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Development and application of a real-time VPCR method for ultra-fast quantitative analysis and Visual Detection of HBV-DNA.

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Τίτλος: Development and application of a real-time VPCR method for ultra-fast quantitative analysis and Visual Detection of HBV-DNA.
Συγγραφείς: Zhao T; School of pharmacy, School of Ethnic Medicine, Chinese Medicine Germplasm Resources Innovation and Effective Uses Key Laboratory of Sichuan Province, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China., Peng T; School of pharmacy, School of Ethnic Medicine, Chinese Medicine Germplasm Resources Innovation and Effective Uses Key Laboratory of Sichuan Province, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China., Cui J; School of pharmacy, School of Ethnic Medicine, Chinese Medicine Germplasm Resources Innovation and Effective Uses Key Laboratory of Sichuan Province, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China., Chen Y; School of pharmacy, School of Ethnic Medicine, Chinese Medicine Germplasm Resources Innovation and Effective Uses Key Laboratory of Sichuan Province, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China., Song W; School of pharmacy, School of Ethnic Medicine, Chinese Medicine Germplasm Resources Innovation and Effective Uses Key Laboratory of Sichuan Province, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China., Chen H; School of pharmacy, School of Ethnic Medicine, Chinese Medicine Germplasm Resources Innovation and Effective Uses Key Laboratory of Sichuan Province, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China., Wu B; Department of Clinical Laboratory, Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China., Chen R; School of pharmacy, School of Ethnic Medicine, Chinese Medicine Germplasm Resources Innovation and Effective Uses Key Laboratory of Sichuan Province, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China.
Πηγή: Journal of clinical microbiology [J Clin Microbiol] 2026 Sep 09; Vol. 64 (9), pp. e0015426. Date of Electronic Publication: 2026 Aug 14.
Τύπος έκδοσης: Journal Article; Evaluation Study
Γλώσσα: English
Στοιχεία περιοδικού: Publisher: American Society for Microbiology Country of Publication: United States NLM ID: 7505564 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1098-660X (Electronic) Linking ISSN: 00951137 NLM ISO Abbreviation: J Clin Microbiol Subsets: MEDLINE
Imprint Name(s): Original Publication: Washington, American Society for Microbiology.
Ιατρικοί όροι (MeSH): Hepatitis B virus*/genetics , Hepatitis B virus*/isolation & purification , DNA, Viral*/genetics , DNA, Viral*/isolation & purification , Hepatitis B*/diagnosis , Hepatitis B*/virology , Real-Time Polymerase Chain Reaction*/methods , Molecular Diagnostic Techniques*/methods , Viral Load*/methods, Humans ; Sensitivity and Specificity ; Reproducibility of Results ; Rapid Diagnostic Tests ; Time Factors
Περίληψη: Hepatitis B virus (HBV) infection remains a major global health challenge, necessitating rapid and accurate diagnostic methods for effective disease management. Although quantitative real-time PCR (qPCR) is widely used, its detection time is relatively long. We developed an ultra-fast HBV-DNA quantification system based on rapid VPCR technology, which, combined with a 10-min nucleic acid extraction process, reduces the entire workflow to approximately 35 min. Crucially, despite this significant increase in speed, the system maintains analytical sensitivity, specificity, and reproducibility comparable to those of conventional qPCR. The system's performance was then evaluated using 143 clinical samples and compared to that of standard qPCR, demonstrating that the developed real-time VPCR system significantly shortened the amplification time while maintaining excellent performance, with 100% sensitivity and 97.09% specificity. Additionally, a cost-effective qualitative endpoint visual detection method based on probe cleavage was established, offering an alternative readout strategy suitable for HBV screening in resource-limited settings without requiring expensive instruments. Collectively, this study presents a rapid quantification system and a qualitative visual detection method that significantly enhance diagnostic efficiency, providing strong technical support for clinical HBV management and screening.IMPORTANCEThe rapid hepatitis B virus (HBV) quantification system established in this study completes the entire workflow within 35 min and delivers excellent performance, meeting the demand for rapid decision-making in time-sensitive scenarios such as emergency care and acute liver failure. Its "test-and-treat" workflow is particularly suitable for patients in remote areas, allowing diagnosis and treatment in a single visit. Meanwhile, the accompanying visual qualitative method provides a low-cost screening strategy for resource-limited settings. This system greatly improves detection efficiency and clinical accessibility, showing important application value for the rapid diagnosis of hepatitis B and other pathogens.
Competing Interests: The authors declare no conflict of interest.
