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Sep 2020
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A Sensitive and Specific PCR-based Assay to Quantify Hepatitis B Virus Covalently Closed Circular (ccc) DNA while Preserving Cellular DNA
一种在保留细胞DNA的同时定量检测乙型肝炎病毒共价闭合环状(ccc)DNA的灵敏、特异PCR方法   

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Abstract

Hepatitis B virus (HBV) is the major cause of liver diseases and liver cancer worldwide. After infecting hepatocytes, the virus establishes a stable episome (covalently closed circular DNA, or cccDNA) that serves as the template for all viral transcripts. Specific and accurate quantification of cccDNA is difficult because infected cells contain abundant replicative intermediates of HBV DNA that share overlapping sequences but arranged in slightly different forms. HBV cccDNA can be detected by Southern blot or qPCR methods which involve enzymatic digestion. These assays are laborious, have limited sensitivity, or require degradation of cellular DNA (which precludes simple normalization). The method described in this protocol, cccDNA inversion quantitative (cinq)PCR, instead uses a series of restriction enzyme-mediated hydrolysis and ligation reactions that convert cccDNA into an inverted linear amplicon, which is not amplified or detected from other forms of HBV DNA. Importantly, cellular DNA remains quantifiable during sample preparation, allowing normalization and markedly improving precision. Further, a second linear fragment (derived from enzymatic digestion of a separate region of the HBV DNA genome and is present in all forms of HBV DNA) can be used to simultaneously quantify total HBV levels.


Graphic abstract:



Selective detection of HBV cccDNA and total HBV DNA using cinqPCR (Reproduced from Tu et al., 2020a).


Keywords: Hepatitis B virus (乙型肝炎病毒), cccDNA (共价闭环DNA), Hepcludex® (Hepcludex®), Bulevirtide (Bulevirtide), Myrcludex B (Myrcludex B), cinqPCR (cinqPCR), DNA nick (DNA缺刻), DNA repair (DNA修复), Viral persistence (病毒持续感染)

Background

Hepatitis B virus (HBV) is a small enveloped virus, which encapsidates a partially double-stranded circular DNA genome, the so-called relaxed circular (rc) DNA. Upon infection of human hepatocytes, the nucleocapsid is transported to the nucleus wherein the rcDNA genome is converted to covalently closed circular (ccc) DNA. This episomal form is highly stable and maintains chronic HBV infection (Tu et al., 2020b). It serves as template for all viral transcripts and the pre-genomic RNA. Elimination of the cccDNA would lead to a complete cure of a chronic hepatitis B infection. Thus, the reliable quantification of cccDNA is key to developing strategies for HBV cure.


In any given infected hepatocyte, HBV DNA exists in different forms (rcDNA, double stranded linear (dsl) DNA, single stranded (ss) DNA, and cccDNA) that share overlapping sequences, but have different structures (Nassal, 2015). Thus, differentiation of cccDNA (which is present in low numbers) from other HBV DNA forms is technically challenging. Southern blot is the gold standard method used to differentiate cccDNA from other forms, though it has low throughput and poor sensitivity. PCR-based methods are more sensitive and can be applied in a medium-throughput manner. Current protocols include an exonuclease treatment (with T5 exonuclease, plasmid-safe DNase, or a combination of ExoI and ExoIII) to digest all DNA species that are not covalently closed (including cellular DNA) (Luo et al., 2017; Allweiss et al., 2018; Qu et al., 2018). By contrast, the method described in this protocol takes advantage of particular restriction sites within the HBV DNA sequence (see graphical abstract) to selectively convert cccDNA into an amplifiable form. Therefore, our method allows the normalization to a single-copy cellular gene and total HBV DNA can be detected from the same sample, dramatically improving precision, sensitivity, and accuracy.

Materials and Reagents

Note: All stored at room temperature unless otherwise stated.

  1. 24-well cell culture plates (Corning, catalog number: 3527)

  2. 1.5 ml Eppendorf tubes (Sarstedt, catalog number: 72706)

  3. Sealing Mats for 96-Well PCR Plates, reusable (Bio-Rad, catalog number: 2239442)

  4. TwinTec PCR plates, 96 semi-skirted (Eppendorf, catalog number: 0030128.575)

  5. Aluminium Foil Seals for PCR and QX100 ddPCR applications (Bio-Rad, catalog number: 181-4040)

  6. P20 racked barrier LTS tips (Rainin, catalog number: RT-L20F)

  7. P200 racked barrier LTS tips (Rainin, catalog number: RT-L200F)

  8. HBV-susceptible cells, e.g., HepG2-NTCP cells (Ni et al., 2014) (maintain in 37 °C incubator at 5% CO2 supply and 95% humidity)

