccDNA gene sequences in the PCR environment. Then, PCR detection of the new strand produced by cccDNA can achieve a certain specificity. However, using sodium bisulfite to treat DNA samples causes the transversion (covalent bond broken) of the cytosine into uracil.
This process is theoretically inefficient and has poor repeatability. Therefore, this method may theoretically have the problems of poor repeatability of cccDNA quantitative results and low sensitivity of qualitative analysis.
[0006] In view of this, the purpose of the present disclosure is to provide a probe composition for detecting HB V-cccDNA, so that the probe composition can specifically detect HBVccc-DNA, effectively distinguishing between rcDNA and cccDNA of HBV, and has good repeatability.
[0007] Another purpose of the present disclosure is to provide use of the above-mentioned probe composition and to provide a method for detecting HBV-cccDNA based on the above- mentioned probe composition.
[0008] In order to achieve the above purposes, the present disclosure provides the following typical technical solutions.
[0009] A probe composition for detecting HBV-cccDNA, comprising a left probe, a middle probe, and a right probe; wherein when all probes are ligated together in accordance with the left probe — the middle probes — the right probe in the 5'—>3' direction, called complete hybridization sequence region, and the complete hybridization sequence region covers the sequence in HBV-cccDNA negative strand genome region which corresponds to the position
DRI at the positive strand of HBV-rcDNA or HBV-cccDNA positive strand genome region which corresponds to the position DR2 at the negative strand of HBV-rcDNA;
[0010] Wherein, the number of the middle probe is > 0, which is composed of hybridization sequence region; the left probe comprises an artificial sequence region located at the 5' end and a hybridization sequence region located at the 3' end; the right probe comprises an artificial sequence located at the 3' end and a hybridization sequence region located at the 5' end; the artificial sequence region is a sequence that is not homologous to the genome sequence of HBV DNA and its subtypes, and is not homologous to the genome of other species that may exist in sample, and the artificial sequence serves as a primer binding region.
[0011] All the probes in the probe composition of the present disclosure are designed to specifically and stably hybridize with a partial region of the negative or positive strand of cccDNA in the sample, and can be chemically ligated to form a chimera chain containing all probes.
[0012] The hybridized region in the negative or positive strand of cccDNA corresponds to the position DR1 or position DR2 of rcDNA respectly. Due to the existence of the breakpoint at the position of DR1 corresponding to rcDNA negative strand (such as positions 1816-1817 bp of NC_003977.1) or the sequence deletion at the position of DR2 corresponding to rc DNA positive strand, not all probes can be stably hybridized and therefor form a chimera chain containing all probes (the aforementioned complete hybridization sequence) under the ligation reaction environment with rcDNA. And then, nucleic acid amplification techniques are employed to detect the obtained nucleic acid product.
[0013] Asa DNA ligase, T4 DNA ligase can rely on ATP to catalyze the binding of 5'-P end and the 3'-OH end of the sticky-end or blunt-end double-stranded DNA or RNA through a phosphodiester bond. In addition, T4 DNA ligase can repair single-stranded gaps in double- stranded DNA, double-stranded RNA, or DNA/RNA hybrids. Utilizing this characteristic of
T4 DNase, when synthesizing a probe, it is necessary to phosphorylate the 5'end of all the probes except for the left probe. After the aforementioned hybridization reaction is stabilized,
T4 DNA ligase is added, taking advantage of its nature to repair single-stranded gaps in double-stranded nucleic acid, to ligate all the probes that are arranged closely in sequence on the cccDNA strand into a chimera chain (the aforementioned complete hybridization sequence). On the other hand, because the probes hybridized with rcDNA containing breakpoint or deletion cannot be closely adjacent, they are unable to be ligated and form the corresponding particular chimera chain (the aforementioned complete hybridization sequence).
[0014] Then denaturation is performed to release the chimera chain formed by hybridization and ligation, and the corresponding PCR primers are designed based on the primer binding region of the upstream and downstream primers of the left and right probes.
These primers are artificially designed sequences and have no homology with the HBV DNA genome. Therefore, when a primer-dependent amplification reaction is applied, no matter how many false cccDNA are synthesized by rcDNA, it will not affect the specific amplification and qualitative and quantitative analysis of the chimera chain by the reaction system, and it is equivalent to the qualitative and quantitative analysis of the true HBV cccDNA in the sample.
The schematic diagram is shown in FIG. 2.
[0015] Preferably, the left probe, the middle probe and the right probe are designed based on the NC_003977.1 standard sequence and other HBV subtype genome sequences.
[0016] Preferably, the middle probe and the right probe are phosphorylated at the 5' end to facilitate the recognition by ligase under suitable conditions to form phosphodiester bonds to tigate multiple probes into one chimera chain.
[0017] In some embodiments of the present disclosure, the number of the middle probe is 0.
In some embodiments of the present disclosure, the number of the middle probe is 1.