References: J Med Virol. 2001 Oct;65(2):250-6. (PMID: 11536230)
Lab Chip. 2021 Jul 13;21(14):2730-2737. (PMID: 34100058)
Clin Infect Dis. 2014 Jul 1;59(1):16-23. (PMID: 24704724)
Nat Rev Dis Primers. 2018 Jun 07;4:18035. (PMID: 29877316)
Theranostics. 2019 Feb 28;9(6):1572-1579. (PMID: 31037124)
J Med Virol. 2023 Oct;95(10):e29144. (PMID: 37796091)
Anal Chim Acta. 2023 Mar 8;1245:340864. (PMID: 36737140)
Clin Microbiol Infect. 2021 Mar;27(3):443-450. (PMID: 32360447)
3 Biotech. 2021 Dec;11(12):493. (PMID: 34881156)
Anal Chem. 2023 Jul 4;95(26):10127-10135. (PMID: 37326604)
Front Bioeng Biotechnol. 2021 Oct 07;9:743322. (PMID: 34692662)
J Virol Methods. 2021 May;291:114094. (PMID: 33549573)
Talanta. 2021 Jan 1;221:121661. (PMID: 33076171)
J Hepatol. 2022 Jun;76(6):1249-1262. (PMID: 35589248)
Emerg Microbes Infect. 2023 Dec;12(1):e2177088. (PMID: 36735916)
BMC Public Health. 2021 Feb 19;21(1):388. (PMID: 33607996)
Front Bioeng Biotechnol. 2022 May 19;10:895236. (PMID: 35662850)
BMC Infect Dis. 2017 Nov 1;17(Suppl 1):702. (PMID: 29143612)
Lancet Glob Health. 2019 Sep;7(9):e1180-e1188. (PMID: 31353061)
Biosens Bioelectron. 2023 Jan 1;219:114790. (PMID: 36274427)
BMC Gastroenterol. 2021 Jul 31;21(1):306. (PMID: 34332532)
Gut. 2023 May;72(5):972-983. (PMID: 36707234)
Grant Information: 82374001 National Natural Science Foundation of China
Contributed Indexing: Keywords: fluorescence visual detection; hepatitis B virus infection; quantitative detection system; real-time VPCR
Substance Nomenclature: 0 (DNA, Viral)
Entry Date(s): Date Created: 20260814 Date Completed: 20260909 Latest Revision: 20260911
Update Code: 20260911
PubMed Central ID: PMC13556246
DOI: 10.1128/jcm.00154-26
PMID: 42599174
Βάση Δεδομένων: MEDLINE
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  Data: Development and application of a real-time VPCR method for ultra-fast quantitative analysis and Visual Detection of HBV-DNA.
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  Data: <searchLink fieldCode="AU" term="%22Zhao+T%22">Zhao T</searchLink>; School of pharmacy, School of Ethnic Medicine, Chinese Medicine Germplasm Resources Innovation and Effective Uses Key Laboratory of Sichuan Province, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China.<br /><searchLink fieldCode="AU" term="%22Peng+T%22">Peng T</searchLink>; School of pharmacy, School of Ethnic Medicine, Chinese Medicine Germplasm Resources Innovation and Effective Uses Key Laboratory of Sichuan Province, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China.<br /><searchLink fieldCode="AU" term="%22Cui+J%22">Cui J</searchLink>; School of pharmacy, School of Ethnic Medicine, Chinese Medicine Germplasm Resources Innovation and Effective Uses Key Laboratory of Sichuan Province, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China.<br /><searchLink fieldCode="AU" term="%22Chen+Y%22">Chen Y</searchLink>; School of pharmacy, School of Ethnic Medicine, Chinese Medicine Germplasm Resources Innovation and Effective Uses Key Laboratory of Sichuan Province, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China.<br /><searchLink fieldCode="AU" term="%22Song+W%22">Song W</searchLink>; School of pharmacy, School of Ethnic Medicine, Chinese Medicine Germplasm Resources Innovation and Effective Uses Key Laboratory of Sichuan Province, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China.<br /><searchLink fieldCode="AU" term="%22Chen+H%22">Chen H</searchLink>; School of pharmacy, School of Ethnic Medicine, Chinese Medicine Germplasm Resources Innovation and Effective Uses Key Laboratory of Sichuan Province, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China.<br /><searchLink fieldCode="AU" term="%22Wu+B%22">Wu B</searchLink>; Department of Clinical Laboratory, Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China.<br /><searchLink fieldCode="AU" term="%22Chen+R%22">Chen R</searchLink>; School of pharmacy, School of Ethnic Medicine, Chinese Medicine Germplasm Resources Innovation and Effective Uses Key Laboratory of Sichuan Province, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China.
