JP2022076831A - Method for detecting hbv-derived cccdna by digital pcr, and reagent for detecting hbv-derived cccdna - Google Patents

Abstract

To provide a method for accurately detecting HBV-derived cccDNA by digital PCR.SOLUTION: The inventive method for detecting HBV-derived cccDNA by digital PCR includes the steps of: the step for allocating a DNA-containing sample into micro compartments, and a detection step for detecting an amplification of HBV-derived cccDNA using a primer, and a hybridization of a resultant amplified product and a probe for HBV-derived cccDNA, for the DNA-containing sample allocated into the micro compartments. The allocated biological sample is a sample untreated with DNA decomposition enzyme.SELECTED DRAWING: None

Description

The present invention relates to a method for detecting HBV-derived ccDNA by digital PCR and a reagent for detecting HBV-derived ccDNA.

It is known that hepatitis B virus (HBV) causes liver cirrhosis and hepatocellular carcinoma when its infection causes the development of acute hepatitis or chronic hepatitis, and if it is prolonged.

HBV usually exists in the state of mature circular DNA (rcc). When HBV infects a cell and invades the nucleus from the cytoplasm, it changes to covalently closed circular DNA (ccDNA), which is a replication intermediate, by an enzyme possessed by the host cell or the like. .. This is transcribed into RNA, and the transcribed RNA is released into the cytoplasm, transcribed again into DNA (relaxed circular DNA: rcDNA) by the reverse transcriptase possessed by the virus, and released extracellularly as mature HBV. .. Current therapeutic agents for HBV infection are generally inhibitors against the reverse transcriptase, which inhibits re-transcription from RNA to DNA (transcription to rcDNA), thereby causing the replicated HBV to be extracellular. It suppresses the release to. However, since the ccDNA present in the nucleus cannot be removed with this therapeutic agent, when administration is interrupted, transcription occurs from ccDNA to RNA, and then reverse transcription from RNA to rcDNA is also resumed. Therefore, it is desired to develop a therapeutic drug that acts upstream from the step by reverse transcriptase, specifically, a therapeutic drug that removes ccDNA in the nucleus, such as inhibition of ccDNA synthesis.

In developing such a new therapeutic agent, it is important to accurately detect ccDNA, which is a replication intermediate. However, the only difference between r cDNA and cc cDNA is that the former is an incomplete circular double-stranded DNA, while the latter is a complete double-stranded DNA, and the base sequences are common. For this reason, the distinction between r cDNA and cc cDNA is performed by using the Southern blotting method or the like, but this method is much more complicated than PCR and requires skillful techniques. In addition, there is a problem that it is difficult to detect and distinguish at several copy levels.

Therefore, at present, detection using PCR is being attempted, and in particular, detection of cccDNA by digital PCR is being attempted because of its excellent ability to separate trace molecules (Non-Patent Document 1).

Gian Paolo Caviglia et ai. Quantitation of HBV cccDNA in anti-HBc-positive liver donors by droplet digital PCR: A new tool to detect occult infection. Journal of Hepatology 2018; vol. 69; 301-307.

Therefore, an object of the present invention is to provide a method for detecting HBV-derived ccDNA with higher accuracy by digital PCR, and further to provide a detection reagent used for the method.

In order to achieve the above object, the method for detecting HBV-derived ccDNA (hereinafter referred to as HBVccDNA) by digital PCR of the present invention includes a step of distributing a DNA-containing sample into micro-compartments and the DNA-containing sample distributed into the micro-compartments. The biological sample to be distributed is a sample not treated with a DNA degrading enzyme, which comprises an amplification using a primer for HBVcc cDNA and a detection step for detecting a hybrid between the obtained amplification product and a probe for HBVcc cDNA. It is characterized by being.

The reagent for detecting HBV-derived cc cDNA (HBVcc cDNA) of the present invention is characterized by containing a probe consisting of the base sequence of SEQ ID NO: 6.