  9. HBV genotype D virus stock (store at -80 °C)

  10. Dulbecco’s Phosphate Buffered Saline (PBS, Sigma-Aldrich, catalog number: D8537)

  11. Dulbecco’s Modified Eagle Medium (DMEM, Life Technologies, catalog number: 41965039, store at 4 °C)

  12. Fetal bovine serum (FBS, Sigma-Aldrich, catalog number: S0615, store at -20 °C)

  13. L-Glutamine (Life Technologies, catalog number: 25030024, store at -20 °C)

  14. Penicillin (10,000 U/ml) Streptomycin (10 mg/ml) (Life Technologies, catalog number: 15140-122, store at -20 °C)

  15. Dimethylsulfoxide (DMSO, Merck, catalog number: 102950)

  16. Polyethylenglycol (PEG 8000, 40% solution in PBS, Sigma-Aldrich, catalog number: 89510)

  17. Trypsin-EDTA solution (Sigma-Aldrich, catalog number: T3924, store at 4 °C)

  18. NucleoSpin® Tissue kit (Macherey-Nagel, catalog number: 740952)

  19. Multichannel pipette (Eppendorf, catalog number: 3125000036)

  20. 10× Cutsmart buffer (NEB, catalog number: B7204, store at -20 °C)

  21. HhaI (NEB, catalog number: R0139, store at -20 °C)

  22. PCR-grade H2O (e.g., B. Braun Melsungen, for injection purposes)

  23. RecJf (NEB, catalog number: M0264, store at -20 °C)

  24. T4 DNA Ligase (NEB, catalog number: M0202, store at -20 °C)

  25. 10 mM Molecular-grade ATP (NEB, catalog number: P0756, store at -20 °C)

  26. XbaI (NEB, catalog number: R0145, store at -20 °C)

  27. DNAZap PCR DNA Degradation Solutions (Thermo Scientific, catalog number: AM9890, store at 4 °C)

  28. VIC-labelled TaqManTM Copy Number Reference Assay for the human RNase P gene (Applied Biosystems, catalog number: 4403328, store at -20 °C)

  29. ddPCR Supermix for Probes (Bio-Rad, catalog number: 1863010, store at -20 °C)

  30. Primers/probe for cccDNA detection (ordered from Eurofins genomics, store at -20 °C)

    1. cccDNA for: 5′-CACTCTATGGAAGGCGGGTA-3′

    2. cccDNA rev: 5′-ATAAGGGTCGATGTCCATGC-3′

    3. cccDNA probe: 5′-FAM- AACACATAGCGCACCAGCA-BHQ1-3′

  31. Primers/probe for total HBV DNA (ordered from Eurofins genomics, store at -20 °C)

    1. Total HBV for: 5′-GTGTCTGCGGCGTTTTATCA-3′

    2. Total HBV rev: 5′- GACAAACGGGCAACATACCTT-3′

    3. Total HBV probe: 5′-FAM-TGAGGCATAGCAGCAGGATG-BHQ1-3′

  32. DG8 cartridges (Bio-Rad, catalog number: 186-4008)

  33. DG8 gaskets (Bio-Rad, catalog number: 186-3009)

  34. Droplet Reader Oil (Bio-Rad, catalog number: 186-3004)

  35. Droplet generation oil for probes (Bio-Rad, catalog number: 186-3005)

  36. Growth medium (see Recipes)

  37. Infection medium (see Recipes)

  38. Ligation mix (see Recipes)

  39. Linearization mix (see Recipes)

Equipment

  1. Pipette for Rainin P20 tips (Rainin, catalog number: 17014392)

  2. Multichannel pipette for Rainin P200 tips (Rainin, catalog number: 17013805)

  3. Thermo cycler (Analytik Jena Biometra, FlexCycler2)

  4. QX200TM Droplet generator (Bio-Rad, catalog number: 1864002)

  5. PX1 PCR plate sealer (Bio-Rad, catalog number: 1814000)

  6. C1000 TouchTM Thermal Cycler with 96-Deep Well Reaction Module (Bio-Rad, catalog number: 1851197)

  7. QX200TM Droplet Reader (Bio-Rad, catalog number: 1864003)

Software

  1. QuantaSoft (Bio-Rad)

  2. Microsoft Excel

  3. Prism (GraphPad)

Procedure

  1. Sample preparation

    1. Seed 2.5 × 105 cells per well in a 24-well format in growth medium one day before infection.

    2. Infect cells overnight with HBV virus stock in infection medium containing 4% PEG 8000 (Ni et al., 2014).

    3. Wash cells twice with PBS at room temperature (RT) and maintain in infection medium until desired harvest time.

    4. Wash cells with 500 μl PBS at RT and add 100 μl trypsin.

    5. Incubate at 37 °C until the cells detach (~5 min).

    6. Resuspend the cells with 900 μl cold medium and transfer to a 1.5 ml Eppendorf tube.

    7. Centrifuge for 5 min at 500 × g at RT and remove the supernatant.

      Optional: Freeze the pellet at -20 °C until further use.

    8. Extract total DNA from the cell pellets using NucleoSpin® Tissue kit according to the manufacturer’s instructions, but elute in 50 μl elution buffer pre-warmed to 70 °C.


  2. Inversion reaction

    1. Clean a silicon sealing mat using the DNA Zap solutions, rinse in deionised water, and let dry on a paper towel.

    2. Prepare the restriction digestion reaction in a 96-well plate containing 10 µl of extracted total DNA, 2 µl of 10× Cutsmart buffer, 0.5 µl HhaI, 0.25 µl RecJf, and 7.25 µl H2O.