Preferably, the length of each middle probe is 10-70 bp; the length of the hybridization region sequence of the left probe and the right probe is 10-70 bp, and the length of the artificial sequence of the left probe and the right probe is 10-40 bp.
[0018] In addition, the probe composition of the present disclosure further comprises a fluorescence-PCR probe (Amplified-probe), and upstream and downstream primers for PCR- based qualitative and quantitative analysis or isothermal amplification-based qualitative and quantitative analysis.
[0019] The Amplified-probe is a complementary probe that spans the middle probe and the hybridization region of the right probe, or the Amplified-probe is a complementary probe that spans the middle probe and the hybridization region of the left probe. The sequence of the upstream primer is identical to the artificial sequence of the left probe, and the sequence of the downstream primer is complementary to the artificial sequence of the right probe. The purpose of the Amplified-probes and the upstream and downstream primers involved in the above qualitative and quantitative analysis techniques is to amplify the complete hybridization 5 sequence ligated by the probe composition of the present disclosure. The Amplified-probe sequence spans the middle probe and the hybridization region of the right/left probe, so that the annealing temperature of the Amplified-probe hybridization with the left or right probe at this step is significantly reduced, thereby avoiding non-specific detection signals generated.
[0020] Preferably, the PCR-based qualitative and quantitative analysis technique is selected from PCR fragment analysis. q-PCR. PCR-SSP, PCR-seq and digital PCR. The isothermal amplification-based qualitative and quantitative analysis technique is selected from loop- mediated isothermal amplification, nicking enzyme amplification reaction technique, rolling circle amplification technique, helicase-dependent DNA isothermal amplification technique, recombinase polymerase amplification and enzymatic recombination isothermal amplification technique.
[0021] In some embodiments of the present disclosure, the present disclosure further provides the TaqMan probes and upstream and downstream primers for q-PCR.
[0022] The present disclosure provides use of the aforementioned probe composition in the manufacture of a kit for detecting HBV-cccDNA or in the detection of HBV-cccDNA.
[0023] The present disclosure also provides a method for quantitatively detecting HBV- cccDNA, comprising hybridizing a sample to be tested with the probe composition in the presence of DNA ligase, and performing a PCR-based qualitative and quantitative analysis or an isothermal amplification-based qualitative and quantitative analysis of the hybridized sample using the upstream and downstream primers complementing to the artificial sequence of the probe composition, and obtaining a result of quantitative detection for HBV-cccDNA.
[0024] Preferably, the sample to be tested is derived from human tissue and/or body fluid. and is obtained by digestion, enrichment, extraction, and purification steps to be a sample with nucleic acid as the main component to be analyzed.
[0025] Preferably, it further comprises denaturing the double-stranded DNA in the sample to be tested into single-stranded DNA before hybridization, so as to facilitate the hybridization of the probe composition with the target nucleic acid.
[0026] Preferably, it further comprises denaturing the DNA to fully release the successfully-ligated, complete hybridization sequence to facilitate the qualitative and quantitative analysis after the probe composition is hybridized and ligated and before the qualitative and quantitative analysis.
[0027] Wherein, the DNA denaturation method is selected from high temperature (temperature in the system >85°C) denaturation, acid/base (when the pH in the system is <3 or > 10) denaturation, and chemical reagent (urea, formamide, formaldehyde) denaturation.
[0028] Compared with the prior art, the benefits of the present disclosure are: 1) By performing hybridization and ligation before the final amplification, the interference of 16 cDNA on the detection of cccDNA is completely avoided; 2) Compared with the aforementioned method of treating samples with sodium bisulfite to break DNA covalent bond to eliminate the double-stranded hybridization characteristics, the method of the present disclosure utilizes hydrogen bond hybridization and uses DNA ligase to generate phosphodiester bond by relying on ATP to synthesize an equivalent detection substance, for the detection of cccDNA, the efficiency and repeatability will be better.
[0029] It can be seen from the above technical solutions that the present disclosure utilizes the sequential arrangement in physical space of two or more hybridization probes and cccDNA after specific hybridization, which can be recognized by ligase to be linked into a chimera chain containing complete hybridization sequence, which can be specifically amplified using artificial sequence primers without being disturbed by the false cccDNA that will be inevitably produced by rcDNA in the PCR reaction environment, in order to specifically detect the presence of cccDNA and/or the load thereof in a sample.
[0030] FIG. 1 shows a schematic diagram of the structural differences between cccDNA and rcDNA.
[0031] FIG. 2 is a schematic diagram showing the principle of detecting cccDNA but not rcDNA using the probe composition of the present disclosure. After denaturation treatment on the sample, the physical distance between the breakpoints of DR1 and DR2 in crDNA becomes greater.
[0032] FIG. 3 is a schematic diagram illustrating the hybridization region and positional relationship of the probe composition of the present disclosure using the positive strand of cccDNA as a template and the sequence in Table 1 as an example.