In the detection of HBVcc cDNA, as described above, it was considered that the coexistence of HBV-derived rcDNA (hereinafter referred to as HBVrcDNA) having the same base sequence in the sample reduces the detection accuracy of HBVcc cDNA. In particular, the abundance of HBVrc cDNA is overwhelmingly higher than that of HBVcc cDNA. Therefore, in the detection of HBVcc cDNA using PCR, pre-degrading HBVrc cDNA with a DNA degrading enzyme has been an indispensable step for those skilled in the art. On the other hand, although the mechanism is unknown, the present inventors have found that HBVcc cDNA can be detected more accurately by performing digital PCR on a DNA-containing sample without pretreatment with a DNase. It came to establish the invention. According to the present invention, since HBVcc cDNA can be detected with high accuracy, it can be effectively used, for example, for evaluation of HBV infection, evaluation of a therapeutic agent for removing HBVcc cDNA, and the like.

Unless otherwise specified, the terms used herein may be used in the meaning commonly used in the art.

(1) Method for detecting HBVcc cDNA by digital PCR The method for detecting HBVcc cDNA by digital PCR of the present invention comprises a step of distributing a DNA-containing sample into micro-compartments and the DNA-containing sample distributed into the micro-compartment as described above. , Amplification using a primer for HBVcc cDNA, and a detection step for detecting hybridization of the obtained amplification product with a probe for HBVcc cDNA, wherein the DNA-containing sample to be distributed is a sample not treated with DNA degrading enzyme. It is characterized by being.

The detection method of the present invention is characterized in that a DNA-containing sample is subjected to digital PCR without being treated with a DNase, and other steps and conditions are not limited. In the present invention, not performing the treatment with the DNA degrading enzyme means, for example, performing digital PCR without adding a DNA degrading enzyme from the outside, and specifically, for example, extracting DNA from a biological sample. Coexistence of foreign DNase at the time, addition of foreign DNase to the DNA content after extraction, foreign DNase does not coexist in the fractionation step and the detection step in the detection method of the present invention. Means that.

The DNA-containing sample used in the detection method of the present invention is, for example, a DNA-containing substance obtained by subjecting the sample to a DNA extraction process. Examples of the sample include biological samples such as tissues or cells. The tissue is, for example, liver tissue, and the cell is, for example, hepatocyte. The sample may be, for example, a tissue or cell collected from a living body, and the sample may be, for example, a cell line collected from a living body and isolated (unified). The cell line may be, for example, a liver cell line or an HBV-infected cell line. In the latter case, the type of the cell line is not particularly limited, and examples thereof include a liver cell line.

The DNA-containing sample can be prepared, for example, by performing a DNA extraction process from a tissue or cell as described above. The method of DNA extraction is not particularly limited, and for example, a commercially available DNA extraction reagent can be used, and processing can be performed according to these instructions.

The DNA-containing sample prepared in this manner is subjected to the distribution step of distributing to a small section without coexisting with a foreign DNA degrading enzyme. Distributing the DNA-containing sample in the digital PCR means, for example, limiting the DNA of the DNA-containing sample to be distributed in the micro-compartment, and in each of the micro-compartments, for example, the target DNA is 0. Generally, it is a molecule or about one molecule. The microsection may be, for example, a well such as a micropore or a droplet. The volume of the well is, for example, 0.7 to 0.9 nL, 0.7 to 0.8 μL, 0.775 to 0.805 nL, and the volume of the droplet is, for example, 0.7 to 0.9 nL. , 0.7-0.8 nL. The DNA-containing sample may be mixed with, for example, a reaction solution containing a reagent necessary for digital PCR, and this mixed solution may be distributed to the wells or introduced into an oil layer to form water-in-oil droplets. Therefore, it can be made into the minute section. The droplets can be prepared using, for example, a microfluidic chip or the like. When droplets are used for the digital PCR, each droplet serves as a reaction system, and thus is also referred to as droplet digital PCR (ddPCR).

The reagents required for the digital PCR are not particularly limited, and general examples include primers, probes, DNA polymerases, dNTPs, and the like. As the primer, for example, a forward primer, a reverse primer, or a set thereof can be used. The primer can be appropriately set based on, for example, the sequence of HBV-derived ccDNA. The probe can also be appropriately set based on, for example, the sequence of HBV-derived ccDNA. Further, since the fluorescent signal can be used for detecting the hybridization between the amplification product and the probe in the detection step described later, the reagent preferably contains a fluorescent reagent. The fluorescent reagent may be, for example, a fluorescent substance that binds to a hybridized double strand (for example, a dye such as SYBR ™ Green) or a fluorescent substance that labels the probe. In the latter case, it is preferable to use a labeled probe labeled with the fluorescent substance as the probe. Specific examples of the primer and the probe will be described later. The reaction solution can be prepared by mixing these reagents with an aqueous solvent such as a buffer solution.

