|
|
Establishment of a single-cell study method for HBV based on droplet digital PCR |
Hua-Jie MA1,MIn Wu2, 3,Zheng-Hong YuanZhigang Yi |
|
|
Abstract The single-cell study of hepatitis B virus (HBV) in liver tissue is very important, but the related research technology is still at an advanced stage and unmet clinical need. Based on droplet digital PCR, the study established a single-cell digital droplet PCR (sc-ddPCR) method for absolute quantification of HBV DNA and RNA at the single cell level. The linearity and lower detection limit of HBV DNA were analyzed by mixing different proportions of HBV positive and negative cells which simulate the liver under chronic HBV infection. Taking episome derived RNA (eRNA) as an example, the study further designed a detection protocol for RNA. By utilizing the structural differences of HBV transcripts, the study designed a specific primer and probe system for eRNA whose specificity was verified in HBV liver cancer cell lines. The linearity and lower detection limit of eRNA were analyzed using gradient diluted standard cell lines. The results showed that the study established a single-cell detection method for HBV DNA and RNA based on ddPCR, which showed good linearity (HBV DNA: R2=0.9998; eRNA: R2=0.9425), and gradient diluted standard cell lines could be accurately detected positive signals with high sensitivity (lower detection limit: HBV DNA=0.16%, eRNA=0.2%). The sc-ddPCR method established in this study can carry out multiple detection for DNA and RNA,which provides a good technical support for further research on HBV virology activities in liver tissue, is conducive to the optimization and development of HBV treatment strategies, and has broad application prospects.
|
Received: 08 May 2023
Published: 01 January 2023
|
|
Corresponding Authors:
Zhigang Yi
|
|
|
|
[1] |
Lee W M.Hepatitis B virus infection[J].N Engl J Med, 1997, 337(24):1733-45
|
[2] |
Nassal M.HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B [Z]. Gut. 2015: 1972-84.10.1136/gutjnl-2015-309809
|
[3] |
Ko C, Chakraborty A, Chou W M, Hasreiter J, Wettengel J M, Stadler D, Bester R, Asen T, Zhang K, Wisskirchen K, Mckeating J A, Ryu W S, Protzer U.Hepatitis B virus genome recycling and de novo secondary infection events maintain stable cccDNA levels[J].J Hepatol, 2018, 69(6):1231-41
|
[4] |
Martinez M G, Boyd A, Combe E, Testoni B, Zoulim F.Covalently closed circular DNA: The ultimate therapeutic target for curing HBV infections [Z]. J Hepatol. 2021: 706-17.10.1016/j.jhep.2021.05.013
|
[5] |
Rydell G E, Larsson S B, Prakash K, Andersson M, Norder H, Hellstrand K, Norkrans G, Lindh M.Abundance of Noncircular Intrahepatic Hepatitis B Virus DNA May Reflect Frequent Integration Into Human DNA in Chronically Infected Patients[J].J Infect Dis, 2022, 225(11):1982-90
|
[6] |
Meier M A, Calabrese D, Suslov A, Terracciano L M, Heim M H, Wieland S.Ubiquitous expression of HBsAg from integrated HBV DNA in patients with low viral load[J].J Hepatol, 2021, 75(4):840-7
|
[7] |
Zhang X, Lu W, Zheng Y, Wang W, Bai L, Chen L, Feng Y, Zhang Z, Yuan Z.In situ analysis of intrahepatic virological events in chronic hepatitis B virus infection[J].J Clin Invest, 2016, 126(3):1079-92
|
[8] |
Bustamante-Jaramillo L F, Fingal J, Blondot M L, Rydell G E, Kann M.Imaging of Hepatitis B Virus Nucleic Acids: Current Advances and Challenges [Z]. Viruses. 2022.10.3390/v14030557
|
[9] |
Peng B, Jing Z, Zhou Z, Sun Y, Guo G, Tan Z, Diao Y, Yao Q, Ping Y, Li X, Ren T, Li B, Li W.Nonproductive Hepatitis B Virus Covalently Closed Circular DNA Generates HBx-Related Transcripts from the HBxEnhancer I Region and Acquires Reactivation by Superinfection in Single Cells[J].J Virol, 2023, 97(1):e0171722-