    3. Mix by pipetting up and down 10 times with a multichannel pipette, taking care to avoid generating bubbles.

    4. Seal with the clean and dry silicon mat.

    5. Run the following program in a thermo cycler: 3 cycles of 15 min at 37 °C and 15 min at 42 °C, 20 min at 80 °C, and hold at 16 °C.

    6. Remove the mat carefully and add 10 μl of the ligation mix.

    7. Mix by pipetting up and down 10 times with a multichannel pipette, taking care to avoid generating bubbles.

    8. Incubate in a thermo cycler at 16 °C for 2 h, 80 °C for 20 min, and hold at 16 °C.

    9. Remove the mat carefully and add 5 μl of the linearization mix.

    10. Mix by pipetting up and down 10 times with a multichannel pipette, taking care to avoid generating bubbles.

    11. Incubate in a thermo cycler at 37 °C for 60 min, 80 °C for 20 min, and hold at 16 °C.

      Optional: Store samples at -20 °C.


  3. Droplet generation and reading

    1. Dilute the sample 2.5-fold by adding 52.5 μl H2O.

    2. Mix by pipetting up and down 10 times with a multichannel pipette, taking care to avoid generating bubbles.

    3. Prepare two reactions for each sample: (1) cccDNA and RNaseP cellular control and (2) total HBV DNA and RNaseP cellular control. Prepare ddPCR mix in a 96-well plate (22 μl per reaction) (see Table 1).


      Table 1. ddPCR mix composition for each reaction
      Reagent Concentration μl
      ddPCR Supermix for probes 11
      TaqManTM Copy Number Reference Assay (VIC-labelled RNaseP) 10× 1.1
      Forward primer (cccDNA or total HBV DNA) 100 μM 0.033
      Reverse primer (cccDNA or total HBV DNA) 100 μM 0.033
      FAM-labelled probe (cccDNA or total HBV DNA) 100 μM 0.033
      H2O 4.5
      Diluted template 5.5


    4. Use microplastic-free Rainin tips. Generate droplets on the ddPCR droplet generator:

      1. Add 20 μl of the ddPCR mix to the sample chambers of a cartridge.

      2. Add 70 μl of droplet generation oil using a multichannel pipette in the oil chamber.

      3. Attach the rubber gasket, place the cartridge in the droplet generator, and run the droplet generation.

      4. Gently transfer 42 μl of the generated droplets using a multichannel pipette to a TwinTec 96-well plate.

      5. Repeat Steps C4a to C4d until all samples are loaded into the TwinTec 96-well plate.

      6. Seal the plate with an aluminum foil seal using the plate sealer.

    5. Directly after sealing, run PCR in deep-well PCR machine with the following program: 10 min at 95 °C; 40 cycles of 10 s at 95 °C, 15 s at 54 °C, and 20 s at 68 °C; 10 min at 95 °C, and hold at 12 °C.

    6. Transfer the TwinTec 96-well plate to the QX200 Droplet Reader.

    7. Set up template using the Quantasoft software (as per manufacturer’s user guide), reading droplets using channels for FAM (Channel 1) and VIC (Channel 2).

    8. Define thresholds for positive and negative droplets based on positive and negative controls, after which the software will automatically quantify values for cccDNA per RNaseP and total HBV DNA per RNaseP.

Data analysis

In the Quantasoft software, we use the 2D graph to draw thresholds for HBV DNA and for the RNaseP positive droplets (Figure 1). The thresholds are gated separately for all the total HBV DNA and all the cccDNA assays. For the data presentation we use the ratios total HBV DNA/RNaseP and cccDNA/RNaseP of each sample with the PoissonRatioMin and PoissonRatioMax values giving a 95% confidence interval of the technical error of the ddPCR.



Figure 1. Data analysis using QuantaSoft. The fluorescence intensity was used to separate droplets containing amplicons detecting: HBV cccDNA (left) or total HBV DNA (right), based on FAM fluorescence (y-axis); and RNaseP, based on VIC fluorescence (x-axis). The thresholds for positive signals (pink) were gated based on positive and negative control reactions (Reproduced from Tu et al., 2020a).

Notes

  1. We have used this protocol with DNA extracted from cells infected in plate formats from 12-well to 96-well. In 12-well plates, we advise to elute the DNA in twice the volume.

  2. For the inversion, we recommend an input of <2 µg of total DNA extract. We also recommend adding 200 ng of carrier DNA (e.g. unrelated plasmid DNA) if the total DNA input is <200 ng.

  3. We usually perform HBV infections with a multiplicity of viral genome equivalents of 200. However, infection can also be performed with a smaller inoculum as the assay is sensitive enough to detect very low numbers of infected cells (Tu et al., 2020a).