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  Data: <searchLink fieldCode="JN" term="%227505564%22">Journal of clinical microbiology</searchLink> [J Clin Microbiol] 2026 Sep 09; Vol. 64 (9), pp. e0015426. <i>Date of Electronic Publication: </i>2026 Aug 14.
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  Data: <i>Publisher: </i><searchLink fieldCode="PB" term="%22American+Society+for+Microbiology%22">American Society for Microbiology </searchLink><i>Country of Publication: </i>United States <i>NLM ID: </i>7505564 <i>Publication Model: </i>Print-Electronic <i>Cited Medium: </i>Internet <i>ISSN: </i>1098-660X (Electronic) <i>Linking ISSN: </i><searchLink fieldCode="IS" term="%2200951137%22">00951137 </searchLink><i>NLM ISO Abbreviation: </i>J Clin Microbiol <i>Subsets: </i>MEDLINE
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  Data: <i>Original Publication</i>: Washington, American Society for Microbiology.
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  Data: <searchLink fieldCode="MM" term="%22Hepatitis+B+virus%22">Hepatitis B virus*</searchLink>/<searchLink fieldCode="MM" term="%22Hepatitis+B+virus+genetics%22">genetics</searchLink> <br /><searchLink fieldCode="MM" term="%22Hepatitis+B+virus%22">Hepatitis B virus*</searchLink>/<searchLink fieldCode="MM" term="%22Hepatitis+B+virus+isolation+%26+purification%22">isolation & purification</searchLink> <br /><searchLink fieldCode="MM" term="%22DNA%2C+Viral%22">DNA, Viral*</searchLink>/<searchLink fieldCode="MM" term="%22DNA%2C+Viral+genetics%22">genetics</searchLink> <br /><searchLink fieldCode="MM" term="%22DNA%2C+Viral%22">DNA, Viral*</searchLink>/<searchLink fieldCode="MM" term="%22DNA%2C+Viral+isolation+%26+purification%22">isolation & purification</searchLink> <br /><searchLink fieldCode="MM" term="%22Hepatitis+B%22">Hepatitis B*</searchLink>/<searchLink fieldCode="MM" term="%22Hepatitis+B+diagnosis%22">diagnosis</searchLink> <br /><searchLink fieldCode="MM" term="%22Hepatitis+B%22">Hepatitis B*</searchLink>/<searchLink fieldCode="MM" term="%22Hepatitis+B+virology%22">virology</searchLink> <br /><searchLink fieldCode="MM" term="%22Real-Time+Polymerase+Chain+Reaction%22">Real-Time Polymerase Chain Reaction*</searchLink>/<searchLink fieldCode="MM" term="%22Real-Time+Polymerase+Chain+Reaction+methods%22">methods</searchLink> <br /><searchLink fieldCode="MM" term="%22Molecular+Diagnostic+Techniques%22">Molecular Diagnostic Techniques*</searchLink>/<searchLink fieldCode="MM" term="%22Molecular+Diagnostic+Techniques+methods%22">methods</searchLink> <br /><searchLink fieldCode="MM" term="%22Viral+Load%22">Viral Load*</searchLink>/<searchLink fieldCode="MM" term="%22Viral+Load+methods%22">methods</searchLink><br /><searchLink fieldCode="MH" term="%22Humans%22">Humans</searchLink> ; <searchLink fieldCode="MH" term="%22Sensitivity+and+Specificity%22">Sensitivity and Specificity</searchLink> ; <searchLink fieldCode="MH" term="%22Reproducibility+of+Results%22">Reproducibility of Results</searchLink> ; <searchLink fieldCode="MH" term="%22Rapid+Diagnostic+Tests%22">Rapid Diagnostic Tests</searchLink> ; <searchLink fieldCode="MH" term="%22Time+Factors%22">Time Factors</searchLink>
– Name: Abstract
  Label: Abstract
  Group: Ab
  Data: Hepatitis B virus (HBV) infection remains a major global health challenge, necessitating rapid and accurate diagnostic methods for effective disease management. Although quantitative real-time PCR (qPCR) is widely used, its detection time is relatively long. We developed an ultra-fast HBV-DNA quantification system based on rapid VPCR technology, which, combined with a 10-min nucleic acid extraction process, reduces the entire workflow to approximately 35 min. Crucially, despite this significant increase in speed, the system maintains analytical sensitivity, specificity, and reproducibility comparable to those of conventional qPCR. The system's performance was then evaluated using 143 clinical samples and compared to that of standard qPCR, demonstrating that the developed real-time VPCR system significantly shortened the amplification time while maintaining excellent performance, with 100% sensitivity and 97.09% specificity. Additionally, a cost-effective qualitative endpoint visual detection method based on probe cleavage was established, offering an alternative readout strategy suitable for HBV screening in resource-limited settings without requiring expensive instruments. Collectively, this study presents a rapid quantification system and a qualitative visual detection method that significantly enhance diagnostic efficiency, providing strong technical support for clinical HBV management and screening.IMPORTANCEThe rapid hepatitis B virus (HBV) quantification system established in this study completes the entire workflow within 35 min and delivers excellent performance, meeting the demand for rapid decision-making in time-sensitive scenarios such as emergency care and acute liver failure. Its "test-and-treat" workflow is particularly suitable for patients in remote areas, allowing diagnosis and treatment in a single visit. Meanwhile, the accompanying visual qualitative method provides a low-cost screening strategy for resource-limited settings. This system greatly improves detection efficiency and clinical accessibility, showing important application value for the rapid diagnosis of hepatitis B and other pathogens.