Next, the detection method of the present invention comprises an amplification step using a primer for HBVcc cDNA and a detection step for detecting hybridization between the obtained amplification product and a probe for HBVcc cDNA in the DNA-containing sample distributed in the micro-compartment. conduct. Amplification using the primer and detection using the probe can be performed by a general digital PCR method. For amplification using the primer, for example, a general PCR method using a thermal cycler can be adopted. Further, the detection using the probe can be performed for each micro-compartment, for example, by measuring a fluorescence signal based on the hybridization between the amplification product and the probe.

By the amplification and the detection of the hybridization, the HBVcc cDNA contained in the DNA-containing sample can be detected. The detection of HBVcc cDNA in the present invention may be, for example, qualitative analysis or quantitative analysis. Conventionally, as described above, treatment with a DNA degrading enzyme has been essential in order to eliminate HBVrcDNA that hindered the detection of HBVcc cDNA, but in the present invention, treatment with a DNA degrading enzyme is not performed. By performing the digital PCR, although the mechanism is unknown, it is possible to accurately detect HBVcc cDNA in the DNA-containing sample. As described above, according to the present invention, it is possible to detect HBVcc cDNA with excellent accuracy, so that it can be said that more accurate evaluation is possible in the development of a new HBV therapeutic agent such as erasing HBVcc cDNA, for example. ..

(2) Primer As the method for detecting HBVcc cDNA of the present invention, for example, a primer designed based on the sequence of HBVcc cDNA can be used. The primer of the present invention may be, for example, only a forward primer or only a reverse primer, but a primer set of a forward primer and a reverse primer is preferable.

The sequence of HBVcc cDNA is stored in the database (GenBank) at Accession No. It is registered in D12980 (SEQ ID NO: 1). The HBVcc cDNA is a closed complete double chain as described above, and SEQ ID NO: 1 is the (+) chain of the HBVcc cDNA.

The primer can be set, for example, to amplify a specific region of HBVcc cDNA. For example, in the case of the (+) chain, the specific region is preferably a region containing the 1592th to 1836th positions in SEQ ID NO: 1 (hereinafter, the (+) chain specific region), and in the case of the (−) chain, the above-mentioned (+). A region complementary to the chain specific region (hereinafter, (-) chain specific region) is preferable, and the length of the specific region is, for example, 30 to 400 bases. As a specific example, among the specific regions, the (+) chain specific region is, for example, the 1550th to 1882th sequence, the 1550th to 1900th sequence, the 1545th to 1882th sequence, 1545 in SEQ ID NO: 1. The 1st to 1900th sequences and the like can be mentioned. The (-) chain specific region may be a complementary sequence to the sequence of the (+) chain specific region.

The complementarity between the primer and the region of interest to be aligned in the template is not particularly limited, as long as the primer can anneal to the template HBVcc cDNA and its amplification in the reaction of digital PCR. The complementarity between the primer and the target region is, for example, 80% or more, 90% or more, 95% or more, and 100%.

When the (+) chain of HBVcc cDNA is used as a template, the forward primer may be, for example, a sequence having a continuous 18 to 30 base lengths at positions 1545 to 1570 of SEQ ID NO: 1. Specific examples include a forward primer consisting of the base sequence of SEQ ID NO: 2 or 3, and a forward primer consisting of the memorized sequence of SEQ ID NO: 2 is preferable.
SEQ ID NO: 2 (1550-1570): 5'-cgtctgtgccttctcatctgc-3'
SEQ ID NO: 3 (1545-1563): 5'-ctccccgtctgtgccttct-3'

When the (+) strand of HBVcc cDNA is used as a template, the reverse primer may be, for example, a sequence having a continuous 18 to 30 base length in the complementary strand to the 1863th to 1900th sequences of SEQ ID NO: 1. Specific examples include a reverse primer consisting of the base sequence of SEQ ID NO: 4 or 5, and a reverse primer consisting of the base sequence of SEQ ID NO: 4 is preferable.
SEQ ID NO: 4 (1882-1863): 5'-gcacagcttggaggcttgaa-3'
SEQ ID NO: 5 (1883-1900): 5'-gccccaaagccacccaag-3'

