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Development of a Novel Bioluminescence Pyrophosphate Assay for the High-Sensitivity Detection of Hepatitis B Virus

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Abstract  

The transmission of bloodborne viruses through transfusion remains a major blood supply–related safety concern, with hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) being the most important pathogens in this context. Real-time bioluminescent pyrophosphate testing has been developed as a means of readily detecting bacterial cells within particular sample types without requiring the use of expensive or complex instrumentation. The sensitivity of this approach, however, is often limited such that it is not compatible with many potential applications. In this study, we sought to overcome the limitations of this pyrophosphate bioluminescent assay format by using 2-deoxyadenosine-5-(α-thio)-triphosphate (dATPαS) in place of dATP for PCR amplification, thereby dramatically reducing background signal levels. We leveraged this combination PCR and bioluminescent pyrophosphate assay approach to facilitate HBV detection. This assay yielded a limit of detection of 500 copies/mL, making it more sensitive than traditional bioluminescent assays, about 1000 times more sensitive than that of PCR product analysis by agarose gel electrophoresis, and roughly as sensitive as qPCR as a means of detecting viral DNA. We then used this assay to analyze 100 serum samples, with qPCR being used for result validation. The assay required 100 min to complete, and was able to detect as few as 500 copies/mL of viral DNA. Overall, our approach was rapid, sensitive, and simple, enabling users to readily detect HBV in a reliable and efficient manner.

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Data availability

The data used or analyzed during the current study are available from the corresponding author on reasonable request.

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Funding

This work was funded by the National Natural Science Foundation of China, grant numbers 61971123 and 61571114, and Jiangsu Commission of Health, grant number LGY2020015.

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Authors and Affiliations

  1. The Second Hospital of Nanjing, Nanjing University of Chinese Medicine, Zhong Fu Lu 1#, Gulou District, Nanjing, 210003, China

    Guolei Tan & Xuping Wu

  2. State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Si Pai Lou 2#, Xuanwu District, Nanjing, 210096, China

    Zhongjie Fei, Rongbin Wei & Pengfeng Xiao

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  1. Guolei Tan
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  2. Zhongjie Fei
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  3. Rongbin Wei
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  4. Xuping Wu
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  5. Pengfeng Xiao
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Contributions

Conceptualization, methodology, software, data curation, writing—original draft preparation: GT, ZF, RW, PX, and XW. Visualization, investigation, validation: GT and ZF. Supervision, project administration, funding acquisition, and writing: PX and XW.

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Correspondence to Xuping Wu or Pengfeng Xiao.

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The authors declare no competing interests.

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Guolei Tan, Zhongjie Fei and Rongbin Wei contributed equally

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Tan, G., Fei, Z., Wei, R. et al. Development of a Novel Bioluminescence Pyrophosphate Assay for the High-Sensitivity Detection of Hepatitis B Virus. Appl Biochem Biotechnol 194, 725–736 (2022). https://doi.org/10.1007/s12010-021-03655-0

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  • DOI: https://doi.org/10.1007/s12010-021-03655-0

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