– Name: Abstract
  Label: Competing Interests
  Group: Ab
  Data: The authors declare no conflict of interest.
– Name: Ref
  Label: References
  Group: RefInfo
  Data: J Med Virol. 2001 Oct;65(2):250-6. (PMID: <searchLink fieldCode="PM" term="%2211536230%22">11536230)</searchLink><br />Lab Chip. 2021 Jul 13;21(14):2730-2737. (PMID: <searchLink fieldCode="PM" term="%2234100058%22">34100058)</searchLink><br />Clin Infect Dis. 2014 Jul 1;59(1):16-23. (PMID: <searchLink fieldCode="PM" term="%2224704724%22">24704724)</searchLink><br />Nat Rev Dis Primers. 2018 Jun 07;4:18035. (PMID: <searchLink fieldCode="PM" term="%2229877316%22">29877316)</searchLink><br />Theranostics. 2019 Feb 28;9(6):1572-1579. (PMID: <searchLink fieldCode="PM" term="%2231037124%22">31037124)</searchLink><br />J Med Virol. 2023 Oct;95(10):e29144. (PMID: <searchLink fieldCode="PM" term="%2237796091%22">37796091)</searchLink><br />Anal Chim Acta. 2023 Mar 8;1245:340864. (PMID: <searchLink fieldCode="PM" term="%2236737140%22">36737140)</searchLink><br />Clin Microbiol Infect. 2021 Mar;27(3):443-450. (PMID: <searchLink fieldCode="PM" term="%2232360447%22">32360447)</searchLink><br />3 Biotech. 2021 Dec;11(12):493. (PMID: <searchLink fieldCode="PM" term="%2234881156%22">34881156)</searchLink><br />Anal Chem. 2023 Jul 4;95(26):10127-10135. (PMID: <searchLink fieldCode="PM" term="%2237326604%22">37326604)</searchLink><br />Front Bioeng Biotechnol. 2021 Oct 07;9:743322. (PMID: <searchLink fieldCode="PM" term="%2234692662%22">34692662)</searchLink><br />J Virol Methods. 2021 May;291:114094. (PMID: <searchLink fieldCode="PM" term="%2233549573%22">33549573)</searchLink><br />Talanta. 2021 Jan 1;221:121661. (PMID: <searchLink fieldCode="PM" term="%2233076171%22">33076171)</searchLink><br />J Hepatol. 2022 Jun;76(6):1249-1262. (PMID: <searchLink fieldCode="PM" term="%2235589248%22">35589248)</searchLink><br />Emerg Microbes Infect. 2023 Dec;12(1):e2177088. (PMID: <searchLink fieldCode="PM" term="%2236735916%22">36735916)</searchLink><br />BMC Public Health. 2021 Feb 19;21(1):388. (PMID: <searchLink fieldCode="PM" term="%2233607996%22">33607996)</searchLink><br />Front Bioeng Biotechnol. 2022 May 19;10:895236. (PMID: <searchLink fieldCode="PM" term="%2235662850%22">35662850)</searchLink><br />BMC Infect Dis. 2017 Nov 1;17(Suppl 1):702. (PMID: <searchLink fieldCode="PM" term="%2229143612%22">29143612)</searchLink><br />Lancet Glob Health. 2019 Sep;7(9):e1180-e1188. (PMID: <searchLink fieldCode="PM" term="%2231353061%22">31353061)</searchLink><br />Biosens Bioelectron. 2023 Jan 1;219:114790. (PMID: <searchLink fieldCode="PM" term="%2236274427%22">36274427)</searchLink><br />BMC Gastroenterol. 2021 Jul 31;21(1):306. (PMID: <searchLink fieldCode="PM" term="%2234332532%22">34332532)</searchLink><br />Gut. 2023 May;72(5):972-983. (PMID: <searchLink fieldCode="PM" term="%2236707234%22">36707234)</searchLink>
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