The combination of the primers of the present invention is not particularly limited, and is, for example, a combination of the forward primer of SEQ ID NO: 2 and the reverse primer of SEQ ID NO: 4, a combination of the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 5, and the sequence. Examples thereof include a combination of the forward primer of SEQ ID NO: 2 and the reverse primer of SEQ ID NO: 5, a combination of the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4, and preferably the forward primer of SEQ ID NO: 2 and the reverse primer of SEQ ID NO: 4. It is a combination with the reverse primer of.

(3) Probe As the method for detecting HBVcc cDNA of the present invention, for example, a probe designed based on the sequence of HBVcc cDNA can be used. The probe of the present invention may be, for example, a sequence that hybridizes to the (+) strand of HBVcc cDNA, or may be a sequence that hybridizes to the (−) strand.

The probe of the present invention is designed based on, for example, a probe designed based on the sequence of the specific region amplified by the primer, specifically, the (+) chain specific region or the (-) chain specific region. Can be used with the probe. The probe of the present invention can be used, for example, with respect to the region 1545 to 1900 (preferably within the region 1592 to 1836 or the region 1602 to 1826) in SEQ ID NO: 1, or said to the region. A probe that hybridizes within a complementary region consisting of a complementary sequence to is preferred. The length of the probe may be, for example, 15 to 35 base lengths.

The complementarity between the probe and the target region in which the probe hybridizes (the region that aligns with the probe in the specific region) is not particularly limited, and the probe is the target region in the amplified product in the detection step. It is only necessary to be able to hybridize to. The complementarity between the probe and the target region is, for example, 80% or more, 90% or more, 95% or more, and 100%.

When the probe hybridizes to the (+) chain specific region, the probe can be exemplified by, for example, a sequence having a continuous 15 to 35 base lengths at positions 1545 to 1900 of SEQ ID NO: 1. Further, more preferably, for example, a region between DR1 and DR2, and as a specific example, a sequence having the continuous base lengths at positions 1592 to 1836 or positions 1602 to 1826 in SEQ ID NO: 1 can be exemplified. As a specific example, as the former, for example, a probe consisting of the base sequence of SEQ ID NO: 6 can be mentioned, and as the latter, for example, a probe consisting of the base sequence of SEQ ID NO: 7 can be mentioned, and the former is preferable. In SEQ ID NO: 7, r is a or g.
SEQ ID NO: 6 (1781-1805): 5'-ctgtaggcataaattggtctgttca-3'
SEQ ID NO: 7 (1545-1563): 5'-cgtcgcatggaraccaccgtgaacgcc-3'

The combination of the forward primer, the reverse primer and the probe is not particularly limited, and for example, a combination of the forward primer of SEQ ID NO: 2 and the reverse primer of SEQ ID NO: 4 and the probe of SEQ ID NO: 6 or 7, SEQ ID NO: 3 The combination of the forward primer of SEQ ID NO: 5, the reverse primer of SEQ ID NO: 5 and the probe of SEQ ID NO: 6 or 7 is mentioned, and among them, the combination of the forward primer of SEQ ID NO: 2 and the reverse primer of SEQ ID NO: 4 and the probe of SEQ ID NO: 6 is mentioned. preferable.

The probe is preferably a labeled probe labeled with a labeling substance, for example, in order to detect hybridization between the obtained amplification product and the probe in the detection step of digital PCR. The labeled substance may be, for example, a substance that emits fluorescence by the hybridization or a substance that eliminates the fluorescence by the hybridization because the change in the intensity of the fluorescence signal due to the hybridization can be used. Further, the labeling substance may be, for example, a combination of a reporter which is a fluorescent substance and a citric acid which quenches the fluorescence of the reporter. In the present invention, the type and labeling location of the labeled substance in the probe are not particularly limited. Examples of the fluorescent substance include dyes such as SYBR ™ Green and FAM. Specific examples of the labeled portion with the fluorescent substance include, for example, the 5'end of the probe or its vicinity (for example, the 2nd base to the 3rd base from the 5'end, the 3rd base, etc.), the 3'end of the probe, or the same thereof. The neighborhood (for example, the second base from the 3'end, the third base, etc.) can be mentioned. In the case of a combination of the reporter and the quencher, for example, it is preferable to bind the reporter to the 5'end of the probe or its vicinity and to bind the quencher to the 3'end of the probe or its vicinity. Examples of the combination of the reporter and the citrate include a combination of FAM and TAMSp. The probe may have, for example, two quenchers, in which case a quencher as described above may be added to the terminal side, and a quencher such as ZEN may be added to the inside of the sequence.