  4. This protocol can also be used with Taqman-based qPCR instead of ddPCR.

  5. This assay is only compatible with HBV sequences from genotype D which is used in most in vitro HBV infection assays. Unfortunately, it does not work with other genotypes due to variation in HhaI restriction sites. Most in vitro assays with HBV are performed with the genotype D strain, this shortcoming does not allow the analysis of clinical samples.

  6. Recommended controls for optimization and quality control:

    1. Positive control to show detection efficiency (total HBV DNA quantification should give similar number as cccDNA quantification): HBV circDNA (Mutz et al., 2018) or HBV 1.1 overlength plasmid spiked into DNA extracted from uninfected cells.

    2. Negative control to show selective quantification of cccDNA: DNA extracted from heparin-purified virus (Seitz et al., 2016) spiked into DNA extracted from uninfected cells.

    3. Negative control to determine signal from virus inoculum during cell infection: Treatment of cells with an entry inhibitor (e.g., Myrcludex B) prior to HBV infection (Donkers et al., 2017).

    4. No template control and uninfected cell control to determine level of non-specific amplification.

Recipes

  1. Growth medium

    500 ml of DMEM

    50 ml of FBS

    5 ml of L-Glutamine

    5 ml of Penicillin (10,000 U/ml) and Streptomycin (10 mg/ml)

  2. Infection medium

    49 ml of Growth medium

    1 ml of DMSO

  3. Ligation mix (10 µl per sample)3 µl of 10 mM ATP

    1 µl of 10× NEB Cutsmart buffer

    0.5 µl of T4 ligase

    5 µl H2O

  4. Linearization mix (5 µl per sample)

    0.5 µl of 10× Cutsmart buffer

    0.5 µl of XbaI

    4 µl H2O

Acknowledgments

This protocol has been published in Tu et al. (2020a). This work received funding from: the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) Project number 240245660 – SFB 1129 (B.Z. and S.U.); the German Centre for Infection Research (DZIF) TTU Hepatitis Projects 5.816 and 5.704 (S.U. and T.T.); and the Australian Centre for HIV and Hepatitis Virology Research (T.T.). We thank Dr. Kathleen Börner for the training and support with using the ddPCR equipment.

Competing interests

Stephan Urban is co-applicant and co-inventor on patents protecting HBV preS-derived lipopeptides (bulevirtide / Hepcludex®, formerly Myrcludex B) for their use as HBV/HDV entry inhibitors. The other authors in this study declare no competing interests.

References

  1. Allweiss, L., Volz, T., Giersch, K., Kah, J., Raffa, G., Petersen, J., Lohse, A. W., Beninati, C., Pollicino, T., Urban, S., Lutgehetmann, M. and Dandri, M. (2018). Proliferation of primary human hepatocytes and prevention of hepatitis B virus reinfection efficiently deplete nuclear cccDNA in vivo. Gut 67(3): 542-552.
  2. Donkers, J. M., Zehnder, B., van Westen, G. J. P., Kwakkenbos, M. J., AP, I. J., Oude Elferink, R. P. J., Beuers, U., Urban, S. and van de Graaf, S. F. J. (2017). Reduced hepatitis B and D viral entry using clinically applied drugs as novel inhibitors of the bile acid transporter NTCP. Sci Rep 7(1): 15307.
  3. Luo, J., Cui, X., Gao, L. and Hu, J. (2017). Identification of an Intermediate in Hepatitis B Virus Covalently Closed Circular (CCC) DNA Formation and Sensitive and Selective CCC DNA Detection. J Virol 91(17): e00539-17.
  4. Mutz, P., Metz, P., Lempp, F. A., Bender, S., Qu, B., Schoneweis, K., Seitz, S., Tu, T., Restuccia, A., Frankish, J., Dachert, C., Schusser, B., Koschny, R., Polychronidis, G., Schemmer, P., Hoffmann, K., Baumert, T. F., Binder, M., Urban, S. and Bartenschlager, R. (2018). HBV Bypasses the Innate Immune Response and Does Not Protect HCV From Antiviral Activity of Interferon. Gastroenterology 154(6): 1791-1804 e1722.
  5. Nassal, M. (2015). HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B.Gut 64(12): 1972-1984.
  6. Ni, Y., Lempp, F. A., Mehrle, S., Nkongolo, S., Kaufman, C., Falth, M., Stindt, J., Koniger, C., Nassal, M., Kubitz, R., Sultmann, H. and Urban, S. (2014). Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes. Gastroenterology 146(4): 1070-1083.
  7. Qu, B., Ni, Y., Lempp, F. A., Vondran, F. W. R. and Urban, S. (2018). T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR. J Virol 92(23): e01117-18.
  8. Seitz, S., Iancu, C., Volz, T., Mier, W., Dandri, M., Urban, S. and Bartenschlager, R. (2016). A Slow Maturation Process Renders Hepatitis B Virus Infectious. Cell Host Microbe 20(1): 25-35.
  9. Tu, T., Zehnder, B., Qu, B., Ni, Y., Main, N., Allweiss, L., Dandri, M., Shackel, N., George, J. and Urban, S. (2020a). A novel method to precisely quantify hepatitis B virus covalently closed circular (ccc)DNA formation and maintenance. Antiviral Res 181: 104865.
  10. Tu, T., Zehnder, B., Qu, B. and Urban, S. (2020b). De novo synthesis of Hepatitis B virus nucleocapsids is dispensable for the maintenance and transcriptional regulation of cccDNA. JHEP Rep 3(1): 100195.

