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A novel one-step quantitative reverse transcription PCR assay for selective amplification of hepatitis B virus pregenomic RNA from a mixture of HBV DNA and RNA in serum

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Abstract

Current antiviral therapies against hepatitis B virus (HBV) infections, such as treatment with nucleos(t)ide analogs (NAs) and interferon alpha, can significantly lower HBV DNA titers, eventually to undetectable levels. However, it is still difficult to completely eliminate the stable template of HBV, the covalently closed circular DNA (cccDNA), and this contributes to viral rebound when treatment is discontinued. HBV pregenomic RNA (pgRNA), which was recently found to be present in the enveloped mature HBV viral particle in blood, is tentatively regarded, with still accumulating clinical evidence, as a novel bona fide virological marker reflecting the amount and status of cccDNA when serum HBV DNA becomes undetectable. HBV pgRNA and DNA share almost identical sequences, and it is therefore difficult to differentiate pgRNA from viral DNA using normal PCR methods. To exclude interference from viral DNA, methods for measuring pgRNA usually require a selective DNA degradation step, which is complicated and time-consuming and also compromises the accuracy of detection. In this study, we developed a simplified quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay with improved accuracy achieved by probing the polyA tail of pgRNA. Using clinical serum samples, we observed that not all patients share the same 3′ sequence, suggesting slight differences between HBV strains in the way they end transcription. We then designed and evaluated a universal primer and probe set for distinguishing HBV pgRNA from HBV DNA. Our results demonstrated that a one-step qRT-PCR assay could selectively amplify HBV pgRNA from a mixture of HBV RNA and DNA, which is valuable for clinical applications.

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Acknowledgments

We thank all of the study participants for their donation of blood samples. This work was supported by the Guangzhou Science and Technology Program (No. 201707010348), by a National Natural Science Foundation of China Grant (No. 81670536 and 81770593) and by the National Grand Program on Key Infectious Disease Control (2017ZX10202203-004-002).

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Author notes

  1. Ming Gao, Chengqian Feng are contributed equally to the work.

Authors and Affiliations

  1. Guangzhou Eighth People’s Hospital, Guangzhou Medical University, 627 Dongfeng Dong Rd, Guangzhou, 510060, Guangdong, China

    Ming Gao, Chengqian Feng, Ruosu Ying, Yuan Nie, Xizi Deng, Ying Zhu, Xiaoping Tang, Yujuan Guan, Fengyu Hu & Feng Li

Authors
  1. Ming Gao
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  2. Chengqian Feng
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  3. Ruosu Ying
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  4. Yuan Nie
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Contributions

Ming Gao and Chengqian Feng performed detection, conditional optimization, and data analysis. Ruosu Ying, Ying Zhu and Yujuan Guan managed patient sample collection from the clinic. Yuan Nie and Xizi Deng helped generate the standard. Fengyu Hu and Feng Li conceived, designed, and supervised the study. Xiaoping Tang contributed critical intelligence to review the study.

Corresponding authors

Correspondence to Fengyu Hu or Feng Li.

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Handling Editor: Zhongjie Shi.

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Gao, M., Feng, C., Ying, R. et al. A novel one-step quantitative reverse transcription PCR assay for selective amplification of hepatitis B virus pregenomic RNA from a mixture of HBV DNA and RNA in serum. Arch Virol 164, 2683–2690 (2019). https://doi.org/10.1007/s00705-019-04372-0

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