|
[10] |
Balagopal A, Hwang H S, Grudda T, Quinn J, Sterling R K, Sulkowski M S, Thio C L.Single Hepatocyte Hepatitis B Virus Transcriptional Landscape in HIV Coinfection[J].J Infect Dis, 2020, 221(9):1462-9
|
[11] |
Seeger C, Mason W S.Molecular biology of hepatitis B virus infection [Z]. Virology. 2015: 672-86.10.1016/j.virol.2015.02.031
|
[12] |
Stadelmayer B, Diederichs A, Chapus F, Rivoire M, Neveu G, Alam A, Fraisse L, Carter K, Testoni B, Zoulim F.Full-length 5'RACE identifies all major HBV transcripts in HBV-infected hepatocytes and patient serum[J].J Hepatol, 2020, 73(1):40-51
|
[13] |
Mason W S, Low H C, Xu C, Aldrich C E, Scougall C A, Grosse A, Clouston A, Chavez D, Litwin S, Peri S, Jilbert A R, Lanford R E.Detection of clonally expanded hepatocytes in chimpanzees with chronic hepatitis B virus infection[J].J Virol, 2009, 83(17):8396-408
|
[14] |
Mason W S, Liu C, Aldrich C E, Litwin S, Yeh M M.Clonal expansion of normal-appearing human hepatocytes during chronic hepatitis B virus infection[J].J Virol, 2010, 84(16):8308-15
|
[15] |
Zhang Y Y, Zhang B H, Theele D, Litwin S, Toll E, Summers J.Single-cell analysis of covalently closed circular DNA copy numbers in a hepadnavirus-infected liver[J].Proc Natl Acad Sci U S A, 2003, 100(21):12372-7
|
[16] |
Grudda T, Hwang H S, Taddese M, Quinn J, Sulkowski M S, Sterling R K, Balagopal A, Thio C L.Integrated hepatitis B virus DNA maintains surface antigen production during antiviral treatment [J]. J Clin Invest, 2022, 132(18).[J].J Clin Invest, 2022, 132(18):-
|
[17] |
Balagopal A, Grudda T, Ribeiro R M, Saad Y S, Hwang H S, Quinn J, Murphy M, Ward K, Sterling R K, Zhang Y, Perelson A S, Sulkowski M S, Osburn W O, Thio C L.Single hepatocytes show persistence and transcriptional inactivity of hepatitis B [J]. JCI Insight, 2020, 5(19).[J].JCI Insight, 2020, 5(19):-
|
[18] |
Zhu Z, Zhang W, Leng X, Zhang M, Guan Z, Lu J, Yang C J.Highly sensitive and quantitative detection of rare pathogens through agarose droplet microfluidic emulsion PCR at the single-cell level[J].Lab Chip, 2012, 12(20):3907-13
|
[19] |
Kumaresan P, Yang C J, Cronier S A, Blazej R G, Mathies R A.High-throughput single copy DNA amplification and cell analysis in engineered nanoliter droplets[J].Anal Chem, 2008, 80(10):3522-9
|
[20] |
Geng T, Novak R, Mathies R A.Single-cell forensic short tandem repeat typing within microfluidic droplets[J].Anal Chem, 2014, 86(1):703-12
|
[21] |
Novak R, Zeng Y, Shuga J, Venugopalan G, Fletcher D A, Smith M T, Mathies R A.Single-cell multiplex gene detection and sequencing with microfluidically generated agarose emulsions[J].Angew Chem Int Ed Engl, 2011, 50(2):390-5
|
[22] |
Clark I C, Wheeler M A, Lee H G, Li Z, Sanmarco L M, Thaploo S, Polonio C M, Shin S W, Scalisi G, Henry A R, Rone J M, Giovannoni F, Charabati M, Akl C F, Aleman D M, Zandee S E J, Prat A, Douek D C, Boritz E A, Quintana F J, Abate A R.Identification of astrocyte regulators by nucleic acid cytometry[J].Nature, 2023, 614(7947):326-33
|
[23] |
Erken R, Loukachov V, Van Dort K, Van Den Hurk A, Takkenberg R B, De Niet A, Jansen L, Willemse S, Reesink H, Kootstra N.Quantified integrated hepatitis B virus is related to viral activity in patients with chronic hepatitis B[J].Hepatology, 2022, 76(1):196-206
|
[24] |
Van Buuren N, Ramirez R, Soulette C, Suri V, Han D, May L, Turner S, Parvangada P C, Martin R, Chan H L Y, Marcellin P, Buti M, Bui N, Bhardwaj N, Gaggar A, Li L, Mo H, Feierbach B.Targeted long-read sequencing reveals clonally expanded HBV-associated chromosomal translocations in patients with chronic hepatitis B[J].JHEP Rep, 2022, 4(4):100449
|
|
|
|