简介

[摘要]乙型肝炎病毒(HBV)是全球范围内肝脏疾病和肝癌的主要原因。感染肝细胞后,病毒建立起稳定的附加体(共价闭合的环状DNA或cccDNA),作为所有病毒转录本的模板。cccDNA的特异性和准确定量非常困难,因为被感染的细胞含有丰富的HBV DNA复制中间体,这些中间体共享重叠序列,但排列形式略有不同。HBV cccDNA可以通过涉及酶消化的Southern印迹或qPCR方法进行检测。这些测定费力,灵敏度有限或需要细胞DNA降解(无法进行简单的标准化)。该协议中描述的方法cccDNA反向定量(cinq)PCR,而是使用一系列限制性酶介导的水解和连接反应,将cccDNA转化为反向线性扩增子,该扩增子无法从其他形式的HBV DNA扩增或检测到。重要的是,细胞DNA在样品制备过程中仍可定量,从而可以进行标准化并显着提高精确度。另外,第二线性片段(源自酶消化HBV DNA基因组的单独区域,并以所有形式的HBV DNA存在)可用于同时定量总HBV水平。

图形摘要:

HBV的cccDNA和总HBV DNA的选择性检测使用cinqPCR (转载自涂等人,2020一)。


[背景]乙型肝炎病毒(HBV)是一种小的有包膜病毒,其encapsidates一个部分双链环状DNA基因组,所谓松弛环状(RC)的DNA。感染人肝细胞后,核衣壳被转运至细胞核,其中rcDNA基因组被转化为共价闭合的环状(ccc)DNA。这种游离形式是高度稳定的,并保持慢性HBV感染(Tu等人,2020b )。它充当所有病毒转录本和基因组前RNA的模板。该cccDNA的消除将导致的完全治愈一个慢性乙型肝炎感染。因此,对cccDNA的可靠的量化是关键,以发展战略为HBV治疗。

在给定的感染肝细胞中,HBV DNA以不同的形式存在(rcDNA,双链线性(dsl)DNA,单链(ss)DNA和cccDNA),它们具有重叠的序列,但结构不同(Nassal ,2015)。因此,将cccDNA与其他HBV DNA形式区别开来(数量很少)在技术上具有挑战性。Southern印迹法是用于将cccDNA与其他形式区分开的金标准方法,尽管它的通量低且灵敏度低。基于PCR的方法更加灵敏,可以以中等通量方式应用。当前的方案包括n核酸外切酶处理(使用T5核酸外切酶,质粒安全的DNase或ExoI和ExoIII的组合),以消化所有未共价封闭的DNA物种(包括细胞DNA)(Luo等人,2017 ; Allweiss等人等人,2018 ;Qu等人,2018)。相比之下,该协议中描述的方法利用了HBV DNA序列内的特定限制位点(请参见图形摘要)将cccDNA选择性转化为可扩增形式。因此,我们的方法允许对单拷贝细胞基因进行标准化,并且可以从同一样品中检测出总的HBV DNA,从而大大提高了精密度,灵敏度和准确性。

关键字:乙型肝炎病毒, 共价闭环DNA, Hepcludex®, Bulevirtide, Myrcludex B, cinqPCR, DNA缺刻, DNA修复, 病毒持续感染



材料和试剂


注意:除非另有说明,否则所有均在室温下保存。


1. 24孔细胞培养板(Corning,目录号:3527)     

2. 1.5毫升Eppendorf管(Sarstedt,目录号:72706)     

3.用于96孔PCR板的密封垫,可重复使用(Bio-Rad ,目录号:2239442)     

4. TwinTec PCR板,半裙96个(Eppendorf,目录号:0030128.575)     

5.用于PCR和QX100 ddPCR应用的铝箔密封件(Bio-Rad,目录号:181-040)     

6. P20机架式屏障LTS尖端(雨淋,目录号:RT-L20F)     

7. P200机架式屏障LTS尖端(雨淋,目录号:RT-L200F)     

8. HBV-易感细胞,例如,HepG2细胞-NTCP细胞(镍等人,2014) (在37℃培养箱中维持在5%CO 2的供应和95%湿度)     

9. HBV基因型D型病毒库存(储存在-80°C)     

10. Dulbecco磷酸盐缓冲盐水(PBS,Sigma-Aldrich,目录号:D8537) 

11. Dulbecco改良的Eagle培养基(DMEM,生命Ť echnologies,目录号:41965039,保存于4℃) 

12.胎牛血清(FBS,Sigma-Aldrich,目录号:S0615,储存在-20°C) 

13. L-Glutam INE(生命Ť echnologies,目录号:25030024,保存于-20℃) 

14.青霉素(10 ,000 U / ml)的链霉素(10毫克/毫升)(生命Ť echnologies,目录号:15140-122,储存在-20℃) 