19833] The embodiment of the present disclosure discloses a probe composition tor detecting HBV-cccDNA, an application and a detection method thereof. Those skilled in the art can learn from the contents of the present disclosure and appropriately improve the process parameters. It should be particularly pointed out that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present disclosure. The probe composition for detecting HBV-cccDNA, application and detection method thereof of the present disclosure has been described by the preferred examples, and © is apparent that related persons can make modifications or proper changes and combination 16 the probe composition for detecting HBV-cccDNA, application and detection method thereof described herein 50 as to achieve and apply the technology of the present disclosure, without departing from the content, spit and scope of the present disclosure. {B034] According to the technical solotion of the present invention, specific probe examples are provided in Table 1.
Tahie 1
TTT 5.3
MP | OP-TCACCAGCACCATGCAACTTTTTCAC; SEQ ID NO: 1 (All are hybridization region sequences)
LP | CACGGCTTCGCTAAGGGTTGGA-
CTAGGAGGCTGTAGGCATAAATTGGTCTGT: SEQ ID NO: 2 (1-22 bp are artificial sequences, the rest are hybridization region sequences)
RP | OP-CTCTGCCTAATCATCTCATGTTCATGTCCTAC-
TCTAGATTGCATCTTGCTCGCAC; SEQ ID NO:3 (33-55 bp are artificial sequences, the rest are hybridization region sequences)
Note: The bolded and underlined part of the sequence is artificial sequence, and the rest are hybridized region sequences.
[0035] In order to describe the hybridization region targeted by the probe of the present disclosure more vividly using the sequences in Table 1 as an example, the present disclosure uses the positive strand of cccDNA as a schematic illustration (the probe was designed based on the negative strand, so the probe sequence is the same as the positive strand). The details are shown in FIG. 3 that the underlined part of the sequence is the MP sequence (middle sequence) in Table 1, and the position of the ellipsis corresponds to the position of the breakpoint of the negative strand of rcDNA, but this position is continuous in the negative strand of cccDNA. The left and right sides are hybridization region sequences of RP and LP probes (right probe and left probe). and the boxed sequence indicates spanning MP and RP 16 hybridization region, which is used to understand the design ideas of subsequent TagMan probes.
[0036] Besides, the present disclosure also provides a TagMan probe and upstream and downstream primers for subsequent TaqMan fluorescence quantification, which were designed according to the design principles of the present disclosure based on the sequence in
Table 1 as an example, see Table 2 below:
Table 2
TagMan MGB probe FAM-ATGATTAGGCAGAGGTGAAAAAG-TARMA+MGB (The underlined part of the sequence is the complementary sequence of the hybridization region sequence of the right probe, and the remaining sequence is the complementary sequence of the middle probe. The probe spans the MP and RP hybridization region, and the overall sequence is shown in SEQ ID NO: 4)
Forward Primer CACGGCTTCGCTAAGGGTTGGA (Identical to the artificial ane a aes
Reverse Primer GTGCGAGCAAGATGCAATCTAGA (Complementary to the artificial sequence of the right hybridization probe, as shown in SEQ
ID NO: 6)
[0037] The basic principle of the present disclosure is to utilize the sequential arrangement in physical space of two or more hybridization probes (The design of the aforementioned three hybridization probes is a relatively feasible scheme, which can facilitate the TaqMan fluorescence quantitative analysis in the later stage, and it is by no means the only hybridization probe design scheme) and cccDNA after specific hybridization, which can recognized by ligase to ligate into a chimera chain containing complete hybridization sequence, which can be specifically amplified using artificial sequence primers without being disturbed by the false cccDNA that will be inevitably produced by rcDNA in the PCR reaction enviroment (PCR techniques used to amplify and detect the chimera chain obviously include but are not limited to TaqMan quantitative technique).
[0038] The above description is merely for understanding the method of the present disclosure and the core concept thereof. It should be noted that, those ordinary skilled in the art can make various improvements and modifications to the present disclosure without departing from the principle of the present disclosure, and these improvements and modifications also fall into the protection scope of the claims of the present disclosure.
SEQLTXT-1
<118> Wang Jun <120> PROBE COMPOSITION FOR DETECTING HBV-CCCDNA, APPLICATION AND
DETECTION METHOD THEREOF
<130> MO211285 <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Probe <400> 1 tcaccagcac catgcaactt tttcac 26 <210> 2 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 cacggcttcg ctaagggttg gactaggagg ctgtaggcat aaattggtct gt 52 <210> 3 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 ctctgcctaa tcatctcatg ttcatgtcct actctagatt gcatcttgct cgcac 55 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220>
Pagina 1
SEQLTXT-1 <223> Probe <400> 4 atgattaggc agaggtgaaa aag 23 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 cacggcttcg ctaagggttg ga 22 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 gtgcgagcaa gatgcaatct aga 23
Pagina 2
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| NL2029623A (en) | 2023-06-06 |
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