(4) Reagent for Detection In the method for detecting HBV-derived ccDNA of the present invention, the types of primers and probes are not particularly limited as described above, but among them, the following reagent for detecting HBVccDNA of the present invention is used. It is preferable because HBVcc cDNA can be detected with high accuracy.

The detection reagent of the present invention preferably contains, as the probe, a probe consisting of the base sequence of SEQ ID NO: 6. As the probe, for example, a labeled probe as described above is preferable.

The detection reagent of the present invention preferably further contains a forward primer consisting of the base sequence of SEQ ID NO: 2 and a reverse primer consisting of the base sequence of SEQ ID NO: 4 with respect to the probe.

Hereinafter, the present invention will be described in detail with reference to Examples and the like, but the present invention is not limited thereto.

(Example 1)
The following reagents 1 and 2 were prepared.
Reagent 1
Forward primer 1: SEQ ID NO: 2
Reverse primer 1: SEQ ID NO: 4
Probe 1: SEQ ID NO: 6
5'- FAM / CT GTA GGC A / ZEN / T AAA TTG GTC TGT TCA / TAMSp -3'
Reagent 2
Forward primer 2: SEQ ID NO: 3
Reverse primer 2: SEQ ID NO: 5
Probe 2: SEQ ID NO: 7 (manufactured by Takara Bio Inc.)
5'-FAM / CG TCG CAT GGA RAC CAC CGT GAA CGC C / TAMSp -3'
In SEQ ID NO: 7, R is A or G, and in this example, a mixture of a probe of R = A and a probe of R = G was used.

Hep38.7-tet cells were used as human hepatocytes. Hep38.7-tet cells were cultured in Dulbecco-modified Eagle's medium (D-MEM) supplemented with tetracycline (0.3 μg / ml) at 37 ° C. for 24 hours, and then replaced with D-MEM supplemented with tetracycline. After culturing for 72 hours, cells were collected. It is known that the Hep38.7-tet cells have 1 copy / 1 copy of HBVcc cDNA and 2 copies / 1 cell of RNaseP gene (Kurume et al., Mathematical analysis). Laboratory Record, 2017, 2043, pp. 95-101, Publisher: Research Institute for Mathematical Sciences, Kyoto University).

The examples were carried out as follows. First, DNA was extracted from the collected cells using a DNA extraction reagent (trade name: SMITEST EX-R & D, MBL Life Science Co., Ltd.), and 30 μL of the obtained extract was used as a DNA sample. A reaction solution having the following composition was prepared without treating the DNA sample with an external DNase, and the reaction solution was distributed to plates with 20,000 wells and subjected to digital PCR. In the reaction solution, the reagent 1 and the reagent 2 were used as the forward primer, the reverse primer and the probe, respectively. For digital PCR, a commercially available device (trade name: QuantStudio (trademark) 3D digital PCR system, Applied Biolad System) was used, and the operating conditions were in accordance with the instruction manual. Then, from the results of the fluorescent signal detected by the digital PCR, the amount of HBVcc cDNA (copy / μL), the amount of RNaseP (copy / μL), and the amount of HBVcc cDNA (copy / cell) are determined based on the instruction manual. Calculated.