15.二甲基亚砜(DMSO,默克(Merck),目录号:102950) 

16.聚乙二醇(PEG 8000,PBS中的40%溶液,Sigma-Aldrich,目录号:89510) 

17.胰蛋白酶-EDTA溶液(Sigma-Aldrich,目录号:T3924,在4 °C下储存) 

18. NucleoSpin ® Tissue试剂盒(马歇雷-Nagel的,目录号:740952) 

19.多通道移液器(Eppendorf,目录号:3125000036) 

20. 10 × Cutsmart缓冲液(NEB,目录号:B7204 ,小号撕在-20 ℃下) 

21. HHA I(NEB ,目录号:R0139 ,小号撕在-20 ℃下) 

22. PCR-级ħ 2 O(例如,贝朗Melsungen的,用于注射目的的) 

23. RecJ ˚F (NEB ,目录号:M0264 ,小号撕在-20 ℃下) 

24. T4 DNA连接酶(NEB ,目录号:M0202 ,小号撕在-20 ℃下) 

25. 10毫分子级ATP(NEB ,目录号:P0756 ,小号撕在-20 ℃下) 

26. XbaI位I(NEB ,目录号:R0145 ,小号撕在-20 ℃下) 

27. DNAZap PCR DNA降解溶液(Thermo Scientific ,目录号:AM9890 ,在4 °C时破裂) 

28. VIC标记的TaqMan TM拷贝数参考测定对于人核糖核酸酶P基因(Applied Biosystems公司,目录号:4403328 ,小号撕在-20 ℃下) 

29. ddPCR超混合液为习题ES(Bio-Rad公司,目录号:1863010 ,小号撕在-20 ℃下) 

30.引物/探针的cccDNA检测(从基因组学欧陆有序,小号撕在-20 ℃下)               

cccDNA的为:5 ' -CACTCTATGGAAGGCGGGTA-3 '
cccDNA的启:5 ' -ATAAGGGTCGATGTCCATGC-3 '
cccDNA的探针:5 ' -FAM- AACACATAGCGCACCAGCA-BHQ1-3 '
31.引物/探针总HBV DNA(从基因组学欧陆有序,小号撕在-20 ℃下) 

HBV总额为:5 ' -GTGTCTGCGGCGTTTTATCA-3 '
总HBV启:5 ' - GACAAACGGGCAACATACCTT-3 '
总HBV探头:5 ' -FAM-TGAGGCATAGCAGCAGGATG-BHQ1-3 “
32. DG8墨盒(伯乐,目录号:186-4008) 

33. DG8垫片(伯乐,目录号:186-3009) 

34.液滴阅读器油(Bio-Rad ,目录号:186-3004) 

35.探针的液滴产生油(Bio-Rad ,目录号:186-3005) 

36.生长培养基(请参阅食谱) 

37.感染介质(请参见食谱) 

38.连接组合(请参阅规则) 

39.线性化混合(请参阅规则) 



设备


用于Rainin P20吸头的移液器(Rainin,目录号:17014392)
用于Rainin P200吸头的多通道移液器(Rainin,目录号:17013805)
热循环仪(Analytik Jena Biometra,FlexCycler 2 )
QX200 TM液滴发生器(伯乐,目录号:1864002)
PX1 PCR平板封口机(Bio-Rad ,目录号:1814000)
C1000触摸TM热循环仪96 -深井反应模块(Bio-Rad公司,目录号:1851197)
QX200 TM液滴读取器(Bio-Rad ,目录号:1864003)


软件


QuantaSoft(Bio-Rad )
微软Excel
棱镜(GraphPad)


程序


样品制备
感染前一天,以24孔格式在生长培养基中每孔播种2.5 × 10 5个细胞。
在含有4%PEG 8000感染介质感染细胞与HBV病毒储过夜(镍等人,2014) 。
在室温(RT)下用PBS洗涤细胞两次,并保持在感染培养基中直至所需的收获时间。
在室温下用500μlPBS洗涤细胞,并添加100μl胰蛋白酶。
在37°C下孵育直至细胞分离(〜5分钟)。
用900μl冷培养基重悬细胞,然后转移至1.5 ml Eppendorf管中。
在室温下以500 × g离心5分钟,然后除去上清液。
ø ptional:˚F reeze在-20粒料℃下直至进一步使用。


提取使用NucleoSpin细胞沉淀的总DNA ®根据制造商的说明Tissue试剂盒,但洗脱50微升洗脱缓冲液预热至70 ℃。


转化反应
使用DNA Zap溶液清洁硅密封垫,用去离子水冲洗,然后在纸巾上晾干。
制备限制性消化反应在96孔板中含有10微升提取的总DNA的,2微升的10 × Cutsmart缓冲液,0.5微升HHA I,0.25微升RecJ ˚F ,和7.25微升ħ 2 O.
用多通道移液器上下吸移10次进行混合,注意避免产生气泡。
用干净干燥的硅垫密封。
在热循环仪中运行以下程序:3个循环,分别在37 °C下15分钟和42 °C下15分钟,在80 °C下20分钟,并保持在16 °C。
雷莫已经垫Ç arefully并添加10微升的连接混合物。
用多通道移液器上下吸移10次进行混合,注意避免产生气泡。
在热循环仪中于16 °C孵育2小时,在80 °C孵育20分钟,并保持在16°C。
删除了仔细垫,并添加5微升的线性组合的。
用多通道移液器上下吸移10次进行混合,注意避免产生气泡。
在热循环仪中于37 °C孵育60分钟,在80 °C孵育20分钟,并保持在16 °C。
可选:S在-20°C下撕碎样品。


