The Development of a Multienzyme Isothermal Rapid Amplification Assay to Visually Detect Duck Hepatitis B Virus

Shuqi Xu ¹, Yuanzhuo Man ¹, Xin Xu ¹

⁰Henan Provincial Engineering Laboratory of Insects Bioreactor, Henan Provincial Engineering and Technology Center of Health Products for Livestock and Poultry, Henan Provincial Engineering and Technology Center of Animal Disease Diagnosis and Integrated Control, Nanyang Normal University, Nanyang 473061, China.

Veterinary Sciences +

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May 24, 2024

View abstract on PubMed

Summary

A new rapid assay combining multienzyme isothermal rapid amplification (MIRA) and lateral flow dipstick (LFD) detects Duck hepatitis B virus (DHBV) in 15 minutes. This fast, sensitive method simplifies DHBV diagnostics for field use.

Area Of Science

Veterinary Virology
Molecular Diagnostics
Biotechnology

Background

Duck hepatitis B virus (DHBV) poses a significant prevalence issue in global duck and Chinese goose populations.
Current detection methods for DHBV are often time-consuming and necessitate specialized laboratory equipment, limiting their field applicability.

Purpose Of The Study

To develop a rapid, efficient, and field-deployable assay for the detection of Duck hepatitis B virus (DHBV).
To overcome the limitations of existing time-consuming and equipment-dependent diagnostic techniques for DHBV.

Main Methods

Development of a novel assay integrating multienzyme isothermal rapid amplification (MIRA) with lateral flow dipstick (LFD) technology.
Optimization of MIRA reaction conditions for DHBV detection at 38 °C for 10 minutes, eliminating the need for a temperature cycler.
Validation of the assay's specificity against common avian pathogens and assessment of its detection limit.

Main Results

The combined MIRA-LFD assay provides DHBV detection results in just 15 minutes, significantly faster than conventional polymerase chain reaction (PCR).
The assay requires no specialized equipment like electrophoresis or gel imaging systems, simplifying its use.
Achieved a detection limit of 45.6 copies per reaction, demonstrating approximately 10-fold higher sensitivity than routine PCR.
Demonstrated high specificity for DHBV, with no cross-reactivity observed against other tested avian viruses.

Conclusions

The developed MIRA-LFD assay offers a simple, rapid, and accurate method for identifying DHBV in clinical serum samples from ducks and geese.
This assay is highly suitable for widespread application in field clinics, improving disease surveillance and management.
The simplified primer and probe design contributes to the assay's efficiency and specificity compared to other isothermal amplification methods.

Keywords:

duck hepatitis B virus lateral flow dipstick multienzyme isothermal rapid amplification rapidity sensitivity visible detection