(Reaction solution composition)
Digital PCR Master Mix 7.5 μL
Forward primer 1.35 μL
Reverse primer 1.35 μL
Probe 0.75 μL
DNA sample 1.5 μL
Distilled water 2.55 μL
15 μL in total

On the other hand, the comparative example was performed as follows. First, DNA was extracted from the collected cells using a DNA extraction reagent (trade name: SMITEST EX-R & D, MBL Life Science Co., Ltd.). Commercially available DNase (trade name: Plasmamid-Safe (trademark) ATP-Dependent DNase: Air Brown Life Science Co., Ltd.) was added to 30 μL of the obtained extract to perform DNAase treatment. The conditions for the DNAase treatment were the addition of 10 units of DNase per 1-30 μL of the extract and the incubation at 37 ° C. for 30 minutes. Then, the extract after the DNA treatment was subjected to the DNA extraction treatment again using the DNA extraction reagent. Digital PCR was performed in the same manner as in Example 1 except that the obtained extract was used as a DNA sample. Then, from the results of the fluorescent signal detected by the digital PCR, the amount of HBVcc cDNA (copy / μL), the amount of RNaseP (copy / μL), and the amount of HBVcc cDNA (copy / cell) were calculated using a calibration curve. .. The calibration curve was prepared by preparing a diluted system of the number of copies of artificial DNA (plasmid) having a structure similar to that of cccDNA, and performing digital PCR under the same conditions.

These results are shown in Table 1. Table 1 shows the number of copies of HBVcc cDNA per 1 μL of the reaction solution, the number of copies of RNase P, and the number of copies of HBVcc cDNA per cell. As shown in Table 1, in the comparative example in which the DNase treatment was performed per 1 μL of the reaction solution, the number of copies of both HBVcc cDNA and RNase was significantly reduced as compared with the example in which the DNase treatment was not performed.

As mentioned above, Hep38.7-tet cells are known to have one copy / cell of HBVcc cDNA. Therefore, it can be evaluated that the closer the result obtained by digital PCR is to 1 copy / cell, the more accurately the HBVcc cDNA can be measured. As shown in Table 1, both when reagent 1 was used and when reagent 2 was used, the examples without DNase treatment had a copy of HBVcc cDNA per cell as compared with the comparative example with DNase treatment. Since the number is close to 1, it was confirmed that the detection accuracy of HBVcc cDNA can be improved by not performing DNase treatment. Further, when the reagent 1 and the reagent 2 are compared with each other in the example not subjected to the DNase treatment, it is 1.03 copy / 1 cell when the reagent 1 is used, and 1.16 copy / 1 using the reagent 2. The number of copies was closer to 1 than that of cells. From this result, it was found that the detection accuracy of HBVcc cDNA can be further improved by using the reagent 1.

Although the present invention has been described above with reference to the embodiments and examples, the present invention is not limited to the above embodiments. Various modifications that can be understood by those skilled in the art can be made to the structure and details of the present invention within the scope of the present invention.

According to the present invention, since HBVcc cDNA can be detected with high accuracy, it can be effectively used, for example, for evaluation of HBV infection, evaluation of a therapeutic agent for removing HBVcc cDNA, and the like.

Claims (7)

The process of distributing the DNA-containing sample to the minute compartments,
The DNA-containing sample distributed in the micro-compartment includes amplification using a primer for HBV-derived ccDNA and a detection step for detecting hybridization between the obtained amplification product and a probe for HBV-derived ccDNA.
A method for detecting HBV-derived ccDNA by digital PCR, wherein the DNA-containing sample to be distributed is a sample that has not been treated with a DNA degrading enzyme. The method for detecting HBV-derived ccDNA according to claim 1, wherein the DNA degrading enzyme is ATP-dependent DNase. The method for detecting HBV-derived ccDNA according to claim 1 or 2, wherein the DNA-containing sample is a DNA sample extracted from liver cells. The detection of HBV-derived ccDNA according to any one of claims 1 to 3, wherein the primer is a set of a forward primer consisting of the base sequence of SEQ ID NO: 2 and a reverse primer consisting of the base sequence of SEQ ID NO: 4. Method. The method for detecting HBV-derived ccDNA according to any one of claims 1 to 4, wherein the probe is a probe consisting of the base sequence of SEQ ID NO: 6. A reagent for detecting HBV-derived ccDNA, which comprises a probe consisting of the nucleotide sequence of SEQ ID NO: 6. The reagent for detecting HBV-derived ccDNA according to claim 6, further comprising a forward primer consisting of the base sequence of SEQ ID NO: 2 and a reverse primer consisting of the base sequence of SEQ ID NO: 4.