液滴的产生和阅读
通过加入52.5稀释样品2.5倍微升ħ 2 O.
用多通道移液器上下吸移10次进行混合,注意避免产生气泡。
为每个样品准备两个反应:(1)cccDNA和RNaseP细胞对照,以及(2)HBV DNA和RNaseP细胞对照。制备ddPCR混合在一个96孔板(22微升每个反应)(见表1)。


表1.每个反应的ddPCR混合物组成


试剂


专注


微升


ddPCR Supermix探针


2 ×


11


TaqMan TM拷贝数参考测定(VIC标记的RNaseP)


10 ×


1.1


正向引物(cccDNA或总HBV DNA)


100微米


0.033


反向引物(cccDNA或总HBV DNA)


100微米


0.033


FAM标记的探针(cccDNA或总HBV DNA)


100微米


0.033


高氧2


4.5


稀释模板


5.5


使用不含塑料的Rainin吸头。在ddPCR液滴生成器上生成液滴:
将20μl的ddPCR混合物添加到试剂盒的样品室中。
使用多通道移液器在油室内添加70μl液滴产生油。
附上橡胶垫圈,将墨盒放入液滴发生器中,然后运行液滴产生过程。
Gentl ÿ转移42微升使用多通道移液管将所生成的液滴的一个TwinTec 96孔板。
重复小号TEPS Ç 4a至Ç 4d中,直到所有的样本被加载到TwinTec 96孔板。
密封用aluminu板米箔密封使用的板盖。
直接密封后,在深阱PCR机与以下程序运行PCR:10分钟在95 ℃; 在95 °C下10 s,在54 °C下15 s和在68 °C下20 s的40个循环; 在95 °C下放置10分钟,并在12 °C下保持。
将TwinTec 96孔板转移到QX200 Droplet Reader。
使用Quantasoft软件(按照制造商的用户指南)设置模板,使用FAM(通道1)和VIC(通道2)的通道读取墨滴。
根据阳性和阴性对照定义阳性和阴性液滴的阈值,之后该软件将自动量化每个RNaseP的cccDNA值和每个RNaseP的总HBV DNA值。


数据分析


在Quantasoft软件中,我们使用2D图形绘制HBV DNA和RNaseP阳性液滴的阈值(图1)。所有HBV DNA总量和所有cccDNA测定的阈值分别设定。对于数据表示,我们使用每个样品的总HBV DNA / RNaseP和cccDNA / RNaseP比率,并具有PoissonRatioMin和PoissonRatioMax值,可得出ddPCR技术误差的95%置信区间。






图1.使用QuantaSoft进行数据分析。荧光强度用于分离含有扩增子的液滴,检测基于FM荧光(y轴)的HBV cccDNA(左)或HBV DNA总数(右);和RNaseP,基于VIC荧光(x轴)。基于阳性和阴性对照反应确定阳性信号(pin k)的阈值。(转自Tu等人,2020年a )。


笔记


我们已将该协议用于从以12孔至96孔板状感染的细胞中提取的DNA提取的DNA。在12孔板中,我们建议以ELUT Ë在两倍体积的DNA。
对于倒置,我们建议输入总DNA提取物<2 µg。如果总DNA输入小于200 ng,我们还建议添加200 ng载体DNA(例如无关的质粒DNA)。
我们通常以200个病毒基因组当量进行HBV感染。但是,由于该检测方法足够灵敏,可以检测到极少量的被感染细胞,因此也可以用较小的接种量进行感染(Tu等人,2020a )。
该协议也可用于基于Taqman的qPCR而不是ddPCR。
该测定仅与大多数体外HBV感染测定中使用的基因型D的HBV序列兼容。不幸的是,由于Hha I限制性位点的变化,它不适用于其他基因型。HBV的大多数体外测定都是使用D型基因株进行的,该缺点无法分析临床样品。
推荐的用于优化和质量控制的控件:
阳性对照以显示检测效率(总HBV DNA定量应与cccDNA定量相近):HBV circDNA (Mutz et al。,2018)或HBV 1.1超长质粒掺入未感染细胞提取的DNA中。
阴性对照显示cccDNA的选择性定量:从肝素纯化的病毒中提取的DNA (Seitz等,2016)掺入未感染细胞提取的DNA中。
阴性对照细胞感染期间确定来自病毒接种信号:细胞治疗进入抑制剂(例如,Myrcludex B)之前,HBV感染(Donkers等人,2017) 。
没有模板对照和未感染的细胞对照来确定非特异性扩增的水平。


菜谱


生长培养基
500毫升的DMEM


50毫升的FBS


5毫升的L-谷氨酰胺


5毫升的青霉素(10 ,000 U / ml)的和链霉素(10毫克/毫升)           

感染培养基
49毫升的生长培养基


1毫升的DMSO


连接混合物(10每个样品微升)
3微升的10毫摩尔ATP
1微升的10 × NEB缓冲Cutsmart


0.5微升的T4连接酶


5微升H 2 O


线性化混合物(每个样品5 µl)
0.5 µl的10 × Cutsmart缓冲液


0.5微升的XbaI位我


4微升H 2 O


致谢


该协议已在Tu等人中发表。(2020一)。Thi的工作获得了以下资金的资助:Deutsche Forschungsgemeinschaft(DFG,德国研究基金会)项目编号240245660 – SFB 1129(BZ和SU);德国感染研究中心(DZIF)TTU肝炎项目5.816和5.704(SU和TT);以及澳大利亚艾滋病毒和肝炎病毒学研究中心(TT)。感谢KathleenBörner博士对使用ddPCR设备的培训和支持。


利益争夺


斯蒂芬城市是共同申请人和共同发明人关于专利保护HBV的preS-衍生脂肽(bulevirtide / Hepcludex ® ,以前Myrcludex B)为IR使用作为HBV / HDV进入抑制剂。这项研究的其他作者宣称没有利益冲突。


参考


奥尔维斯(美国),沃尔兹(美国),吉尔施(K.),卡西(Kah),拉法(Raffa),彼得森(J.) ,M.和Dandri,M.(2018)。原代人肝细胞和预防乙型肝炎病毒再感染的扩散中有效消除核cccDNA的体内。肠道67(3):542-552。
Donkers,JM,Zehnder,B.,van Westen,GJP,Kwakkenbos,MJ,AP,IJ,Oude Elferink,RPJ,Beuers,U.,Urban,S.和van de Graaf,SFJ(2017)。使用临床应用的药物作为胆汁酸转运蛋白NTCP的新型抑制剂,可减少乙型和丁型肝炎病毒的进入。科学代表7(1):15307。
罗建。,崔旭。,高升,和胡建(2017)。鉴定乙型肝炎病毒中的一种中间体,其共价闭合环状(CCC)DNA形成以及灵敏和选择性的CCC DNA检测。J Virol 91(17):e00539-17。
Mutz,P.,Metz,P.,Lempp,FA,Bender,S.,Qu,B.,Schoneweis,K.,Seitz,S.,Tu,T.,Restuccia,A.,Frankish,J.,Dachert ,C.,Schusser,B.,Koschny,R.,Polychronidis,G.,Schemmer,P.,Hoffmann,K.,Baumert,TF,Binder,M.,Urban,S. and Bartenschlager,R.(2018) 。HBV绕过了先天的免疫反应,并没有保护HCV免受干扰素的抗病毒活性。胃肠病学154(6):1791-1804 e1722。
纳萨尔·M。(2015)。HBV cccDNA:病毒的持久性储存库和治愈慢性乙型肝炎的主要障碍。肠道64(12):1972-1984。
Ni,Y.,Lempp,FA,Mehrle,S.,Nkongolo,S.,Kaufman,C.,Falth,M.,Stindt,J.,Koniger,C.,Nassal,M.,Kubitz,R.,Sultmann ,H。和Urban,S.(2014)。乙型和丁型肝炎病毒利用牛磺胆酸钠共转运多肽使物种特异性进入肝细胞。胃肠病学146(4):1070-1083。
Qu,B.,Ni,Y.,Lempp,FA,Vondran,FWR和Urban,S.(2018)。乙型肝炎病毒复制中间体的T5核酸外切酶水解可通过PCR对共价闭合的环状DNA进行可靠的定量和快速药效测试。J Virol 92(23):e01117-18。
Seitz,S.,Iancu,C.,Volz,T.,Mier,W.,Dandri,M.,Urban,S.和Bartenschlager,R.(2016)。缓慢的成熟过程会导致乙肝病毒感染。细胞宿主微生物20(1):25-35。
Tu,T.,Zehnder,B.,Qu,B.,Ni,Y.,Main,N.,Allweiss,L.,Dandri,M.,Shackel,N.,George,J.和Urban,S.( 2020年a )。一种精确定量乙肝病毒共价闭合环状(ccc)DNA形成和维持的新方法。Antiviral Res 181:104865。
Tu,T.,Zehnder,B.,Qu,B. and Urban,S.(2020 b )。乙肝病毒核衣壳的从头合成对于cccDNA的维持和转录调控是必不可少的。JHEP报告3(1):100195。
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引用:Zehnder, B., Urban, S. and Tu, T. (2021). A Sensitive and Specific PCR-based Assay to Quantify Hepatitis B Virus Covalently Closed Circular (ccc) DNA while Preserving Cellular DNA. Bio-protocol 11(8): e3986. DOI: 10.21769/BioProtoc.3986.
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